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					                        Roche Applied Science
                       Gene Knockdown




                           Focus on Your Target –
Integrated Solutions
                                Effective and Specific Gene Knockdown
          Integrated Solutions for Gene Knockdown




          The RNA interference (RNAi) pathway was first discovered when injection
    of double-stranded RNA (dsRNA) homologous to a gene in Caenorhabditis
    elegans resulted in the down-regulated expression of this target gene. Due to its
    exceptional specificity and efficiency, gene knockdown by RNAi has now become
    a powerful functional genomics tool widely used for assigning gene function,
    pathway analysis, as well as target identification and validation.




                    Integrated Solutions for Gene Knockdown                   2

                    What is RNA Interference ?                                4

                    Gene Knockdown Workflow                                   6

                      Overview                                                6

                    siRNA Generation                                          8

                    Transfection                                             12

                    Knockdown Verification                                   14

                      mRNA Quantification                                    14

                      Protein Detection                                      19

                    Functional Assays                                        20

                    Ordering Information                                     25

                      Disclaimers and Trademarks                             29

                    References and Abbreviations                             31




2
   siRNA Generation                     Transfection               Knockdown Verification        Functional Assays

   esiWay Silencing Resources (RZPD)*   X-tremeGENE siRNA          LightCycler® 2.0 System       Cell Proliferation
   X-tremeGENE siRNA Dicer Kit          Transfection Reagent                                     Reagent WST-1



                                                                                                                             Integrated
                                                                                                                            Solutions for
Benefit                                                                                                                         Gene
                                                                                                                            Knockdown

      from reliable high-quality products and technologies designed to work
together optimally, facilitating the success of your gene knockdown experiment
and providing consistent results.

Accelerate siRNA generation                                    Measure cell viability with high sensitivity
Save time by using the X-tremeGENE siRNA Dicer                 Use the Cell Proliferation Reagent WST-1
Kit to synthesize pools of diced siRNA for highly              to determine cell viability after knocking down
effective gene knockdown.                                      your target gene.

Perform effective gene knockdown                               * esiWay Silencing Resources are available from the RZPD
Benefit from the high efficiency and low cytotoxicity          German Resource Center for Genome Research.
of the X-tremeGENE siRNA Transfection Reagent                  For further information, visit www.rzpd.de/products/esiRNA
to deliver siRNA into a variety of cell types.

Rely on accurate real-time PCR technology
Confirm successful silencing of target-gene
expression by relative quantification with the
LightCycler® 2.0 System.




                                                                                                                            3
                        What is RNA Interference?




                RNA interference (RNAi), also known as post-           This is because dsRNA longer than 30 base pairs
                transcriptional gene silencing, is initiated by long   induces a non-specific, interferon-mediated
                double-stranded RNA (dsRNA) molecules that             response that shuts down translation altogether and
                are homologous to a gene sequence. These long          initiates cellular apoptosis (7). Before using dsRNA
                dsRNA molecules trigger a cascade of events that       for RNAi analysis in mammalian cell lines, the long
                lead to the degradation of the corresponding           dsRNA must first be cleaved into 21–23 base pair
                mRNA, and subsequently to reduced levels of the        siRNA duplexes using the Dicer enzyme. This mimics
                corresponding protein (1, 2, 3, 4, 5, 6).              the first part of the cellular RNAi pathway in vitro
                                                                       and facilitates the transfection of the gene-targeted
                1. The long dsRNA is cleaved into 21–23 base pair      siRNA without initiating an apoptotic response
                  small interfering RNAs (siRNAs) by a cytoplasmic     from the cell (8).
                  enzyme called Dicer.                                 Introduction of such diced siRNA (d-siRNA)* into
                                                                       the cells triggers the endogenous RNAi pathway,
                2. The siRNA duplexes are unwound and assembled
                                                                       which inhibits target-gene expression (Figure 1). This
                  into a multi-component nuclease, RNA-induced
                                                                       effect may be analyzed by monitoring the mRNA
                  silencing complex (RISC).
                                                                       level through real-time PCR or northern-blot analysis,
                3. The activated RISC targets the complementary        and by examining protein levels using western-blot
What is RNA       mRNA for degradation.                                analysis or by performing functional assays.
Interference?
                RNAi can be used to suppress the function of a
                gene in a variety of organisms and cell lines, if
                the sequence of the gene is known. However, in
                mammalian cell lines long dsRNA duplexes
                cannot be used directly for RNAi analysis.             * also called esiRNA for endoribonuclease-prepared siRNA




            4
                                                                             What is RNA
                                                                             Interference?




Figure 1: Overview of RNAi pathway induced by transfection of diced siRNA.




                                                                             5
                Gene Knockdown Workflow
                                                                  Overview

                siRNA Generation                         Transfection                          Knockdown Verification


                                                                                               mRNA Quantification

                 Generation of diced siRNA              siRNA Transfection                     Manual RNA Isolation

                 Accelerate the generation of high-     Use one single reagent for             Isolate high-quality RNA using
                 purity pools of diced siRNA.           siRNA- and co-transfection-            High Pure RNA Kits.
                                                        based gene knockdown
                    esiWay Silencing Resources*                                                   High Pure RNA Isolation Kit
                                                        experiments.
                    X-tremeGENE siRNA Dicer Kit
                                                           X-tremeGENE siRNA                   Isolate mRNA directly from variable
                                                           Transfection Reagent                amounts of cultured cells.

                                                                                                  mRNA Isolation Kit

                 Plasmid Purification                   Plasmid Transfection                   Use one reagent for the simulta-
                 Purify transfection-grade plasmids     Efficiently transfect a wide variety   neous isolation of total RNA, DNA,
                 expressing short hairpin RNA           of eukaryotic cells with plasmids      and proteins.
                 (shRNA).                               expressing shRNA.                         TriPure Isolation Reagent
                    Genopure Plasmid Kits                  FuGENE 6 Transfection
                                                           Reagent                             Automated RNA Isolation

                                                                                               Fully automate your RNA
                                                                                               isolation for up to 32 samples using
                                                                                               the MagNA Pure LC System.

  Gene                                                  Selection Antibiotics                     MagNA Pure LC
Knockdown                                                                                         Instrument
                                                        Select your stably
 Workflow
                                                        transfected cells with                    MagNA Pure LC RNA
                                                                                                  Isolation Kit –
                                                           Geneticin (G418)
                                                                                                  High Performance
                                                           Hygromycin B
                                                                                                  MagNA Pure LC mRNA
                                                                                                  Isolation Kit I




                                                                                               Protein Detection

                                                                                               Protein Stabilization

                                                                                               Protect your protein by using
                                                                                               an optimized mixture of
                                                                                               different protease inhibitors.

                                                                                                  Complete Protease Inhibitor
                                                                                                  Cocktail Tablets
                *available from RZPD German
                 Resource Center for Genome Research,
                 www.rzpd.de/products/esiRNA




            6
                                            Functional Assays




Northern Blotting                           Cell Proliferation Assays

Quantify your mRNA using sensitive,         Measure cell viability after
nonradioactive labeling methods.            transfection.

   DIG Northern Starter Kit                    Cell Proliferation Reagent WST-1




                                            Apoptosis Assays
cDNA Synthesis &
Quantitative Real-Time PCR                  Choose one of the various sensitive
                                            assays for the study of apoptosis-
Quantify your mRNA by fast and
                                            related targets.
accurate real-time PCR.
                                               Cell Death Detection
   Transcriptor First Strand cDNA
                                               ELISA PLUS
   Synthesis Kit

   LightCycler® 2.0 System
   • LightCycler® 2.0 and                   Cytotoxicity Assays
     1.5 Instruments
   • LightCycler® Probe Design Software     Quantify cytotoxicity/cytolysis using
   • LightCycler® Relative Quantification   fast and precise assays.                      Gene
     Software                                                                           Knockdown
                                               Cytotoxicity Detection Kit (LDH)
   • LightCycler® FastStart PLUS Kits                                                    Workflow

   Universal ProbeLibrary




Western Blotting

Detect your protein by sensitive
chemiluminescence methods.

