Purifying RNA and mRNA by ridzzz

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									Purifying RNA and mRNA
Purification Requirements                                    Do you need total RNA or poly(A)+ RNA for your
                                                             application? For most applications, total RNA is
The purity and integrity of RNA is critical to the success   sufficient, but in some
of any RNA-based analysis. Purification methods must          cases the added
                                                                                                RN
                                                                                        Ribonucle asin
                                                                                                                ®
protect RNA during and after purification.                    sensitivity of poly(A)+
                                                             RNA is beneficial. This               ase
                                                                                         protects Inhibitor
To successfully isolate intact RNA by any procedure,
the methods must:                                            is especially true if the
                                                             RNA is intended for        RNA in      purified
1. Disrupt cells or tissue.                                                                      downs
                                                                                            applicatio tream
                                                             cDNA library
2. Denature the nucleic acid:protein complexes.              construction or
3. Inactivate endogenous ribonucleases.                      detection of very
                                                                                                      ns.
                                                                                           See page
4. Purify the RNA from contaminating DNA and                 rare messages.                            3
   protein.



 Look for these symbols to find the system                    Promega has a variety of solutions for your RNA
 right for your application.                                 purification needs. All systems produce high-quality
                                                             RNA ready for use from a variety of starting materials.
                                                             The PureYield™ RNA Midiprep System uses a novel
                                  Animal Tissue              procedure for removal of genomic DNA from the RNA
                                                             prep without DNase treatment. The RNA is ideally suited
                                                             for any application influenced by the presence of
                                                             genomic DNA, such as quantitative, real-time RT-PCR.
                                  Cultured Cells             The SV, SV 96 and MagneSil® Total RNA Isolation
                                                             Systems include DNase for in-process genomic DNA
                                                             removal. Both the SV 96 and MagneSil RNA Systems
                                                             can be automated for high-throughput purification. The
                                  Blood                      RNAgents® System is a total scalable solutions-based
                                                             system that uses organic extraction to produce high
                                                             quality RNA.

                                   Plant Tissue

                                                             Please note, in the flow charts that follow, Promega systems
                                                             are listed based on the protocols are provided in the Technical
                                                             Literature sent with each system. Other applications with other
                                   Bacteria
                                                             source materials may exist.
                                                             Please contact Promega Technical Services if you have
                                                             questions: techserv@promega.com
                                   Yeast
                              5584MA




www.promega.com • techserv@promega.com                                                                                    7
         Purifying RNA and mRNA
                                                                                            Unique p
                                                                                                    r
                                              Animal                                     Novel cle oduct!
                                              Tissue                                    removes aring agent
                                                                                                all
                                                                                           genomic detectable
                                                                                                    DN
                                                                                             No DNas A.
                                                                                        treatme      e
                                                                                               nt need
                                         Automated                      Manual                         ed!


                                                                                             PureYield™ RNA
                                                                                             Midiprep System
                 MagneSil® Total RNA                 SV 96 Total RNA
                 mini-Isolation System               Isolation System

                                                                                               SV Total RNA
                                                                                             Isolation System
                                                                         o
      Can proc                                                   cluded t
    2mg of
                ess up t
                         o                              DNase in reduce
             tissue ly                                    greatly
                       sate                                           -over.
     in 200µ
              l of                                       gDN A carry                        RNAgents® Total

       buffer in lysis
                                                                                           RNA Isolation system

                 t
     96-well he
               format
                                                                                            PolyATtract® System
                                                                               d
                                                                     tion-base                     1000




                                                                                                                  5585MA
                                                       Sca lable solu
                                                                     that can
                                                          reagent
                                                                        o 1g of
                                                          proc ess up t          p
                                                                        ingle pre               Go from
                                                         tissue in a s                                    tiss
                                                                                                 poly(A)+ ue to
                                                                                                           RNA
                                                                                                    directly
                                                                                                            .


         The SV Total RNA Isolation System has been cited for total
         RNA isolation from a wide variety of mammalian tissues
         including heart, lung, kidney, brain, eye, colon, small intestine,
         muscle, cochlea, stomach, placenta and thymus. Some more
         exotic tissues include:
                            snake venom sacs & glands
                                    insect larvae
                                   whole water flea
                                    zebrafish gut
                                      carp eggs
                                 frog liver & gonads
                             chicken heart, leg & brain
                                 marine polychaetes
         For more information, check the online citations
         database at: www.promega.com/citations or contact
         Technical Services at: techserv@promega.com



8                                                                                    Promega RNA Analysis Notebook
Purifying RNA and mRNA
                                                                                           Unique p
                                                                                                   r
                                   Cultured                                             Novel cle oduct!
                                      Cells                                            removes aring agent
                                                                                               all
                                                                                          genomic detectable
                                                                                                   DN
                                                                                            No DNas A.
                                                                                       treatme      e
                                                                                              nt need
                                Automated                      Manual
                                                                                                      ed!


                                                                                              PureYield™ RNA
                                                                                              Midiprep System
        MagneSil® Total RNA                 SV 96 Total RNA
        mini-Isolation System               Isolation System

                                                                                                SV Total RNA
                                                                                              Isolation System
                                               DNase in
          ess: ~1
                  05                            greatly cluded to
       oc                                               reduce
Can pr             ll
           96-we                              gDNA c                                         RNAgents® Total
 c ells in 3 cells in                                 arry-ove
                                                              r.                            RNA Isolation system
        ; s10
format         format
  38  4-well
                                                 Scalable                                   PolyATtract® System
                                                          solution-b
                                                                    ased