   Lumi-Light PLUS Western
   Blotting Kit




                                                                                    7
                           siRNA Generation
                                    Production of highly effective siRNA pools from
                                    long dsRNA using Dicer




                 Several methods are available for producing          In an alternative approach, siRNA can be expressed
                 siRNA in vitro:                                      in vivo in cultured cells. A siRNA expression
                                                                      cassette is introduced into either a plasmid or a viral
                 ½ Chemical synthesis
                                                                      vector. From this expression cassette, a short hairpin
                 ½ In vitro transcription of small RNAs               RNA (shRNA) is expressed, resembling the endo-
                                                                      genous microRNA (miRNA) which is the natural
                 ½ In vitro transcription of long RNAs which are
                                                                      substrate for the Dicer enzyme. After transfection
                   subsequently digested with Dicer enzyme
                                                                      of the construct, the expressed shRNA is then converted
                                                                      into effective siRNA by the cellular machinery.
                 With the technologies available today, chemical
                 synthesis of siRNA is a straightforward procedure.
                 Alternatively, the small RNA molecules that make      Generation of diced siRNA
                 up the siRNA double strand can be generated by
                                                                      The X-tremeGENE siRNA Dicer Kit mimics the
                 in vitro transcription with T7 RNA polymerase
                                                                      first part of the cellular RNAi pathway and
                 (e.g., using small synthetic DNA oligonucleotides
                                                                      efficiently generates ready-to-transfect pools of
                 including a T7 promoter region as template).
                                                                      target-gene-specific d-siRNA (Figure 2). An
                 However, one drawback of both methods is that not
                                                                      optimized T7 in vitro transcription system is utilized
                 all sequences complementary to the target mRNA
                                                                      to synthesize high yields of long dsRNA from a
                 are equally well suited for gene knockdown. This
  siRNA                                                               gene-specific DNA template containing T7 promoter
                 necessitates careful and time-consuming design of
Generation                                                            sequences at both ends. Use your cDNA and two
                 the optimal siRNA.
                                                                      PCR primers consisting of a gene-specific 3' part and
                 One way of increasing the probability of obtaining   the T7 promoter sequence each, to generate this
                 knockdown is by using a target-gene-specific         DNA template by PCR amplification; or, employ a
                 mixture of different siRNAs more likely to contain   ready-to-use target-gene-specific esiWay Silencing
                 effective sequences. These siRNA mixtures can        Resource available from the RZPD German Resource
                 be produced by in vitro transcription from a gene-   Center for Genome Research.
                 specific DNA template containing two opposing
                 T7 promoter sequences (e.g., esiWay Silencing Re-    Recombinant Dicer enzyme is then applied to digest
                 sources from RZPD). The resulting long double-       the dsRNA into a complex mixture of 21–23 base
                 stranded RNA (dsRNA) is subsequently digested        pair siRNA. Finally, residual undigested dsRNA is
                 into 21–23 base pair siRNA by Dicer enzyme           removed to yield highly pure d-siRNA (Figure 2).
                 (Figure 2). The new X-tremeGENE siRNA Dicer Kit      Purity of d-siRNA is of major importance, since the
                 from Roche Applied Science has been specially        remaining long dsRNA causes a non-specific inter-
                 developed to generate highly pure diced siRNA        feron response in mammalian cells.
                 (d-siRNA) for effective gene knockdown.




             8
                                                                                    Figure 2: Overview of d-siRNA
                                                                                 (esiRNA) generation using
                                                                                 the X-tremeGENE siRNA Dicer
                                                                                 Kit and an esiWay Silencing
                                                                                 Resource as template.

                                                                                 * esiWay Silencing Resources are
                                                                                 available from the RZPD German
                                                                                 Resource Center for Genome
                                                                                 Research.
                                                                                 For further information, visit
                                                                                 www.rzpd.de/products/esiRNA




                                                                                                                          siRNA
                                                                                                                        Generation

Rely on d-siRNA generated using the X-tremeGENE siRNA Dicer Kit to:

½ Ensure effective gene knockdown by applying                ½ Use the kit’s control DNA template (hypo-
  a mixture of siRNAs generated analogous to the               xanthine phosphoribosyl transferase [HPRT])
  in vivo process which is more likely to silence              to synthesize a positive control d-siRNA.
  target-gene expression than a single siRNA.                  Knockdown of the HPRT gene is conveniently
                                                               measured using the LightCycler® h-HPRT
½ Minimize off-target effects due to the high
                                                               Housekeeping Gene Set and the LightCycler®
  purity and low concentration of each individual
                                                               2.0 Instrument (9, 10).
  siRNA in the pool.
½ Save time and resources by using a pool of
  diced siRNA instead of screening individual
  siRNA sequences to find an effective one.

For detailed information, visit www.roche-applied-science.com/geneknockdown




                                                                                                                    9
              siRNA Generation
                  Production of highly effective siRNA pools from
                  long dsRNA using Dicer


              Choose esiWay Silencing Resources from RZPD as                 Knockdown of the anti-apoptotic gene
              starting templates, to streamline the generation               AKT2 using RNAi
              of diced siRNA targeting human and/or mouse genes.
                                                                             To illustrate a typical gene knockdown
              esiWay Silencing Resources are gene-specific PCR
                                                                             experiment, silencing of the anti-
              fragments with an average length of 300 base pairs
                                                                             apoptotic gene AKT2 by AKT2-specific
              guaranteed not to contain any repetitive or conserved
                                                                             siRNA is described. The methods and
              sequences. They carry T7 promoters at both ends
                                                                             results of this experiment demonstrate
              enabling the immediate in vitro transcription of
                                                                             the following:
              dsRNA and subsequent dicing to prepare d-siRNA
                                                                             - how an AKT2-specific esiWay Silencing
              using the X-tremeGENE siRNA Dicer Kit.
                                                                             Resource is used as a template for
                                                                             the generation of diced siRNA with the
                                                                             X-tremeGENE siRNA Dicer Kit;
                                                                             - how AKT2-specific siRNA is delivered
              esiWay Silencing Resources from RZPD
                                                                             into PC-3 cells using X-tremeGENE
              German Resource Center for
                                                                             siRNA Transfection Reagent;
              Genome Research
                                                                             - how reduced AKT2 expression is
                                                                             analyzed using relative quantification with
                                                                             the LightCycler® 2.0 System, and by
              ½ Gene-specific PCR fragments containing
                                                                             western blotting;
                T7 promoter sequences at both ends
                                                                             - how effects of AKT2 knockdown on cell
              ½ Available for nearly 5,000 human genes* and                  proliferation and apoptosis are measured.
                14,000 mouse genes
  siRNA
                                                                             AKT2 is a member of the Akt subfamily
Generation    ½ Average size 300 base pairs
                                                                             of serine/threonine protein kinases
              ½ No repetitive or conserved sequences                         whose genes are activated by different
                                                                             stimuli such as growth factors, protein
              ½ Serve as template for generation of d-siRNA
                                                                             phosphatase inhibitors, and stress (11).
                (esiRNA) with the X-tremeGENE siRNA Dicer Kit
                                                                             Akt protein kinases phosphorylate a
                                                                             variety of cellular proteins containing
              *the collection is continuously expanding and is expected to   the Akt phosphorylation consensus
              cover the entire human transcriptome by the end of 2005
                                                                             sequence R-X-R-X-X-S/T-F/L and con-
              For detailed information and to order, visit                   tribute to the survival of cells, during
              www.rzpd.de/products/esiRNA                                    stress by blocking the apoptotic pathway.




         10
                                            Synthesis of AKT2-specific siRNA


This is supported by the observation        To generate siRNA for AKT2 gene
that the administration of antisense        knockdown in PC-3 cells, the AKT2-
RNA directed against one Akt isoform        specific esiWay Silencing Resource
causes apoptosis. Known or suspected        RZPDp3000A01530 (398 base pairs) was
targets of Akt phosphorylation include      used in combination with the X-tremeGENE
GSK3, BAD, caspase-9, and transcription     siRNA Dicer Kit. Long dsRNA was
factor FKHRL1. Due to its anti-apopto-      synthesized by in vitro transcription using
tic activity, AKT2 has been implicated in   the AKT2-specific esiWay Silencing
human neoplasia. Amplification or over-     Resource as template. This long dsRNA was
expression of AKT2 has been detected in     subsequently digested into d-siRNA by
10%–20% of ovarian carcinomas and           Dicer enzyme (Figure 3, lane 2). Finally, the
pancreatic cancers. Akt expression also     remaining undigested dsRNA was removed
correlates with disease progression in an   using High Pure spin filter tubes yielding
experimental model of prostate cancer       a prominent band of highly pure d-siRNA
(12). In the experiments described here,    suitable for transfection (Figure 3, lane 3).
the effect of blocking AKT2 activity in
the prostate cancer cell line PC-3, which
                                                                         Figure 3: AKT2-specific
was shown to express the AKT2 protein,
                                                                     diced siRNA, before and after
has been analyzed.                                                   purification with High Pure
                                                                     spin filter tubes (included in
                                                                                                          siRNA
                                                                     the X-tremeGENE siRNA
                                                                                                        Generation
                                                                     Dicer Kit).
                                                                     Aliquots of the dicing reaction
                                                                     were analyzed on a 4%
                                                                     agarose gel.
                                                                     Lane 1: 100 bp ladder;
                                                                     Lane 2: 300 ng unpurified
                                                                     dicer reaction;
                                                                     Lane 3: 300 ng purified
                                                                     d-siRNA.
                                                1     2      3




                                                                                                       11
               Transfection
                         Delivery of siRNA into cells with
                         high knockdown efficiency and low cytotoxicity




               siRNA delivery into cultured cells by transfection is          siRNA Transfection
               a crucial step within the RNAi workflow. To study
                                                                             Choose X-tremeGENE siRNA Transfection Reagent
               the consequences of gene knockdown, it is essential
                                                                             from Roche Applied Science to effectively study the
               to have the highest possible transfection efficiency.
                                                                             cellular and functional consequences of gene
               Otherwise, residual gene activity in untransfected
                                                                             knockdown in many commonly used cell types, and
               cells can greatly interfere with the effects caused by
                                                                             in several hard-to-transfect cell lines such as HT-29
               the down-regulated expression of this particular
                                                                             (human adenocarcinoma cell line).
               gene.
                                                                             X-tremeGENE siRNA Transfection Reagent is a
               A transfection reagent suitable for RNAi experiments
                                                                             multi-component reagent that forms a complex with
               must:
                                                                             siRNA as well as with mixtures of siRNA and plasmid
               1) mediate the delivery of the nucleic acid into the
                                                                             DNA. This transfection complex efficiently delivers
               cells with a very high efficiency;
                                                                             siRNA into mammalian cells to induce gene
               2) exhibit as little cytotoxicity as possible since a
                                                                             silencing. Since X-tremeGENE siRNA Transfection
               cytotoxic effect caused by the transfection reagent
                                                                             Reagent functions exceptionally well in the presence
               could interfere with the detection and monitoring
                                                                             or absence of serum, and demonstrates low
               of the targeted knockdown effect.
                                                                             cytotoxicity, it eliminates the need to change media.
               To exclude transfection-specific off-target effects,
                                                                             When used for co-transfection-based gene knock-
               use a high quality transfection reagent like
                                                                             down, the high transfection efficiency of
               X-tremeGENE siRNA Transfection Reagent or
                                                                             X-tremeGENE siRNA Transfection Reagent enables
               FuGENE 6 Transfection Reagent.
                                                                             significant protein expression and effective
Transfection
                                                                             knockdown levels.
               X-tremeGENE siRNA Transfection Reagent has
               been specially developed for the efficient transfection
                                                                             ½ Achieve over 90% gene knockdown in many
               of siRNA molecules. It should be used if siRNA is
                                                                               cell types using siRNA.
               employed alone and if siRNA is delivered together
               with a plasmid in a co-transfection experiment.               ½ Enjoy maximum flexibility using one single
               FuGENE 6 Transfection Reagent has been optimized                reagent to perform both siRNA- and
               for plasmid transfections and should be used for                co-transfection experiments.
               introducing plasmids expressing shRNA.
                                                                             ½ Benefit from high efficiency and low cytotoxicity.
                                                                             ½ Save time as there is no need for media changes.
                      Transfection of siRNA          Transfection of shRNA
                Co-transfection of siRNA + Plasmid     expression plasmid


                      X-tremeGENE siRNA                   FuGENE 6           For detailed information, visit
                      Transfection Reagent           Transfection Reagent
                                                                             www.roche-applied-science.com/geneknockdown



          12
    Transfection of AKT2-specific siRNA into PC-3 cells

   In a 24-well plate, exponentially growing PC-3        specific siRNA using 2.5 µl X-tremeGENE
   prostate cancer cells (4 x 104 cells/well) were       siRNA Transfection Reagent and 0.5 µg of the
   seeded in RPMI 1640 medium containing                 respective siRNA, according to the package
   10% (v/v) fetal calf serum and 2 mM glutamine.        insert. Verification of AKT2 knockdown and
   After growing the cells overnight, transfection       analysis of RNAi-induced effects were carried
   was carried out with either AKT2- or luciferase-      out two days after transfection.




 Transfection of plasmids expressing shRNA
 (Plasmid Transfection)

FuGENE 6 Transfection Reagent is ideally suited          ½ Rely on an established transfection reagent pro-
for the effective delivery of plasmids expressing           ven to successfully transfect more than 700 cell
small hairpin RNA (shRNA) to study the effects of           types.
gene knockdown in cell culture. It is an advanced
                                                         ½ Achieve high transfection efficiencies in tran-
non-liposomal formulation that transfects an
                                                            sient and stable transfections.
extremely wide variety of eukaryotic cells, including
primary cultures and hard-to-transfect cell lines,       ½ Obtain results you can trust due to the
with high efficiency and minimal cytotoxicity.              reagent's exceptionally low cytotoxicity.
FuGENE 6 Transfection Reagent can be used for
                                                         For detailed information, visit                            Transfection
both transient transfection experiments and              www.roche-applied-science.com/transfection
the generation of permanently transfected cell lines
in case of long-term gene silencing. For the selection   High-quality plasmid DNA is an important pre-
of such lines, Roche Applied Science provides            requisite for the optimal formation of the transfection
the commonly used selection antibiotics Geneticin        complex and, as a consequence, for the successful
(G 418) and Hygromycin B.                                delivery of the DNA into the cell. The Genopure
                                                         Plasmid Kits, based on ion-exchange chromato-
                                                         graphy, are specially designed for the purification of
                                                         transfection-grade plasmid DNA.
                                                         For detailed information, visit
                                                         www.roche-applied-science.com/napure




                                                                                                                   13
                   Knockdown Verification
                            Confirmation of gene knockdown by
                            mRNA quantification and protein detection




                                                                           Gene knockdown verification is recommended,
                Gene knockdown can be analyzed at both the mRNA            both by mRNA quantification and protein detection,
                and protein level. Quantification of mRNA necessi-         to unambiguously correlate the down regulation of
                tates primers and probes for RT-PCR or northern            protein expression to siRNA-induced gene silencing.
                blotting. Protein detection requires knowledge about       With the LightCycler® 2.0 System, Roche Applied
                the protein identity, and availability of specific anti-   Science provides state-of-the-art technology for the
                bodies; or proteins with detectable biochemical            sensitive and accurate determination of mRNA
                activities; or tagged proteins.                            levels by relative quantification.


                mRNA Quantification


                 Knockdown verification at the mRNA level requires         ½ Fully automate your RNA
                 the isolation of pure, intact RNA from transfected           isolation for up to 32 samples.
                 cells. Roche Applied Science provides many kits,
                                                                           ½ Rely on proven magnetic bead
                 reagents, and even a fully automated device – the
                                                                              technology for the isolation of
                 MagNA Pure LC System, enabling the isolation of
                                                                              highly pure RNA.
                 high-quality RNA with a minimum of effort and time.
                                                                           ½ Choose from ready-to-use
                                                                              system reagents optimized for
                   MagNA Pure LC System                                       efficient isolation of total
                                                                              RNA or mRNA.                         Figure 4: MagNA
                 The MagNA Pure LC Instrument is a robotic work-
                                                                                                                Pure LC Instrument.
Knockdown        station for the rapid and cross-contamination-free
Verification     isolation of nucleic acids based on magnetic bead         For detailed information, visit www.magnapure.com
                 technology. After RNA purification, the MagNA Pure
                 LC Instrument precisely performs RT-PCR setup             The High Pure RNA Isolation Kit is recommended
                 (e.g., for combined use with the LightCycler® 2.0         for the manual isolation of high-quality total RNA.
                 System). With the MagNA Pure LC RNA Isolation             The resulting RNA eluate can be directly applied in
                 Kit - High Performance and the MagNA Pure LC              first-strand cDNA synthesis.
                 mRNA Isolation Kit I, dedicated system reagents           When mRNA and protein analyses are required from
                 are provided for the isolation of highly pure total       the same sample, use TriPure Isolation Reagent to
                 RNA and mRNA from cells.                                  simultaneously isolate RNA, DNA, and proteins.