                                                                                                                    5586MA
                                                                                                   1000
                                                   reagent
                                                            that can
                                               process
                                                       up to 108
                                                  in a sing        cells
                                                           le prep
                                                                                              Go from tissue to
                                                                                               poly(A)+ RNA
Precipitation of RNA Samples                                                                       directly.
Precipitation of RNA is commonly used for purification
or concentration purposes. To ensure maximum
recovery when precipitating small quantities (<1µg) or
dilute solutions of RNA (<1µg/µl), it is helpful to add a
carrier such as RNase-free glycogen. Glycogen does not
participate in reactions that use nucleic acids as
substrate, thus eliminating the background that can
occur from the use of nucleic acid carriers during
precipitation. In addition to maximizing the precipitation        The SV 96 Total RNA Isolation System is most commonly
of RNA, the glycogen will add mass to the RNA pellet              cited for total RNA isolation from cultured cells. The system
(making it easier to see). Simply add 20µg of molecular-          is ideally suited for researchers desiring to go from a 96-well
biology-grade glycogen to your sample prior to                    cell culture plate to 96 purified total RNA samples. The
precipitation. RNA can also be selectively precipitated           system is vacuum dependent and does not require a
from DNA by adding an equal volume of 5M ammonium                 centrifuge and the protocol can be performed manually or
acetate and incubating on ice for 20 minutes. Lithium             can be automated on a variety of liquid handlers.
chloride can be substituted for Ammonium Acetate as
LiCl exhibits preferential precipitation of RNA in the
presence of DNA and protein (5).




www.promega.com • techserv@promega.com                                                                                         9
     Purifying RNA and mRNA
                                                                                                                    ld
                                             Blood                                                         RNA yieod
                                                                                                           from blo ith
                                                                                                           varies w count
                                                                                                                 ell
                                       Automated                           Manual                         white c


              MagneSil® Total RNA                   PureYield™ RNA                             SV Total RNA
              mini-Isolation System                 Midiprep System                          Isolation System




                                                                                                                      luded to
                                                                        RNAgents® Total
                                                                      RNA Isolation System
                                                                                                              Nase inc duce




                                                                                                 5587MA
                                                                                                            D
                                      roduct!                                                                 greatly
                                                                                                                      re
                            Unique p          nt                                                                         y-over.
                                      ring age                                                                  NA carr
                          Novel clea detectable                                                              gD
                                   all
                          removes mic DNA.                         Scalable
                                                                             solut
                              geno          e                        reagent ion-based
                                 No DNas eded!
                                          ne                      process that can
                                       nt
                             treatme                                       up
                                                                 tissue in to 1gm of
                                                                           a single
                                                                                    prep
                        20µl
                 rocess in
            Can p blood
              whole l format;
                   el
             96-w le blood in
                   ho         t
             5µl w ell forma
                    w
               384-
                                                                                Protocols require
                                                                                                blood
                                                                              removal of red
                                                                                                Red
                                                                               cells with SV n
                                                                                                  io
                                                                                Cell Lysis Solut
                                                                                                   ion
                                                                                prior to purificat




     In addition to RNA isolation from mammalian
     blood, the SV Total RNA Isolation System has
     been used on related non-mammalian sources,
     including marine invertebrate hemolymph and
     fish red blood cells.




10                                                                                  Promega RNA Analysis Notebook
Purifying RNA and mRNA

                                              Plant
                                             Tissue



                                                         Manual




                  PureYield™ RNA                        SV Total RNA                  RNAgents® Total




                                                                                                             5588MA
                  Midiprep System                     Isolation System              RNA Isolation System




                      duct!
           Un ique pro agent                                                             Scalable
                                                                                                   solut
                   aring                                 luded to                          reagent ion-based
          ovel cle ll detectable               DNase inc uce
        N
                 sa                                         d
                                                 greatly re ver.                        process that can
        removenomic DNA.                                   ry-o
                                                                                                 up
                                                                                       tissue in to 1gm of
             ge         ase                     gDNA car                                         a single
                 No DNneeded!                                                                             prep
                    ent
            treatm




 The SV Total RNA Isolation System has been cited for total RNA purification from tissues of the following:
    Arabidopsis thaliana                   Lotus japonica                         Pisum sativum
    Beta vulgaris                          Lycospersicon esculentum               Prunus cerasifera
    Brassica napus                         Manihot exculenta                      Trifolium repens
    Cichorium intybus                      Medicago truncatula                    Vigna sp.
    Hordeum vulgare                        Nicotiana tabacum                      Vitis vinifera

 Tissues used for isolation include root, stem, leaves, seedlings and berries. For more information, check the online
 citations database at: www.promega.com/citations or contact Technical Services at: techserv@promega.com




www.promega.com • techserv@promega.com                                                                                  11
         Purifying RNA and mRNA

                                                                     Protoco
                          Bacteria                                  Gram P ls for
                                                                          os
 Protocols require                                                  Gram N itive &
                                                                           egat
 lysozyme and/or                                                      bacteria ive
     lysostaphin
                                        Manual




                       SV Total RNA               PureYield™ RNA               The SV Total RNA Isolation System has been




                                                                      5589MA
                     Isolation System             Midiprep System              cited for total RNA purification from many
                                                                               bacteria. Some examples are:
                                                                               Alteromonas               Pseudomonas
                             o
                     cluded t
            DNase in reduce                                                    Bacillus                  Rhizobium
              greatly ry-over.                                                 Chlamydia                 Rhodopseudomonas
                     ar
             gDNA c                                                            Escherichia               Salmonella
                                                                               Klebsiella                Simkania
                                                                               Lactobacillus             Streptococcus
                                            Unique p
                                                      r                        Microcystis               Streptomyces
                                         Novel cle oduct!                      Mycobacterium             Synechocystis
                                        removes     aring ag
                                                 all detec ent                 Neisseria                 Vibrio
                                           genomic         table
                                                      DNA.                     Orchrobactrum             Yersinia
                                             N
                                        treatmo DNase
                                               ent nee
                                                                               Photorhabdus
                                                        ded!                   For more information, check the online
                                                                               citations database at:
                                                                               www.promega.com/citations/
                                                                               or contact Technical Services at:
                                                                               techserv@promega.com


                     require
          Protocols or                           Yeast &
                     e
              lyticas se                         Fungi
                zymola                                                         The SV Total RNA Isolation System has been
                                                                               cited for total RNA purification from:
                                                                               Saccharomyces             Bierkandera sp.
                                                                                cerevisiae                Gigaspora margarita
                                        Manual
                                                                               Blumeria grominis         Trichophyton rubrum




                       SV Total RNA              PureYield™ RNA
                                                                     5590MA




                     Isolation System            Midiprep System




12                                                                                        Promega RNA Analysis Notebook
Purifying RNA and mRNA                                                                                                                    RNAgent ®
                                                                                                                                         Isolation S
                                                                                                                                                     s Total
                                                                                                                                                     ystem
                                                                                                                                                                 RNA
                                                                                                                                        Protocol
                                                                                                                                                   available
                                                                                                                                                              at:
Scalable Total RNA Isolation                                                                                                            www.pro
                                                                                                                                       tb087.ht
                                                                                                                                                  mega.com
                                                                                                                                                              /tbs/tb08
Promega’s original solution-based system, the                                                                                                     ml                   7/
                                                                                                                                       Cat.# Z
RNAgents® Total RNA Isolation System is based on the                                                                                             5110
classic method described by Chomczynski and Sacchi                                                                                    Citations
                                                                                                                                                  available
(1). We improved the method by developing a novel                                                                                     www.pro                at:
                                                                                                                                                mega.com
Denaturation Solution that reduces DNA carryover.                                                                                                          /citations
This scalable system can be used with mammalian
                                                                                                                                                                     /
                                                                                                                   10
tissue, cultured cells and plant tissue and can be
adapted to any sample size.                                                                                             8




                                                                                                       RNA Yield (mg)
                            1    2    3    4     5    6
                                                                                                                        6


                                                                                                                        4




                                                                                                                                                                  1270MA11_5B
                                                           – 28S                                                        2


                                                                                                                        0
                                                           – 18S                                                            0   0.2    0.4    0.6   0.8     1.0
                                                                                                                                       Tissue (g)
                                                                                           Purification of total RNA from mouse liver with RNAgents® Total RNA
                                                                                           Isolation System.
                                                           0349TA11_5A




Formaldehyde gel electrophoresis of total RNA. Five micrograms of total RNA
isolated with RNAgents® System from HeLa cells (lane 1), mouse intestine (lane 2), mouse
spleen (lane 3), mouse lung (lane 4), mouse kidney (lane 5) and mouse liver (lane 6).              Add Sodium
                                                                                                     Acetate to
                                                                                                prepared lysate.



  Table 1. Yields and A260/A280 Ratios of Total RNA purified                                         Add Phenol:                                           Add Denaturing
  with the RNAgents® System.                                                                         Chloroform:
                                                                                                 Isoamyl Alcohol
                                                                                                                                                          Solution. Vortex.

                                                                                                    to tube, mix
                                                                                                                                                          Add Isopropanol.
                                                                                                        and chill.
  Source                             Yield of Total RNA                   A260/A280                                                                       Centrifuge, then
                                                                                                                                                          discard supernatant.
  HeLa Cells                               1.6mg/108 cells                       1.85
                                                                                                Transfer mixture
                                                                                                 and centrifuge.                                          Add ice-cold
  Human WBC                                1.3mg/10 cells
                                                       8
                                                                                 1.72                                                                     75% ethanol.

  Mouse Intestine                           2.3mg/g tissue                       1.75      Transfer top aqueous
                                                                                             phase to new tube.
  Mouse Spleen                              8.3mg/g tissue                       1.67
  Mouse Lung                                1.9mg/g tissue                       1.75                                                                     Centrifuge, then
                                                                                                                                                          discard supernatant.
  Mouse Liver                               6.6mg/g tissue                       1.99
                                                                                                Add Isopropanol.
  Mouse Kidney                              3.1mg/g tissue                       1.70         Incubate at –20°C.
                                                                                                                                                          Air-dry the RNA
                                                                                                                                                          pellet. Dissolve in
                                                                                                                                                          water or TE buffer.
                                                                                                                                                          Store.
                                                                                                Centrifuge, then
                                                                                            discard supernatant.
                                                                                                                                                                                 3513MA09_2B




                               naturing
                                                                                           For RT-PCR go to "RNA Wash"



              RNAge  nts® De         separate
                                              ly
                       is  available                                                       The RNAgents® Procedure.

              Solution               ombined
                        be   easily c        ing
               and can reagents provid
                        ur
                with yo cal choice for
                        omi
                an econ ion.
                          lat
                 RNA iso



www.promega.com • techserv@promega.com                                                                                                                                                         13
     Purifying RNA and mRNA
     Small-scale Single-prep                                                                            Homogenize sample (tissues,


     Total RNA Isolation
                                                                                                        cultured cells or white blood cells)
                                                                                                        in RNA Lysis Buffer.



     The single-prep SV Total RNA Isolation System offers
     speed and convenience. The column-based method                                                      Transfer 175µl to a fresh tube. Add
                                                                                                         RNA Dilution Buffer; mix and centrifuge.
     can isolate total RNA from up to 60mg of mammalian
     tissue. Denaturation and inactivation of RNases are
     accomplished without the use of phenol. Promega was
                                                                                                         Transfer the cleared lysate to a fresh tube;
     the first to offer a system that eliminates residual DNA                                            add 95% Ethanol and mix.

     by performing DNase digestion directly on the column
     membrane, greatly reducing DNA carryover.
                                                                                                        Transfer to Spin Column Assembly.                DNase
                                                                                                                                                        included
                                 System
                      Isolation
             l RNA
     SV Tota             at:
               vailable                8/
      rotocol a
                                                                    Spin                                                                                Vacuum
     P                       bs/tm04
                  ga.com/t
                                                                Spin for one minute.                                                                Remove lysate

              ome                                                                                                                                   by vacuum.

      www.pr                                                    Wash with RNA
                                                                Wash Solution.                                                                      Wash with RNA
                tml
      tm048.h
                                                                                                                                                    Wash Solution.
                                                                DNase Treatment.

                 3100
                                                                Add DNase                                                                           DNase Treatment.

       Cat.# Z
                                                                Stop Solution.                                                                      Add DNase Stop Solution.
                               t:
                     ailable a
                                                                Spin for one minute.

            tions av
                                                                                                                                                    Wash 2X with RNA
                                       s/
        Cita                  /citation
                                                                Wash 2X with                                                                        Wash Solution.

                    ega.com
                                                                RNA Wash Solution.
                 om
        www.pr                                                                   Elute RNA into                                  Remove Spin Basket and place
                                                                                 Elution Tube.                                   in collection tube. Centrifuge.
                                                                                                                                 Place Spin Basket in Elution Tube.




                                                                                                                                                                           1766MB04_1A
                                  Max. Amount     Avg.         DNase es                           Total time: 60-70 minutes.