                                                                           For detailed information, visit
                                                                           www.roche-applied-science.com/napure




           14
 LightCycler® 2.0 System

Real-time PCR is the technology of choice to confirm
knockdown of target gene expression at the mRNA          ½ Confirm gene knockdown by accurate and
level. Using the LightCycler® 2.0 Instrument, you          reproducible real-time PCR using relative
can monitor the amplification of the PCR product           quantification.
in real-time and online, utilizing up to six different
                                                         ½ Rely on high sensitivity for quantification of
detection channels. The amplification occurs in
                                                           silenced target-gene expression.
specially designed glass capillaries which provide a
high surface-to-volume ratio enabling extremely          ½ Choose from a variety of detection formats.
fast thermal transfer. This feature combined with
the air-driven temperature control of the instrument
                                                         Use the Transcriptor First Strand cDNA Synthesis
enables rapid PCR: an entire 35-cycle run can be
                                                         Kit for two-step RT-PCR with the LightCycler® 2.0
performed in as little as 30 minutes. Additional
                                                         System to generate high yields of full-length cDNA.
benefits are high reproducibility and a minimized
                                                         Depending on the detection format selected for
risk of primer dimer generation.
                                                         the successive amplification step,
For sensitive and specific PCR product detection,
                                                         LightCycler® FastStart DNA MasterPLUS HybProbe,
HybProbe probe and hydrolysis probe formats
                                                         LightCycler® FastStart DNA MasterPLUS SYBR
are recommended. Other detection formats like
                                                         Green I, or LightCycler® TaqMan® Master ensure
SYBR Green I are also supported.
                                                         highly specific and sensitive hot-start PCR. The
Analysis of gene knockdown results is substantially
                                                         robust performance of the hot-start polymerase
improved with the LightCycler® Relative Quanti-
                                                         included in these master mixes leads to highly
fication Software/LightCycler® Software 4.0. This
                                                         reproducible results. An optimized MgCl2 concen-
software calculates target gene expression in relation
                                                         tration eliminates the need for further titration.
to a non-regulated reference gene and corrects for
experimental variances. Even minimal changes
in gene expression levels are shown directly with the
highest accuracy independent of sample amount
used. Consequently, the software helps generate
meaningful quantification data.


                                                                                    Figure 5: LightCycler ® 2.0
                                                                                 Instrument.


         mRNA Quantification using quantitative real-time PCR
                                                                                                                   Knockdown
                                                                                                                   Verification




                                                                                                                  15
                Knockdown Verification
                        Confirmation of gene knockdown by
                        mRNA quantification and protein detection

                LightCycler® 2.0 System

                 LightCycler ® 2.0 Instrument

                 LightCycler ® 1.5 Instrument

                 LightCycler ® FastStart DNA Master PLUS Kits

                 LightCycler ® TaqMan ® Master

                 LightCycler ® h-Housekeeping Gene Selection Set

                 LightCycler ® Probe Design Software 2.0
                                                                       For detailed information, visit
                 LightCycler ® Relative Quantification Software        www.lightcycler-online.com




                     Confirmation of AKT2 knockdown in PC-3 cells by quantitative real-time PCR

                    To quantitate mRNA expression in PC-3 cells        with luciferase-specific siRNA was nearly at
                    transfected with different siRNAs by real-         the same level (Figure 6). In contrast, for
                    time PCR, RNA was isolated using the High Pure     cells transfected with AKT2-specific siRNA, a
                    RNA Isolation Kit two days after transfection.     shift of 2.7 in the crossing point (CP) values
                    50 µl of total RNA was eluted from the columns.    was observed, corresponding to a knockdown
                    For each sample, 5 µl total RNA was reverse        efficiency of approximately 85%.
                    transcribed into cDNA using the Transcriptor
                    First Strand cDNA Synthesis Kit with random
                    primers. The cDNA was diluted tenfold in
                    PCR-grade water prior to quantification using
                    the LightCycler® 2.0 System.
                    Quantitative real-time PCR was performed with
Knockdown           the LightCycler® 2.0 Instrument and the
Verification        LightCycler® FastStart MasterPLUS SYBR Green I
                    using 0.5 µM of each primer, and 4 mM MgCl2.
                    AKT2-specific primers were designed with
                    the LightCycler® Probe Design Software 2.0.
                    To normalize the results, primer/probe pairs for
                    the housekeeping gene 5-aminolevulinate
                    synthase (h-ALAS), included in the LightCycler®
                    h-Housekeeping Gene Selection Set, were used.          Figure 6: Quantitative real-time PCR.
                                                                       First-strand cDNA was synthesized from RNA isolated
                    When PC-3 cells were transfected with AKT2         from PC-3 cells transfected with different siRNAs.
                    siRNA, a specific decrease of the correspon-       Quantitative real-time PCR was performed using the
                    ding mRNA was detected. AKT2 expression in         LightCycler® 2.0 System, and fluorescence signals are
                    untransfected cells and in cells transfected       shown.



           16
 Universal ProbeLibrary Sets

Choose a Universal ProbeLibrary Set or single probes                Universal ProbeLibrary Workflow
for the specific and convenient quantification of
target-gene-expression levels by real-time PCR using                                                       Day 1
                                                                           Identify gene or target
the LightCycler® 2.0 System.                                                sequence of interest
Universal ProbeLibrary is a combination of 165 probes
and unique assay design software that delivers                           Design custom assay at
                                                                       www.universalprobelibrary.com
real-time PCR assays for virtually any transcript of
an organism (human, primates, mouse, rat, Drosophila,                 Order assay-specific primers from
C. elegans, and Arabidopsis). The probes are                           your preferred oligo supplier for
                                                                              overnight delivery
designed and prevalidated to provide transcriptome-
wide coverage for the specific organisms. Each                        Select the appropriate probe from    Day 2
                                                                      the Universal ProbeLibrary Set in
probe can be ordered separately, or as an organism-                              your freezer
specific set of 90 probes. The probes are dual-
labeled with a reporter fluorophore and dark quencher.                      Perform real-time PCR
Universal ProbeLibrary probes use a unique nucleotide
chemistry called LNA (Locked Nucleic Acid). This
                                                                               Evaluate results
allows to shorten the length of the probes to 8 or 9
bases. The shortened LNA PCR probes maintain
the high melting temperature required for real-time
PCR, plus single-mismatch discrimination.
                                                         ½ Benefit from significantly reduced assay design
Therefore, Universal ProbeLibrary probes are fully
                                                            time and increased flexibility.
compatible with commonly used PCR conditions
and the hydrolysis probe detection format. Specific      ½ Achieve high assay specificity by using
amplification of the target sequence is assured             sequence-specific probes together with carefully           Knockdown
by carefully selected primers calculated by the assay       selected primer pairs.                                     Verification
design software.
                                                         ½ Conveniently integrate assays into your
Performance of the assay with the selected
                                                            workflow as no special hardware or unique
Universal ProbeLibrary probe follows established
                                                            reaction conditions are required.
real-time PCR protocols using the LightCycler®
TaqMan® Master and the LightCycler® 2.0 Instrument.
Gene-specific expression quantification assays           For detailed information on the Universal ProbeLibrary and
are easily designed using the ProbeFinder software       to design your next qPCR assay, visit
accessible at www.universalprobelibrary.com              www.universalprobelibrary.com




                                                                                                                      17
                Knockdown Verification
                    Confirmation of gene knockdown by
                    mRNA quantification and protein detection



                 Northern blotting

                Silencing of target gene expression can also be          In addition, DIG-labeled probes can be stored for
                determined by northern blotting. The use of positively   several months without any loss in performance.
                charged nylon membranes and nonradioactive               Choose the DIG Northern Starter Kit containing all
                digoxigenin-labeled probes combined with chemilu-        the reagents required for the preparation of DIG-
                minescence detection is widely acknowledged as a         labeled RNA probes, and for the chemiluminescence
                technology equivalent to radioactive methods. The        detection of the corresponding transcripts with
                DIG System developed by Roche Applied Science            CPD-Star substrate.
                offers signal detection with high sensitivity and low
                                                                         For detailed information, visit
                background, but also safe and convenient handling.       www.roche-applied-science.com/dig




                    Confirmation of AKT2 knockdown in PC-3 cells by northern blotting


                    To detect suppressed AKT2 mRNA expression,                                                 M: RNA Molecular
                    total RNA was isolated from PC-3 cells two                                                 Weight Marker II,
                    days after transfection with different siRNAs                                              digoxigenin-labeled
                                                                                                               Lane 1: Untransfected
                    using the High Pure RNA Isolation Kit. A
                                                                                                               PC-3 cells
                    northern blot was performed using 5 µg total                                               Lane 2: PC-3 cells
                    RNA, according to the package insert of the                                                transfected with AKT2
                    DIG Northern Starter Kit. For hybridization, a                                             siRNA
Knockdown           digoxigenin-labeled AKT2 DNA probe                                                         Lane 3: PC-3 cells
Verification        synthesized with the PCR DIG Probe Synthesis                                               transfected with
                    Kit was used. Hybridization with DIG-labeled                                               Luciferase siRNA
                    Actin RNA Probe served as loading control.
                    Expression of AKT2 in untransfected cells
                    (Figure 7, lane 1) and in cells transfected with        M            1   2   3

                    luciferase-specific siRNA (Figure 7, lane 3)
                                                                            Figure 7: Analysis of mRNA expression by northern
                    was nearly at the same level. However, in cells      blotting. RNA isolated from cells transfected with different
                    transfected with AKT2-specific siRNA, AKT2           siRNAs was used for northern blotting and hybridized with
                    mRNA expression was below the detection              digoxigenin-labeled probes. Hybridization against ß-Actin
                    limit, showing efficient knockdown (Figure 7,        was used as loading control.
                    lane 2).




           18
Protein Detection
 Western blotting
                                                              ½ Benefit from high sensitivity and detect
To monitor the effect of siRNA transfection at the              antigens in the range of 1–5 picograms.
protein level, cells are usually lysed and the protein
                                                              ½ Perform multiple exposures due to signals
of interest is detected by western blotting. Western
                                                                stable for more than 9 hours.
blotting is a well-established technique for the
detection of even minute amounts of a protein. It             ½ Save precious sample material as reduced
requires a specific antibody that binds to the                  amounts of antigen are required.
denatured or partially denatured form of the pro-
tein to be detected. Alternatively, if a tagged protein
                                                              Protease inhibitors may be required depending on
has been expressed in a co-transfection experiment,
                                                              the endogenous protease content of the cells, and
a tag-specific antibody can be used. Chemilumines-
                                                              the vulnerability of the protein of interest to pro-
cence has become the method of choice for protein
                                                              tease digestion. Use of complete tablets containing
detection providing high sensitivity.
                                                              a mixture of protease inhibitors for complete
                                                              protein protection is recommended. These tablets
Lumi-LightPLUS Western Blotting Kit                           ensure that the effect of gene knockdown is not
(Mouse/Rabbit)                                                obscured by non-specific proteolytic degradation.
The substrates included in the Lumi-LightPLUS                 For detailed information on kits and reagents for western
Western Blotting Kit (Mouse/Rabbit) are optimi-               blotting, visit www.proteomic-science.com
zed not only to retain high sensitivity levels, but           For detailed information on complete Protease Inhibitor Cocktail
also to emit light for much longer time periods.              Tablets, visit www.roche-applied-science.com/proteaseinhibitor



      Confirmation of AKT2 knockdown in PC-3 cells by western blotting

    To confirm AKT2 knockdown at the protein                                                        Figure 8: Analysis of
    level, cells were lysed in the presence of complete                                         AKT2 protein expression by
    Protease Inhibitor Cocktail Tablets two days                                                western blotting. Equal amounts
                                                                                                                                     Knockdown
                                                                                                of proteins isolated from cells
    after transfection. The lysate was used to perform                                                                               Verification
                                                                                                transfected with different siRNAs
    western-blot analysis according to standard                                                 were applied to SDS PAGE and
    procedures using PVDF Western Blotting Mem-                                                 subsequent western-blot analysis
    branes. After incubation with a goat anti-AKT2                                              with an AKT2-specific antibody.
    antibody, detection was carried out using an                                                The band at 40 kDa is an un-
    anti-goat IgG antibody horseradish peroxidase                                               specific reaction of the first
    conjugate in combination with blocking reagent                  1       2       3           antibody and is used as loading
    and substrate, included in the Lumi-LightPLUS                                               control.
                                                                                                Lane 1: PC-3 cells transfected
    Western Blotting Kit (Mouse/Rabbit).
                                                                                                with AKT2 siRNA
    Expression of AKT2 protein was reduced by 85%                                               Lane 2: PC-3 cells transfected
    after transfection with AKT2-specific siRNA                                                 with Luciferase siRNA
    (Figure 8, lane 1), while transfection with luciferase-                                     Lane 3: Untransfected PC-3
    specific siRNA (Figure 8, lane 2) had no effect.                                            cells



                                                                                                                                    19
              Functional Assays
                     Analysis of cellular responses to gene knockdown with siRNA




              After detecting successful knockdown of a target        Cell Proliferation Reagent WST-1
              gene at both the mRNA and protein level, the
                                                                      Choose Cell Proliferation Reagent WST-1 to assay
              resulting downstream effects on cell growth are of
                                                                      for the metabolic activity of viable cells with
              particular interest.
                                                                      high sensitivity. WST-1 is a modified tetrazolium
              Roche Applied Science provides a range of highly
                                                                      salt that is reduced to a colored, water-soluble
              sensitive and reliable assays to study the effects of
                                                                      formazan by viable cells (Figure 9). The formazan
              suppressing a gene on cell proliferation and
                                                                      can be easily and rapidly quantified.
              apoptosis induction.

               Cell Proliferation Assays
                                                                      ½ Benefit from the high sensitivity of WST-1 and
              Colorimetric microplate-format assays (96-well)           detect low cell numbers.
              have been developed to analyze cell viability and
                                                                      ½ Generate accurate results with the absorbance
              proliferation using an ELISA plate reader. Cellular
                                                                        measured, strongly correlating to the cell number.
              damage inevitably results in the cells’ inability
              to maintain and provide metabolic energy for cell       ½ Save time with a ready-to-use solution used in
              function and growth. Metabolic activity assays            a convenient one-step assay.
              measure this effect.
                                                                      For detailed information on cell proliferation assays, visit
                                                                      www.roche-applied-science.com/apoptosis

                 Figure 9: Molecular structure of WST-1 and
              the corresponding reaction product.




Functional
 Assays




         20
 Determination of PC-3 cell viability                            Apoptosis Assays
 after AKT2 knockdown
                                                                Apoptosis or “programmed” cell death is a highly
                                                                regulated process with a defined sequence of events.
To investigate the effect of AKT2 knockdown on
                                                                Roche Applied Science has developed assays to
the viability of PC-3 cells, a cell proliferation assay
                                                                monitor progression of apoptosis at the different
was performed with transfected or untransfected
                                                                stages. Membrane alterations characterized by
PC-3 cells two days after transfection using
                                                                phosphatidylserine exposed on the outside of the
Cell Proliferation Reagent WST-1. The extent of
                                                                cell membrane in apoptotic cells, for example,
colored formazan formed during the reaction
                                                                can be detected using labeled Annexin-V. The
directly correlates with the metabolic activity of
                                                                hallmark of apoptosis is the fragmentation of the
the cells. Absorbance was measured at 437 nm
                                                                genomic DNA. This irreversible event commits
against medium.
                                                                the cell to die and can be confirmed by the detection
Transfection with AKT-2 specific siRNA signifi-
                                                                of mono- or oligonucleosomes. Given the funda-
cantly decreased the proliferation rate and
                                                                mental role of the apoptotic pathway, test systems
viability of PC-3 cells, whereas luciferase-specific
                                                                which measure apoptosis are valuable tools for
siRNA had only a small effect (Figure 10).
                                                                studying gene functions by RNAi.
This is to be expected since blocking of AKT2
gene expression results in apoptosis.
                                                                ½ Choose from a wide range of kits and reagents,
                                                                  each optimized for the detection of a particular
                                                                  stage in the apoptosis process.
                  100
                                                                ½ Rely on established assays for the sensitive
  Viability [%]