                                                                  nt giv
     Sample Type                   Processed      Yield
                                                          treatme esults
     Mouse Liver                          30mg    131µg     better r
     Mouse Kidney                         20mg    44µg
     Mouse Spleen                         15mg    79µg                            +DNase                       –DNase
     Mouse Brain                          60mg    39µg
     Mouse Muscle                         30mg    22µg
     Rat Pancreas                         30mg    100µg                                                                           –1.2Kb
     Rat Heart                            60mg    16µg
     Rat Lung                             60mg    36µg
     Tomato Leaf                          30mg    5µg                                                                             –400bp
     E. coli                         109 cells    36µg
     S. cerevisiae                4 × 107 cells   19µg
     Taken from Technical Manual #TM048
                                                                                                                                     3548TA09_1A




                                                                   RT-PCR amplification from total RNA purified from mouse liver lysate with or
                                                                   without DNase treatment. Amplification primers used for RT-PCR were designed to
                                                                   amplify both mRNA and DNA in the same reaction. Amplification products specific for the
                                                                   mRNA are 400bp, and amplification products for the gene are 1.2kb. Both the SV and SV
                                                                   96 Total RNA Isolation Systems include DNase for on-membrane DNase treatment to
                                                                   remove genomic DNA below detectable levels.




14                                                                                                Promega RNA Analysis Notebook
Purifying RNA and mRNA
Mid-scale, Single-prep Total                                                                                                                                                                                              1.
                                                                                                                                                                                                                          2.
                                                                                                                                                                                                                               Prepare lysate.
                                                                                                                                                                                                                               Add RNA Dilution Buffer. Mix.

RNA Isolation                                                                                                                                                                                                             3.
                                                                                                                                                                                                                          4.
                                                                                                                                                                                                                               Add Clearing Agent. Mix and vortex.
                                                                                                                                                                                                                               Incubate at 70°C for 5 minutes.
                                                                                                                                                                                                                          5.   Cool for 5 minutes.
As standard molecular biology applications become more
sensitive, it is increasingly important for RNA isolations
to be free of contaminating genomic DNA. The
PureYield™ RNA Midiprep System uses a novel combi-
nation of reagents, membrane, and protocol to achieve                                                                                                                                                                     1. Transfer mixture to PureYield™
                                                                                                                                                                                                                             Clearing Column in collection tube.
pure RNA with undetectable genomic DNA contam-                                                                                                                                                                            2. Centrifuge to clear the lysate.
ination. The isolation is completed without the use of a
                                                                                                                                                                                                                                                                e
                                                                                                                                                                                                                                                       No DNas t
DNase treatment, organic solvents, protease digestions,
or alcohol precipitations. The eluted RNA is ready for                                                                                                                                                                                                          n
sensitive downstream applications such as quantitative
                                                                                                                                                                                                                                                       treatme
RT-PCR, RT-PCR, and microarray analysis.                         ctable
                                                                                                                                                                                                      1. Add isopropanol to the                          required
                                                          No dete
                                                                                                                                                                                                         cleared lysate.

                                                                                                                                                                   y qPCR
                                                                                                                                                                                                      2. Mix and transfer to

                                                                                                                                                             gDNA b
                                                                                                           Amplification Curves                                                                          PureYield™ Binding Column.
             0




                                                                                                                                                                         Centrifuge (Spin)                                                       Vacuum (Vac)
             -1.1E5 -1.0E5 -9.0E5 -8.0E5 -7.0E5 -6.0E5 -5.0E5 -4.0E5 -3.0E5 -2.0E5 -1.0E5




                                                                                                                                                                            1. Centrifuge.                                                        1. Apply vacuum.
                                                                                                                                                                            2. Wash 2X.                                                           2. Wash 2X.
                                                                                                                                                                            3. Centrifuge                                                         3. Dry 3 minutes.
                                                                                            DNA Standards                                                                      10 minutes
                                                                                            Competitor I                                                                       to dry.
       RFU




                                                                                            No-Template Control
                                                                                            PureYield™
                                                                                            RNA Midiprep



                                                                                                                                                                                                                          1. Transfer PureYield™ Binding
                                                                                                                                                 5360TA




                                                                                                                                                                                                                             Column to 50ml collection tube.
                                                                                            5      10      15      20     25      30   35   40
                                                                                                                                                                                                                          2. Add Nuclease-Free Water
                                                                                                                  Cycle                                                                                                      and incubate 2 minutes.
                                                                                                                                                                                                                          3. Centrifuge 3 minutes to elute




                                                                                                                                                                                                                                                                      5124MB
RNA purified with the PureYield™ RNA Midiprep System has no detectable                                                                                                                                                        pure RNA.
genomic DNA contamination. RNA was purified from 1 × 108 HEK293T using the
PureYield system and competitor “I’s” solution-based purification method. 100ng of                                                                                        Schematic representation of the PureYield™ Total RNA Isolation System.
purified total RNA was analyzed using the Plexor™ qPCR System to determine the
quantity of gDNA using primers specific for the human thyroid peroxidase gene
(TPOX). Human Genomic DNA (Cat.# G3051) in quantites of 104, 103, 102 and Average                                                                                     Yields of Total RNA Isolated from Tissues and Cells.
101 copies was used as a standard.                                                                                                                                             Sample
                                                                                                                                                          Sample            Amount / Maxium                 Average Yield               Average           Average
                                                                                                                                                          Type              Sample Capacity                 per Prep (µg)1              A260/A230         A260/A280
                                                                                                                                                          Rat Tissues
                                                                                                                                                          Liver                     300mg                         990.7                    1.8                 1.9
       PureYield                                                                                                                                          Lung                      300mg                         193.9                    2.0                 2.1
                ™ RNA
      System              Midipre                                                                                                                         Kidney                    200mg                         329.1                    2.3                 2.1
                                 p
                                                                                                                                                          Spleen                    150mg                         430.9                    2.3                 2.1
     Protocol                                                                                                                                             Brain                     300mg                         305.5                    2.3                 2.1
              available
     www.pr             at:                                                                                                                               Heart                     300mg                         255.3                    2.2                 2.1
            omega.c
    279.htm          om/tbs/t                                                                                                                             Muscle                    300mg                         115.1                    2.1                 2.1
            l                m279/t
                                   m                                                                                                                      Bacteria
    Cat. #                                                                                                                                                E. coli              1 × 1010 cells                     872.7                    2.5                 2.1
           Z3740
                     and Z3                                                                                                                               Plant Tissue
                            741                                                                                                                           Canola                    300mg                          87.8                    1.5                 2.1
                                                                                                                                                          Cell Lines
                                                                                                                                                          HEK 293T              5 × 107 cells                     453.3                    2.1                 1.9
                                                                                                                                                          HeLa                  5×    107   cells                 329.2                    1.8                 2.0
                                                                                                                                                          Blood              20ml (10ml/tube)                     ~10*                       *                 *
                                                                                                                                                          1. The values represent means of results achieved at Promega. Yields will depend on the metabolic state of
                                                                                                                                                             the sample, culture conditions, harvesting conditions and sample preparations. The average total RNA yield
                                                                                                                                                             shown is from a 1ml elution.
                                                                                                                                                          * Varies by white cell count. A white cell count of ~5 × 106 cells/ml yields ~10µg of total RNA.