                  80                                              detection of cells undergoing apoptosis.
                  60
                                                                ½ Select from multiple detection formats – light
                  40
                                                                  or fluorescence microscopy, flow cytometry, or
                   20
                                                                  ELISA with colorimetric or chemiluminescent
                    0
                                                                  substrates.
                        Untrans-   Luciferase   AKT2
                         fected      siRNA      siRNA


   Figure 10: Cell proliferation assay.                         For detailed information on apoptosis assays, visit
Viability of PC-3 cells transfected with different siRNAs was   www.roche-applied-science.com/apoptosis
analyzed using Cell Proliferation Reagent WST-1.
                                                                                                                         Functional
                                                                                                                          Assays




                                                                                                                        21
              Functional Assays
                 Analysis of cellular responses to gene knockdown with siRNA

              Cell Death Detection ELISAPLUS
                                                                         ½ Specifically detect cells undergoing apoptosis
              Analyze histone-associated DNA fragments (mono-
                                                                           as histone-associated DNA fragments are
              and oligonucleosomes), which are known to be
                                                                           measured from cell lysate (apoptotic cells) or
              present in the cytoplasm of cells undergoing
                                                                           from cell culture supernatant (necrotic cells).
              apoptosis, with the Cell Death Detection ELISAPLUS.
              This assay applies mouse monoclonal antibodies             ½ Achieve high sensitivity and detect apoptosis
              directed against DNA and histones. The sample is             in as few as 600 cells.
              incubated in a streptavidin-coated microplate
                                                                         ½ Obtain reproducible relative quantification
              with a mixture of anti-histone-biotin and anti-DNA-
                                                                           by numerical measurements of nucleosomal
              peroxidase antibody conjugates. Nucleosomes are
                                                                           particles in a convenient one-step assay.
              captured via their histone component by the
              anti-histone-biotin antibody, while binding to the
              streptavidin-coated microplate. Anti-DNA-peroxidase        For detailed information on apoptosis assays, visit
              antibody simultaneously binds to the DNA part of           www.roche-applied-science.com/apoptosis
              the nucleosomes. After washing and incubating with
              peroxidase substrate ABTS (2,2’-Azino-di[3-ethyl-
              benzthiazoline sulfonate]), the amount of peroxidase
              retained in the immunocomplex is photometrically
              determined in an ELISA plate reader.

                                                  Figure 11: Principle
                                              of the Cell Death
                                              Detection ELISA PLUS.




                   Analysis of apoptotic effects of AKT2 knockdown in PC-3 cells


                   To further analyze the apoptotic effect of AKT2          DNA fragments released into the supernatant
                   knockdown, an apoptosis assay was carried out            was much lower (data not shown).
                   with transfected and untransfected PC-3 cells
                   using the Cell Death Detection ELISAPLUS.                              1,500

                   The cells were spun down in the culture plates
                   and the supernatant was completely removed.                            1,000
                                                                             Absorption




                   The remaining cells were subsequently lysed with
                   lysis buffer from the kit. Then equal aliquots                          500
Functional         from the lysed cells and the culture supernatant
 Assays            were tested according to the protocol. In PC-3                            0
                   cells transfected with AKT2-specific siRNA, a                                  Untrans-   Luciferase   AKT2
                                                                                                   fected      siRNA      siRNA
                   high level of intracellular fragmented DNA was
                   detected compared with untransfected cells                  Figure 12: Analysis of apoptosis-induced DNA
                   or cells transfected with luciferase-specific siRNA      fragmentation. DNA fragmentation was analyzed in lysa-
                   (Figure 12). The effect was mainly due to                tes of PC-3 cell transfected with different siRNAs using
                   apoptosis and not necrosis, since the number of          the Cell Death Detection ELISA PLUS .



         22
 Cytotoxicity Assays

In contrast to necrosis and apoptosis, cytotoxicity
does not define a specific cellular death mechanism.
Consequently, assays that measure cytotoxicity
give information about the proportion of dead cells
at a given timepoint. In RNAi experiments such
assays are used to test whether the down-regulation
of a gene has caused a cytotoxic effect.

                                                            Figure 13: Biochemistry of the Cytotoxicity Detection
                                                         Kit (LDH). In the first enzymatic reaction LDH reduces
                                                         NAD+ to NADH + H + by oxidation of lactate to pyruvate. In
Cytotoxicity Detection Kit (LDH)                         the second enzymatic reaction, the catalyst (diaphorase)
Measure cytotoxicity and cell lysis by detecting         transfers H/H+ from NADH + H + to the tetrazolium salt INT.

lactate dehydrogenase (LDH) activity using the
Cytotoxicity Detection Kit (LDH). LDH is a stable
                                                         ½ Rely on accurate results strongly correlating to
cytoplasmic enzyme present in all cells. It is
                                                           the number of lysed cells.
rapidly released into the cell culture supernatant
when the plasma membrane is damaged. LDH                 ½ Avoid the hazards of 3H-thymidine-
activity in the supernatant is determined by a coupled     uptake/release techniques or 51chromium-
enzymatic reaction in which the tetrazolium salt           release assays.
INT is reduced to formazan (Figure 13). The water-
                                                         ½ Benefit from a convenient assay with no prela-
soluble formazan can be easily quantified in an
                                                           beling and washing steps required.
ELISA plate reader.
                                                         For detailed information on cytotoxicity assays, visit
                                                         www.roche-applied-science.com/apoptosis




                                                                                                                        Functional
                                                                                                                         Assays




                                                                                                                       23
              Functional Assays
                 Analysis of cellular responses to gene knockdown with siRNA



               Analysis of cytotoxic effects of AKT2 knockdown              Summary
               in PC-3 cells


               At a very late stage of apoptosis, apoptotic cells          A specific and highly efficient knockdown
               finally lyse, and the subsequent release of the             of the AKT2 gene was achieved using
               cytoplasmic enzyme LDH into the culture medium              X-tremeGENE siRNA Transfection Reagent for
               can be monitored. Two days after transfection,              delivery of AKT2-specific siRNA. Knockdown
               LDH release from transfected and untransfected              was verified at the mRNA level by relative
               PC-3 cells was analyzed. 100 µl of culture super-           quantification using the LightCycler® 2.0 System,
               natant was mixed with the same volume of reaction           or by northern blotting and detection with
               mix from the Cytotoxicity Detection Kit (LDH)               DIG-labeled probes. These results were further
               and incubated. Absorbance was measured at                   confirmed at the protein level using western
               490 nm against medium.                                      blot analysis with the Lumi-LightPLUS substrate.
               Transfection with luciferase-specific siRNA did             It was shown that the knockdown of the anti-
               not change the rate of cell lysis compared to               apoptotic protein AKT2 has dramatic effects
               untransfected cells. However, with AKT2-specific            on PC-3 cells. Proliferation of the cells was
               siRNA a significant increase in lysis of PC-3               severely influenced by the initiation of apoptosis,
               cells by induction of the apoptotic process was             as demonstrated with the WST-1 assay.
               observed (Figure 14).                                       When the cells were further analyzed with the
                                                                           Cell Death Detection ELISAPLUS, DNA frag-
                                                                           mentation and membrane reorganization
                                2,500
                                                                           (data not shown) were detected. These changes
                                                                           finally led to the complete disintegration of
                                2,000                                      the cell membrane. This process was monitored
                                                                           by the release of the cytoplasmic LDH into
                                1,500                                      the culture medium with the Cytotoxicity
                   Absorption




                                                                           Detection Kit (LDH).
                                1,000



                                 500


Functional                         0
 Assays                                 Untrans- Luciferase   AKT2
                                         fected    siRNA      siRNA


                  Figure 14: Cytotoxicity assay. Release of LDH from
               PC-3 cells transfected with different siRNAs was analyzed
               using the Cytotoxicity Detection Kit (LDH).




         24
                       Ordering Information




siRNA Generation

Product                                     Cat. No.         Pack Size

d-siRNA Generation

   X-tremeGENE siRNA Dicer Kit *            04 579 020 001   1 Kit (10 reactions)

Plasmid Purification
   Plasmid Purification

   Genopure Plasmid Midi Kit                03 143 414 001   1 Kit (20 preparations)

   Genopure Plasmid Maxi Kit                03 143 422 001   1 Kit (10 preparations)

   Genopure Buffer Set                      04 634 772 001   Buffer for 20 Maxi Preps
                                                             or 60 Midi Preps




Transfection

Product                                     Cat. No.         Pack Size

siRNA Transfection
Transfection

   X-tremeGENE siRNA Transfection Reagent   04 476 093 001   1 ml (400 transfections in a 24-well plate)
                                            04 476 115 001   Multi-pack 5 x 1 ml (2,000 transfections)

Plasmid Transfection

   FuGENE 6 Transfection Reagent            11 815 091 001   0.4 ml (120 transfections)
                                            11 814 443 001   1 ml (300 transfections)
                                            11 988 387 001   5 x 1 ml (1,500 transfections)

   Geneticin (G 418)                        11 464 981 001   1 g non-sterile
                                            11 464 990 001   5 g non-sterile

   Hygromycin B                             10 843 555 001   1 g (20 ml) sterile




                                                                                                              Ordering
                                                                                                            Information




                                                                                                           25
               Ordering Information


               Knockdown Verification

                Product                                                        Cat. No.         Pack Size

                RNA Stabilization
                  RNA Stabilization

                   Protector RNase Inhibitor                                   03 335 399 001   2,000 U
                                                                               03 335 402 001   10,000 U (5 x 2,000 U)

                Automated RNA Isolation
                   Automated RNA Isolation

                   MagNA Pure LC Instrument                                    12 236 931 001   -

                   MagNA Pure LC RNA Isolation Kit - High Performance ✚        03 542 394 001   1 Kit (192 isolations)

                   MagNA Pure LC mRNA Isolation Kit I ✚                        03 004 015 001   1 Kit (192 isolations)

                Manual RNA Isolation
                  Manual RNA Isolation

                   High Pure RNA Isolation Kit *                               11 828 665 001   1 Kit (50 purifications)

                   mRNA Isolation Kit                                          11 741 985 001   1 Kit

                   TriPure Isolation Reagent                                   11 667 157 001   50 ml
                                                                               11 667 165 001   200 ml

                Real-Time PCR
                   Real-Time PCR

                   Transcriptor First Strand cDNA Synthesis Kit ✜              04 379 012 001   1 Kit (50 reactions)

                   LightCycler® 2.0 Instrument #                               03 531 414 201   -

                   LightCycler® 1.5 Instrument ◗                               04 484 495 001   -
                                                                           ●
                   LightCycler®   FastStart DNA   MasterPLUS    HybProbe       03 515 575 001   1 Kit (96 reactions)
                                                                               03 515 567 001   1 Kit (480 reactions)

                   LightCycler® FastStart DNA MasterPLUS SYBR Green I ▼        03 515 869 001   1 Kit (96 reactions)
                                                                               03 515 885 001   1 Kit (480 reactions)

                   LightCycler® TaqMan® Master †                               04 535 286 001   1 Kit (96 reactions)

                   LightCycler® h-Housekeeping Gene Selection Set *            03 310 159 001   5 x 16 reactions

                Universal ProbeLibrary

                   Universal ProbeLibrary Set, Human ◆                         04 683 633 001   1 Set

                   Universal ProbeLibrary Set,   Mouse ◆                       04 683 641 001   1 Set

                   Universal ProbeLibrary Set,   Rat ◆                         04 683 650 001   1 Set

                   Universal ProbeLibrary Set, Primates ◆                      04 683 617 001   1 Set
  Ordering         Universal ProbeLibrary Set, Drosophila ◆                    04 683 625 001   1 Set
Information
                                                            ◆
                   Universal ProbeLibrary Set, C. elegans                      04 683 609 001   1 Set

                   Universal ProbeLibrary Set, Arabidopsis      ◆              04 683 595 001   1 Set

                   Universal ProbeLibrary Control Set                          04 696 417 001   1 Set




          26
Knockdown Verification

Product                                                          Cat. No.         Pack Size

Northern Blotting
   Northern Blotting

   DIG RNA Labeling Kit (SP6/T7)                                 11 175 025 910   1 Kit (2 x 10 labeling reactions)

   DIG Luminescent Detection Kit                                 11 363 514 910   1 Kit (50 blots)

   DIG Northern Starter Kit                                      12 039 672 910   1 Kit (10 labeling reactions and
                                                                                  detection of 10 blots of 10 x 10 cm)

   DIG Easy Hyb                                                  11 603 558 001   500 ml

   Nylon Membranes, positively charged                           11 209 272 001   10 Sheets (20 x 30 cm)
                                                                 11 209 299 001   20 Sheets (10 x 15 cm)
                                                                 11 417 240 001   1 Roll (0.3 x 3 m)

Protein Stabilization
   Protein Stabilization

   Complete Protease Inhibitor Cocktail Tablets                  11 697 498 001   20 Tablets
                                                                 11 836 145 001   3 x 20 Tablets

   Complete, EDTA-free Protease Inhibitor Cocktail Tablets       11 873 580 001   20 Tablets

Western Blotting
  Western Blotting

   Lumi-LightPLUS Western Blotting Kit (Mouse/Rabbit)            12 015 218 001   1 Kit

   Lumi-LightPLUS Western Blotting Substrate                     12 015 196 001   100 ml

   Western Blocking Reagent                                      11 921 673 001   100 ml

   PVDF Western Blotting Membranes                               03 010 040 001   1 Roll (0.3 x 3 m)

Anti-Tag Antibodies
   Anti-Tag Antibodies

   Anti-HA High Affinity (rat monoclonal antibody; clone 3F10)   11 867 423 001   50 µg
                                                                 11 867 431 001   500 µg

   Anti-c-myc (mouse monoclonal antibody; clone 9E10)            11 667 149 001   200 µg (lyoph.)
                                                                 11 667 203 001   5 mg (1 ml)

   Anti-His6 (mouse monclonal antibody; clone BMG-His-1)         11 922 416 001   100 µg

Reporter Gene Assays
   Reporter Gene Assays

   ß-Gal Reporter Gene Assay, chemiluminescent                   11 758 241 001   1 Kit (500 assays, microplate format;
                                                                                  250 assays, tube format)

   Luciferase Reporter Gene Assay, high sensitivity              11 669 893 001   200 Assays
                                                                 11 814 036 001   1,000 Assays                               Ordering
   hGH ELISA                                                     11 585 878 001   1 Kit (192 tests)                        Information

   SEAP Reporter Gene Assay, chemiluminescent                    11 779 842 001   1 Kit (500 assays, microplate format;
                                                                                  250 assays, tube format)

   CAT ELISA                                                     11 363 727 001   1 Kit (192 tests)




                                                                                                                          27
               Ordering Information



               Functional Assays

                Product                                                Cat. No.         Pack Size

                     Proliferation Assays
                CellCell Proliferation Assays

                   Cell Proliferation Reagent WST-1                    11 644 807 001   2,500 Tests

                   Cell Proliferation ELISA, BrdU (chemiluminescent)   11 669 915 001   1 Kit (1,000 tests)

                   Cell Proliferation Kit I (MTT)                      11 465 007 001   1 Kit (2,500 tests)

                   Cell Proliferation Kit II (XTT)                     11 465 015 001   1 Kit (2,500 tests)

                Apoptosis Assays
                  Apoptosis Assays

                   Cell Death Detection ELISAPLUS                      11 774 425 001   1 Kit (96 tests)

                   Cell Death Detection ELISAPLUS, 10x                 11 920 685 001   1 Kit (10 x 96 tests)

                   Annexin-V-FLUOS Staining Kit                        11 858 777 001   50 Tests
                                                                       11 988 549 001   250 Tests

                Cytotoxicity Assays
                   Cytotoxicity Assays

                   Cytotoxicity Detection Kit (LDH)                    11 644 793 001   1 Kit (2,000 tests)