www.promega.com • techserv@promega.com                                                                                                                                                                                                                                         15
      Purifying RNA and mRNA
                                                                                                                              SV 96 T
                                                                                                                                         o
                                                                                                                             System tal RNA Isolation
      96-Well Total RNA Isolation                                                                                            Protocol
                                                                                                                                      available
      As your RNA isolation needs grow, Promega has the                                                                     www.pro              at:
      tools to increase your RNA isolation throughput. All the                                                                        mega.com
                                                                                                                            tb294.ht             /tbs/tb29
      advantages of the single-prep SV Total RNA Isolation                                                                            ml                     4/
      System are included in a 96-well format of the SV 96                                                                 Cat.# Z
                                                                                                                                     3500, Z
      Total RNA Isolation System. The system uses a vacuum                                                                 Automate             3505
                                                                                                                                    d protoco
      manifold to allow the isolation of RNA from an entire                                                                                    ls availab
      plate of 96 samples as efficiently as possible. Isolations
                                                                                                                                                         le.
      can be scaled up for benchtop purification or for use
      with liquid-handlers. Use the Vac-Man® 96 Vacuum
      Manifold for benchtop and automated methods unless
      your robotic system requires a specific manifold.                                                                                             Apply sample lysate


                                                                                                             Binding Plate

                                                                                                                                                         Bind RNA.




                                                                                                                                                             Wash.
                                                                                                                                                                               DNase
                                                                                                                                                        DNase treat.          included

                                                                                                                                                             Wash.


                                                                                                                                                       Elute total RNA.
                                                                                 3446CA06_1A




                                                                                                              Elution Plate




                                                                                                                                                                              2651MB03_1A
                                                                                                                                                  Highly pure total RNA.



      Comparison of SV and SV 96 Total RNA Isolation
                                                                                                                                       tly from
      Yields                                                                                                                   Go direc NA to
                             0.8           1 × 105 Cells                                                                        eluted R R!
                             0.7                                                                                                   RT-PC
                             0.6
          Total Yield (µg)




                             0.5
                             0.4                                                               RT-PCR from a Decreasing Number of SH-SY5Y
                             0.3
                                                                                               Human Neuroblastoma Cells
                             0.2
                                                                                                                                     SH-SY5Y/SV96
                             0.1
                                                                   3806MA08_2A




                                                                                                                                 0
                                                                                                                                         5
                                                                                                                                               2
                                                                                                                           k
                                                                                                                       .5
                                                                                                                               25
                                                                                                                                     12
                                                                                                                                             56

                                                                                                                                                   1
                                                                                                                                                          1
                                                                                                                                                               5
                                                                                                           k

                                                                                                                  k
                                                                                                         50

                                                                                                               25
                                                                                                                      12




                                                                                                                                                  78
                                                                                                                                                        39
                                                                                                                                                              19

                                                                                                                                                                   98
                                                                                                                                                                        49
                                                                                                                                                                             24




                              0
                                                                                                                            6,
                                                                                                                                    3,
                                                                                                                                          1,




                                   SV 96      Vacuum       Spin
                                                                                                                                                                                            3344TA03_1A




                                                                                               β-actin –
                                               Standard SV Total
                     SV,                                                                       RNA was isolated from individual wells containing the indicated number of
      If it works in
                                                  RNA preps
                                                                                               cells using the SV 96 Total RNA Isolation System. Four microliters of each eluate
                    V 96!
     it’ll work in S
                                                                                               were amplified with the Access RT-PCR System (Cat.# A1250) and β-Actin Primer Pair
                                                                                               (Cat.# G5740).


16
                                                                                                                           Promega RNA Analysis Notebook
Purifying RNA and mRNA
                                  RNA iso
                              SV 96        lated w                                                  5.0
                                      Total RN ith the                                              4.0
                               go direc          A Syste
                                        tly into           m c
                                                  quantitat an                                      3.0




                                                                                              ∆Rn
                                  RT-PC                    ive
                       0.7
                                           R react                                                  2.0
                       0.6                           ions.
                       0.5
                                                                                                    1.0
    µg




                       0.4                                                                              0
                       0.3
                                                                                                            0            5       10     15      20      25         30         35      40
                       0.2
                                                                                                                                             Cycle
                       0.1
                        0                                                                                   Number of HeLa Cells




                                                                                                                                                                                           4068TA03_3B
                                                        3467MA06_1A
                             HeLa   NIH 3T3     CHO
                                                                                                              50,000      12,500                         3,125
                                     SV96                                                                     25,000      6,250                          1,563
                       2.5                                                                Real-time quantitative RT-PCR analysis of lamin A/C message. Total RNA
                                                                                          isolated from indicated number of HeLa cells and eluted in 100µl of Nuclease-Free Water.
                                                                                          Twenty microliters were used in a 100µl RT reaction and 5µl transferred to a 50µl
                       2.0                                                                quantitative, real-time PCR reaction. Further details in Brisco, P. and Hooper, K. (2003)
                                                                                          Quantitative, real-time RT-PCR expression using the SV 96 Total RNA Isolation System.
                                                                                          Promega Notes 84, 23–26.
     Ratio A260/A280