  Ordering
Information




          28
Disclaimers and Trademarks
✚                                                                   ▼  Purchase of this product is accompanied by a limited license
   The purchase of this product does not convey any licenses
or other rights for the performance of PCR.                         to use it in the Polymerase Chain Reaction (PCR) process,
                                                                    including homogeneous PCR methods described in U.S. Patents
* This product is optimized for use in the Polymerase Chain         Nos. 5,994,056, 6,171,785, 6,569,627 and corresponding
Reaction (PCR) process covered by patents owned by
                                                                    patents outside the United States, for life science research in
F. Hoffmann-La Roche Ltd. No license under these patents to
                                                                    conjunction with a thermal cycler whose use in the automated
use the PCR process is conveyed expressly or by implication
                                                                    performance of the PCR process is covered by the up-front
to the purchaser by the purchase of this product.
                                                                    license fee, i.e., an authorized thermal cycler.
◗                                                                   No real-time apparatus or system patent rights or any other
   This LightCycler® Instrument is licensed under U.S. Patent
No. 6,814,934 and non-U.S. counterpart patent claims for use in     patent rights owned by Applera Corporation are conveyed
life science research. It is also an Authorized Thermal Cycler.     expressly, by implication or by estoppel. No rights for any other
Purchase and use of this LightCycler® Instrument, in conjunction    application, including any in vitro diagnostic application, are
with Authorized Reagents, provides a limited license for use of     conveyed expressly, by implication or by estoppel under U.S.
the PCR process in life science research. No rights are conveyed    Patents Nos. 6,174,670 and 6,245,514 and corresponding
expressly, by implication or by estoppel under any other patent     patent claims outside the United States, or any other patents
claims or for any other application.                                owned by Roche Molecular Systems, Inc. and
                                                                    F. Hoffmann-La Roche Ltd, claiming real-time amplification
# This LightCycler® 2.0 Instrument is licensed under U.S. Patent    and detection methods.
No. 6,814,934 and corresponding claims in its non-U.S. counter-
                                                                    ✜  Purchase of this product is accompanied by a limited, non-
parts for use in research, in vitro diagnostics and other applied
fields. It is also an Authorized Thermal Cycler. Purchase and use   transferable license to use it in the Polymerase Chain Reaction
of the LightCycler Instrument, in conjunction with Authorized       (PCR) process for the purchaser’s life science research in
Reagents, provide a limited license for use of the PCR process      conjunction with a thermal cycler whose use in the automated
in life science research. No rights are conveyed expressly, by      performance of the PCR process is covered by the up-front
implication or by estoppel under any other patent claims or for     license fee, either by payment to Applied Biosystems or as
any other application.                                              purchased, i.e., an authorized thermal cycler.
                                                                    †
●  Purchase of this product is accompanied by a limited               Purchase of this product is accompanied by a limited license
license to use it in the Polymerase Chain Reaction (PCR)            to use it in the Polymerase Chain Reaction (PCR) process
process for the purchaser’s life science research in                with 5’ nuclease detection for the purchaser’s life science
conjunction with a thermal cycler whose use in the automated        research in conjunction with Licensed Probe and a thermal
performance of the PCR process is covered by the up-front           cycler whose use in the automated performance of the PCR
license fee, i.e., an authorized thermal cycler. No real-time       process is covered by the up-front license fee, i.e., an
apparatus or system patent rights or any other patent rights        authorized thermal cycler. This product is an Authorized Core
owned by Applera Corporation, and no rights for any other           Kit without Licensed Probe. Its purchase price includes a
application, including any in vitro diagnostic application          limited, non-transferable immunity from suit under certain
under patents owned by Roche Molecular Systems, Inc. and            patents owned by Roche Molecular Systems, Inc. or
F. Hoffmann-La Roche Ltd claiming homogeneous or                    F. Hoffmann-La Roche Ltd, for using only this amount of the
real-time amplification and detection methods are conveyed          product in the practice of the 5' nuclease process solely for          Ordering
expressly, by implication or by estoppel.                           the purchaser's life science research in conjunction with            Information
                                                                    Licensed Probe. No rights are conveyed expressly, by
                                                                    implication or by estoppel under any other patent claims or
                                                                    for any other application.




                                                                                                                                        29
               ◆  NOTICE TO PURCHASER: LIMITED LICENSE                           NOTICE TO PURCHASER: DISCLAIMER OF LICENSE
               A license to perform the 5' nuclease process for research         This product is compatible for use in the Polymerase Chain
               requires the use of a Licensed 5' Nuclease Kit (containing        Reaction (PCR) process claimed in patents owned by Roche
               Licensed Probe), or the combination of an Authorized Core         Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. No
               Kit plus Licensed Probe, or license rights that may be            license under these patents is conveyed expressly, by
               purchased from Applied Biosystems. This product contains          implication, by estoppel or otherwise to the purchaser by the
               Licensed Probe. Its purchase price includes a limited,            purchase of this product. A license to use these patented
               non-transferable immunity from suit under U.S. Patents            processes for certain research and development activities
               Nos. 6,214,979 and 5,804,375 (claims 1-12 only) and               accompanies the purchase of certain reagents of Applied
               corresponding patent claims outside the United States,            Biosystems and other licensed suppliers when used in
               owned by Roche Molecular Systems, Inc. or                         conjunction with an authorized thermal cycler, or is available
               F. Hoffmann-La Roche Ltd (“Roche”), for using only this           from Applied Biosystems.
               amount of probe in the practice of the 5’ nuclease process
               solely for the purchaser’s own internal research and
                                                                                 ProbeLibrary™ is covered by US and other patent applications
               development activities. This product is also a Licensed Probe
                                                                                 owned by Exiqon A/S. Locked Nucleic Acids (LNA™) are
               for use with service sublicenses available from Applied
                                                                                 covered by U.S. patents No. US 6,794,499, US 6,670,461, US
               Biosystems. This product conveys no rights under U.S.
                                                                                 6,268,490 and US 6,770,748 and other patents and patent
               Patents Nos. 5,210,015 and 5,487,972, which claim 5’ nuclease
                                                                                 applications owned by Exiqon A/S and Prof. Takeshi Imanishi.
               processes, or U.S. Patents Nos. 5,476,774 and 5,219,727,
                                                                                 The quencher used in the probes is covered by patent
               which claim quantification methodology, and corresponding
                                                                                 applications owned by Exiqon A/S.
               patent claims outside the United States of any of the
               foregoing patents and no right under any other patent claims
               (such as apparatus or system claims in U.S. Patent                COMPLETE, FASTSTART, GENOPURE, HIGH PURE, HYBPROBE,
               No. 6,814,934) and no right to perform commercial services        LIGHTCYCLER, MAGNA PURE, TAQMAN, and X-TREMEGENE
               of any kind, including without limitation reporting the results   are trademarks of Roche.
               of purchaser's activities for a fee or other commercial           esiWay is a trademark of RZPD.
               consideration, is hereby granted expressly, by implication, or    FuGENE is a registered trademark of Fugent, L.L.C., USA.
               by estoppel. This product is for research purposes only.          SYBR is a registered trademark of Molecular Probes. Inc.
               Diagnostic uses require a separate license from Roche.            PROBELIBRARY and LNA are registered trademarks of
               Further information regarding the 5’ nuclease licensing           Exiqon A/S, Vedbaek, Denmark.
               program may be obtained from the Director of Licensing,           Other brands or product names are trademarks of their
               Applied Biosystems, 850 Lincoln Centre Drive, Foster City,        respective holders.
               California 94404, USA.




  Ordering
Information




          30
References and Abbreviations Headline 04
                      Subline 05




 1. Hannon, G.J. (2002) Nature 418: 244-251.
 2. McManus, M. T., Sharp, P. A. (2002)
    Nat Rev Genet. 3: 737-747.
 3. Shi, Y. (2003) Trends Genet. 19: 9-12.
 4. Hannon, G.J., Rossi, J.J. (2004) Nature 431: 371-378.
 5. Meister, G., Tuschl. T. (2004) Nature 431: 343-349.
 6. Mello, C.C., Conte, D.Jr. (2004) Nature 431: 338-342.
 7. Kaufman, R. J. (1999) Proc. Natl. Acad. Sci.
    USA 96: 11693-11695.
 8. Bernstein, E. et al. (2001) Nature 409: 363-366.
 9. Biochemica 1: 4–6 (2004).
10. Biochemica 1: 7–8 (2004).
11. Liu, A. X. et al. (1998) Cancer Res 58: 2973–2977.
12. Graff, J. R. et al. (2000) J Biol Chem 275:24500–24505.

RNAi = RNA Interference
dsRNA = double-stranded RNA
siRNA = small interfering RNA
d-siRNA = diced small interfering RNA
esiRNA = endoribonuclease-prepared small interfering RNA
shRNA = short hairpin RNA
RISC = RNA-induced silencing complex




                                                              References and
                                                               Abbreviations




                                                              31
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