                       1.5


                       1.0
                                                                                              Storage of purified RNA
                       0.5
                                                                                              RNA should be stored in a dedicated container at
                                                                                              –70°C or below. To avoid multiple freeze-thaw cycles
                         0
                                                                                              and contamination, dispense the purified RNA into
                                                                      3468MA06_1A




                             HeLa     NIH 3T3     CHO                                         convenient volumes. For long-term storage or storage
                                       SV96                                                   of critical samples, precipitate RNA aliquots by adding
                                                                                              1/10th volume of 3M sodium acetate and 2 volumes
                                                                                              of ethanol. Store the precipitated RNA under ethanol
Ribosomal RNA Sizes                                                                           at –70°C or below.
Species                                  rRNA                         Size (kb)
Human                                    18S                                        1.9
                                         28S                                        5.0
                                                                                                                                                            NA
Mouse                                    18S
                                         28S
                                                                                    1.9
                                                                                    4.7                                                      SV 96 Total R d
                                                                                                                                                             an
Drosophila                                                                                                                                   works on plant
                                         18S
                                         28S
                                                                                    2.2
                                                                                    2.8                                                       mammalian tissues.
Tobacco                                  16S                                        1.5
                                         18S                                        1.9
                                         23S                                        2.9
                                         25S                                        3.7
                                                                                               Tomato            Corn        Tobacco   Barley   Wheat    Alfalfa            Arabid. Soy Rice
Yeast                                    18S                                        2.0   M    L   S            L    S        L    S   L    S   L   S    L     S        M      L     L    L
                                         26S                                        3.8
E. coli                                  16S                                        1.5
                                         23S                                        2.9
                                                                                                                                                                                                         3509TA07_1B




                                                                                          Isolation of total RNA from either leaf (L) or stem (S) tissues of various plant
                                                                                          species. Bands indicate 28S, 18S and chloroplast rRNAs. Twenty microliters of each
                                                                                          elution were loaded on a 1% gel stained with ethidium bromide. Protocol detailed in
                                                                                          Grunst, T. (2001) High-throughput isolation with the SV 96 Total RNA Isolation System.
                                                                                          Promega Notes 79, 29–32.




                                                                                                                                                                                                                       17
www.promega.com • techserv@promega.com
     Purifying RNA and mRNA
     96-Well or 384-Well Total
     RNA Isolation                                                                                                                      Add sample lysate to
                                                                                                                                        MagneSil® RNA PMPs. Mix.

     The MagneSil® Total RNA mini-Isolation System
     provides a high-throughput 96- or 384-well format for
     fast, simple preparation of intact, purified total RNA
     from small amounts of cultured cells, tissue, or fresh
                                                                                                                                        Capture PMPs.
     whole blood samples. The system is designed to                                                                                     Discard supernatant.
     perform four plates worth of purification in a 96- or                                                                               Wash PMPs.
     384-well plate format. Total RNA isolation is achieved
     without the need for vacuum filtration, centrifugation or
     precipitation. The procedure is specifically geared for
     automated liquid handlers.                                                                                                         Incubate PMPs with
                                                                                                                                        DNase 10 minutes.
                                                                                                                                        Add DNase Stop Solution.
                                               Elute R
                                                         NA from
                                              96-well                a
                                                         purificat
                                              in as lit           io
                                                       tle as 1 n
                                                                5µl                                                                     Capture PMPs.
                                                                                                                                        Discard supernatant.
                                                                                                                                        Wash PMPs.
             Concentration (ng/µl)




                                      30




                                                                                                                                                                        4316MB11_3A
                                      20
                                                                                                                                        Elute RNA.
                                      10
                                                                                                   Schematic diagram of the MagneSil® Total RNA mini-Isolation System
                                       0                                                           protocol.
                                        10   15   20   25 30   35   40   45 50
                                                  Elution Volume (µl)

                                     0.6                                                           Maximum starting material for MagneSil® Total RNA
                                                                                                   mini-isolation System protocols:
             Yield (µg)




                                     0.4

                                     0.2                                                           Sample                          96-well                  384-well
                                                                                                   Tissue Lysate                                             Not
                                     0.0                                                           (in 100µl lysis buffer)           2mg                recommended
                                                                                     4377MA11_3A




                                        10   15   20   25 30   35   40   45 50
                                                                                                   Cultured Cells                     105                      103
                                                  Elution Volume (µl)
                                                                                                   Whole Blood                       20µl                      5µl
     Flexibility in elution volume to control sample concentration. Total RNA isolated
     from 2 × 104 HeLa cells/well using MagneSil Total RNA mini-Isolation System in a 96-well
     format. Titration of elution volume shown. Total RNA concentration and yield calculated by
     measurement of isolated total RNA with Molecular Probes RiboGreen® assay.




                                      The mobile stationary phase
                                      of magnetic particles allows
                                     elution in smaller volumes than
                                          membrane-based RNA
                                           purification systems



18                                                                                                                        Promega RNA Analysis Notebook
Purifying RNA and mRNA
Ready for quantitative,                                                                                        Automated Methods for:
                                                                                                               • Beckman Coulter Biomek® 2000
real-time RT-PCR                                                                                               • Beckman Coulter Biomek® FX

  A.         102                                                                                               • Tecan Te-MO™
             101                                                   103                                         • Xiril X100
                                                        104
             10   0                                                                        102                 Only the Biomek® FX and Te-MO can apply the 384-well
       ∆Rn




                                            105                                            10
             10–1                                                                                              method. Some instruments may need extra hardware to
             10–2
                                                                                                               automate this system For more information go to:
                                                                          negative control                     www.promega.com/automethods/
             10–3
                      0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40                                If you don’t have one of the instruments listed, you may
                                              Cycle                                                            be able to adapt the system to your instrument. Our
                                             GAPDH                                                             reagent manuals include information such as how much
                40
                                                                                                               of each reagent are required at each step to assist with
                30                                                                                             the adaptation.
         Ct




                20

                10

                      0
                                                                                                                     MagneS ®
                       1

                              10


                                        0

                                                   0

                                                               00


                                                                              0

                                                                                       6




                                                                                                                              il
                                      10

                                               00




                                                                         00

                                                                                   +0




                                                                                                                    mini-isola Total RNA
                                                              ,0
                                             1,




                                                                         0,

                                                                                  1E
                                                        10

                                                                    10




                                                                                                                              tion Sys
                                         Cell Number                                                               available          tem Pro
                                                                                                                             a               tocol
                                                                                                                   www.pr t:
  B.         101
                                                                                                                            om
                                                                   103                                            tb328.h ega.com/tbs/tb
             100                                        104                                                                tml              328/
                                                                                                                  Cat.# Z
                                                                                                                           3351
       ∆Rn




             10–1                                 105                                      102                    Automa  tio
                                                                                   10                              required n and magnetic
             10–2                                                                                                             for use.     stands

             10–3
                      0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
                                              Cycle
                                              c-myc
                40
                30
          Ct




                20
                10
                      0
                       1

                               10


                                        0


                                                    0


                                                                 0


                                                                             0


                                                                                         6
                                      10


                                                  00


                                                               00


                                                                           00


                                                                                      +0

                                                                                                 4378TA11_3A
                                              1,


                                                             ,

                                                                       0,

                                                                                   1E
                                                          10

                                                                     10




                                         Cell Number
Real-time RT-PCR analysis of purified RNA. 20µl aliquots of total RNA isolated
from a 10X dilution series of HeLa cells seeded in a 96-well plate were used as template
for reverse transcription (100µl reaction). Panel A: 5µl aliquots (n=3) of the RT reaction
were used for PCR of GAPDH target. Panel B: 5µl aliquots (n=1) of the RT reaction were
used for PCR of a c-Myc target. GAPDH signal (abundant mRNA) and c-Myc (rare mRNA)
could be detected in as few as 10 HeLa cells.




www.promega.com • techserv@promega.com                                                                                                                                    19
       Purifying RNA and mRNA
                                                                              From T
                                                                                     otal R
       Poly(A)+ mRNA Isolation                                               PolyAT
                                                                                   tra ®
                                                                                           NA...
                                                                                    ct m
       Many applications are improved by isolating poly(A)+             Protoco             RNA I
                                                                                ls availa           solation
       RNA either directly from the eukaryotic source material         www.p             ble at:             System
       or from total RNA previously isolated from the source
                                                                               romega
                                                                       Cat.#           .com/t
       material. Some methods use oligo(dT) immobilized on a                  Z520              bs/tm0
                                                                                      0, Z5            21/tm0
       support such as cellulose or sepharose. These methods          Citatio                 210, Z           21.htm
                                                                             ns avai                   5300,         l
       are prone to ribosomal RNA carryover. Promega’s                www.p          lable a                  Z5310
                                                                            romega           t:
       PolyATtract® Systems use solution hybridization of                           .com/c
                                                                                            itations
       Poly(A)+ RNA to a biotinylated oligo(dT) followed by                                          /
       capture with a streptavidin-coated paramagnetic                                 total RNA containing
       particle. The rapid hybridization and magnetic                                      mRNA fraction
                                                                            5′
                                                                              m7G                               AAAAAAA3 ′
       separation greatly improve the speed, efficiency and
                                                                                                   Hybridize with
       quality of mRNA isolations.                                                                 biotin-oligo(dT).
                                                                                                                                 5′
                                                                                                                                   B-T T T T T T T 3′

       The PolyATtract® mRNA Isolation Systems can be used                  5′
                                                                                 m7G                            AAAAAAA3 ′
       if you are starting with total RNA. The kits are designed                                                3′
                                                                                                                   T T T T T T T-B5′

       to purify Poly(A)+ RNA from 1–5mg or 0.1–1mg of total                                       Add streptavidin PMPs.
       RNA. Reactions are scalable to meet your needs.
                                                                                                         PMP

       To save time, isolate poly(A)+ RNA directly from starting
       material using the PolyATtract® System 1000. The                     5′
                                                                                 m7G                            AAAAAAA3 ′
       system uses a guanidine solution to disrupt the tissue                                                    T T T T T T T-B       PMP


       and inhibit RNases, followed by the PolyATtract®                                            Magnetize.

       methodology for the isolation of Poly(A)+ RNA.
                                                                                       5′                                  AAAAAAA3 ′




                                                                                                                                                         MAGNET
                                                                                         m7G
       Promega has published applications for mammalian                                                                     T T T T T T T-B      PMP

       and plant tissues.                                                                          Wash and elute.



                                                                            5′                                 AAAAAAA3 ′




                                                                                                                                                         MAGNET
                                                                                 m7G

                     ...




                                                                                                                                                                  0369MA03_1A
            Isolation
                                                                                                                               T T T T T T T-B     PMP


     Direct         ystem 1
                           000                                                              (aqueous)                                    (solid)
             ract S
                ®
     PolyATt         able at:              28.html
      Protocols avail            m2  28/tm2                              Solution-based
                        om/tbs/t                                                     oligo(dT )
       www.p  romega.c                                             hybridization to
                            5400                                                      nt than
                 5420, Z                                             is more efficie
       Cat.# Z            :                                             hybridization to
                                                                                          an
                ations at                                                            igo(dT )
        See cit              /citation
                                      s/
                                                                      immobilized ol
                 om ega.com
         www.pr                                                                                               µg total RNA                    µg mRNA
                                                                                                        12      6      3     1.5          2        1     0.5 M


         Prokaryotic mRNA enrichment
         Bacterial mRNA does not have the poly(A) tail
         characteristic of eukaryotic mRNA. Other tactics
         must be employed if you wish to get at the bacterial
                                                                      Northern blot analysis
         mRNA. Su and Sordillo (1998) used a probe directed           comparing total RNA and
         to bacterial rRNA to remove the rRNA from a total            poly(A)+ RNA isolated
                                                                      from mouse liver. The
         bacterial RNA prep to enrich the sample for the              Northern blot was probed for
         bacterial mRNA.                                              low abundance α-1-proteinase
                                                                      inhibitor message. Poly(A)+
         Su, C. and Sordillo, L.M. (1998) A simple method to          RNA was isolated directly from
                                                                      the tissue with the
         enrich mRNA from total prokaryotic RNA. Mol.
                                                                                                                                                                                0417TA06_2A




                                                                      PolyATtract® System 1000.
         Biotechnol. 10, 83–85.


20                                                                                                Promega RNA Analysis Notebook
Purifying RNA and mRNA
Downstream Applications                                   Nuclease Protection
                                                          The nuclease protection assay is a sensitive technique
Expression Profiling                                       used for the detection and quantitation of target RNA
See chapter 5, Analyzing RNA with Microarrays.            sequences and related RNAs (6). A radiolabeled DNA or
                                                          RNA probe is allowed to hybridize to target RNA in
See chapter 3, Quantifying RNA with qRT-PCR.
                                                          solution. After hybridization, the remaining single-
RT-PCR                                                    stranded probe is removed from the reaction by
                                                          incubation with either a ribonuclease, if the probe was
See chapter 4, Amplifying RNA with RT-PCR.
                                                          RNA (RNase protection), or S1 nuclease, if the probe
Primer Extension Analysis                                 was DNA (S1 assay). Reaction products are resolved by
Primer extension is used to quantitate and determine      polyacrylamide gel electrophoresis to quantitate the
the location of the 5′-end of specific RNAs. With this     amount of protected probe. The technique is extremely
technique, a radiolabeled DNA primer complementary to     sensitive, doesn’t require transfer of RNA to a solid
the RNA being studied is hybridized to the target and     support, and multiple RNA species can be probed in a
extended using the enzymatic properties of reverse        single reaction. See the chapter called “Making RNA in
transcriptase. The DNA primer is designed to anneal to    vitro” for information about making RNA probes.
sequences near the 5′-end of the RNA target, and the      Promega also offers
extension reaction terminates when the reverse            RNase ONE™
                                                          Ribonuclease, a
                                                                                         RNase
transcriptase reaches the extreme 5′-end of the RNA.                                              ONE™
                                                          highly efficient               Cat.#               Ribonuc
The length of the cDNA product accurately defines the                                             M4261              lease
distance between the 5′-end of the radiolabeled primer    ribonuclease that            Citation
                                                          cleaves after each                     s availa
and the 5′-end of the RNA (transcriptional start site).                               www.pr              ble
The quantity of cDNA produced is proportional to the      base in RNA so you                    omega.c at:
                                                                                                          om/cita
amount of target RNA.                                     won’t have to use                                       tions/
                                                          mixtures like RNase A
                           m                              and RNase T1.
                    Syste
             ension
      r Ext           t:                                  Northern Blotting
Prime           able a
        ol avail       /tbs/                              Northern blotting is the classic technique used to
 Protoc        ga.com                                     examine the expression profile of mRNA following a
         prome
  www. b113.html                                          specific treatment. Northern analysis has given way to
          /t
   tb113                                                  techniques like RT-PCR and microarray analysis. For a
              3030
    C at.# E                                              complete method for Northern blotting, see the
                                                          Expression Analysis chapter of the Protocols and
                                                          Applications Guide, available online at:
                                                          www.promega.com/paguide.
         Need information on other
                applications?
    Contact Promega Technical Services                                         Protocols & App
                                                                                              lications
           techserv@promega.com                                                      Guide at:
                                                                              www.promega.c
                                                                                            om/paguide




www.promega.com • techserv@promega.com                                                                                21
     Purifying RNA and mRNA
     Total RNA Purification Products                                                                            Size       Cat.#
     SV Total RNA Isolation System(d)*                                                                        10 preps      Z3101
                                                                                                              50 preps      Z3100
     PureYield™ RNA Midiprep System(c,e)                                                                      10 preps      Z3740
                                                                                                              50 preps      Z3741
     SV 96 Total RNA Isolation System*                                                                          1 × 96      Z3500
                                                                                                                5 × 96      Z3505
     RNAgents® Total RNA Isolation System*                                                                    Scalable      Z5110
     MagneSil®       Total RNA mini-Isolation         System(f)                                                 4 plate     Z3351

     Items Available Separately
     SV RNA Lysis Buffer*                                                                                        50ml       Z3051
     SV RNA Red Cell Lysis Buffer*                                                                              200ml       Z3141
     Used to lyse red blood cells prior to isolation of RNA from the nucleated white blood cells.

     Vac-Man® Laboratory Vacuum Manifold                                                                        1 each     A7231
     For use with the SV Total RNA System in a vacuum format.
     Twenty samples can be processed at once.

     Vacuum Adapters                                                                                           20 each     A1331
     Required for use with the Vac-Man® Laboratory Vacuum Manifold and the
     SV Total RNA Isolation System in a vacuum format.

     Vac-Man® 96 Vacuum Manifold                                                                                1 each     A2291
     Required for use of the SV 96 Total RNA Isolation System.

     RNAgents® Denaturing Solution                                                                              120ml       Z5651

     mRNA Purification Products                                                                                 Size       Cat.#
     PolyATtract® mRNA Isolation System I*                                                                 3 isolations     Z5200
     1–5mg total RNA; Magnetic Stand included

     PolyATtract® mRNA Isolation System II*                                                                3 isolations     Z5210
     Refill system for Z5200; no magnetic stand

     PolyATtract® mRNA Isolation System III*                                                              15 isolations     Z5300
     0.1–1mg total RNA; Magnetic Stand included

     PolyATtract® mRNA Isolation System IV*                                                               15 isolations     Z5310
     Refill system for Z5300; no magnetic stand

     PolyATtract® System 1000 with Magnetic Stand*                                                         3 isolations     Z5420
     PolyATtract®       System 1000 without Magnetic Stand*                                                3 isolations     Z5400
     *For Laboratory Use.




     References
     1. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by
        acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem.
        162, 156–159.
     2. Marcus, L. et al. (1996) PolyATtract® Systems for mRNA purification. Promega
        Notes 60, 14–16.
     3. Murillo, I. et al. (1995) Isoaltion of total RNA and mRNA from plant tissues.
        Promega Notes, 2–5.
     4. Rhodes, R. and Kephart, D. (1996) Automated Robotic Isolation of Poly(A)+
        mRNA Using PolyATtract® mRNA Isolation Reagent. Promega Notes 75, 10–13.
     5. Barlow, J.J. et al. (1963) A simple method for quantitative isolation of
        undegraded high molecular weight ribonucleic acid. Biochem. Biophys. Res.
        Comm. 13, 61–66.
     6. Ausubel, F.M. et al. (2000) Current Protocols in Molecular Biology, Vol. 2, John
        Wiley & Sons, New York, 4.6–4.7.




22                                                                                                  Promega RNA Analysis Notebook

								
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