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					Real-Time quantitative PCR:
Choices and Decisions

Dr Sandrine Javorschi-Miller
R&D program Manager

European Functional Genomic Seminar
May 2005
                 Why using real-time PCR?




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                                           Gene Expression Analysis Technologies



                               >10,000

                                                            Real-time PCR

                                           100
                       Number of samples




                                                                       Low-density
                                                                         Arrays
                                            10

                                                       RPA                       High-density
                                                     Northern                       Arrays
                                                 1                                           SAGE

                                                     2-10           100-1000         >10,000
                                                             Number of genes

       To consider: Ratio samples / genes, needs for accuracy
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                                 Comparison of Quantitative Assays (RNA/DNA):
                         Sensitivity                               Dynamic Range

                                100            Real-Time PCR              108
                                101                                       107
                                102             Amplicor/TMA              106
                                                   NASBA
                                103                                       105
                                                   bDNA
                                104                                       104
                                                  XPLORE
                                105                                       103
                                                 Microarrays
                                106                                       102
                                                    RPA
                                107                                       101
                                                  Northern
                                108                                       100

NASBA: nucleic acid seq based amplification             TMA: transcription mediated amplification
      bDNA: branched DNA assay                              RPA: RNAse protection assay
   Xplore: based on Invader technology

                                          Advantage: Real-Time PCR
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             Real-time PCR in more details




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                                                                  Phases of amplification




                                          •   Exponential phase
                                          •   Plateau phase


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                                                    Exponential phase vs. plateau

        • At some time or another, all reactions regardless of initial
          amount reach the same plateau!
           – Plateau is not quantitative
           – Exponential phase is quantitative


               Amplicon
                amount




                                                               Cycles



                                Plateau information is not quantitative
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                                          Before the Real-Time era: End-point PCR
 • Examples of semi-quantitative PCR
    – End point analysis after reduce number of cycles (exponential phase)
                 • Not accurate            GOI (pg)   ?   0   0.1   1   10
                 • Not sensitive
                                                                             PCR condition
                                                                                95oC for 2 min
                                                                             # of cycles 30
                                                                                95oC for 15 sec
                                                                                62oC for 30 sec
                                                                                72oC for 30 sec




         – Competitive PCR
                 • Time and reagent consuming




                                 End Point is at best semi-quantitative
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                           Choices and Decisions




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                      One Step or Two Step RT-PCR?




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                                                        One Step or Two Step RT-PCR?

One Step RT-PCR                                                                     Two Step RT-PCR
   (One tube)                                           RNA                           (Two tubes)
         1 target                                                                      X targets
                                                                      Oligo dT
                                           GS primers              Random Primers
                                                                    (GS Primers)




                                                               cDNA                   Target pool




      1 amplicon                          Amplicon            Amplicon                X amplicons



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                                           One Step or Two Step RT-PCR?


  One-Step RT-PCR                            Two-Step RT-PCR
  • Highly defined conditions to             • Separate conditions for cDNA
    support RT and Taq                         synthesis & PCR
  • Requires gene specific primer            • Flexible choice of primers
  • Ideal for quantification of 1 or 2       • Ideal for quantification of
    messages from a large number               multiple genes from a limited
    of RNA samples                             number of RNA samples


               Perfect for:                       Perfect for:
               - Lot of samples                   - Few samples
               - Small amount of targets          - Large amount of targets




            Two-step RT-PCR is more convenient and cost effective
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                      Which Reverse Transcriptase?




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                                                               What RT enzyme?

• No RNA template degradation > Higher
  cDNA yield
   – Improved sensitivity
• Reduced 5’ / 3’ bias due to mispriming
   – Increased reliability
• Greater percentage of full-length cDNA                      RNAse H reduced
   – complete sequence representation                          Thermostable
                                                            ReverseTranscriptase:
• Improved thermostability
   – high temperature cDNA synthesis                       SuperScript III ™ RT
   – relaxes secondary structure
   – improved primer specificity
• Consistent cDNA synthesis efficiency
   – wide template diversity
   – wide range of template amount

                              Good Reverse transcriptase is essential!
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                                                                       SuperScript™ III Platinum® One-Step RT-PCR

                                                                      B-Actin TaqMan primers (RNA concentrations from 100ng to 0.1pg)


                                       1.00E+01

                                                      Company A one step kit
                                                      SSIII one step qRT-PCR
             Normalized Fluorescence




                                       1.00E+00




                                       1.00E-01
                                                  0          5         10           15           20            25            30         35   40
                                                                                                Cycle




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                                          Which Polymerase?




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                                                               Which Taq polymerase?

Hot Start                                                Antibody Hot Start
- Increased specificity                                  - Fast activation
- Reduced artifacts (less Primer Dimers)                 - Maximum activity in early cycles




                                                                  Platinum® Taq DNA
                                                                       Polymerase




                                           Hot Start Taq is a must!
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                                              PLATINUM® Taq automatic hot start


                                          Initial Template
                PCR Assembly                                  Temperature Cycling
                                           Denaturation



                                          94oC, 30s - 3 min




               Inactive                                           Fully Active
         Taq DNA Polymerase                                   Taq DNA Polymerase



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                                          UDG or no UDG?




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                                                                    Carry-over protection/comparison
                                           Mix with UDG and dUTP
         DNA                                                                                                               Amplicon with dUTP

Amplicon with dUTP                                X



                                                           Uracil DNA Glycosylase Decontamination
                                                           Done by Nested PCR with pCR2.1 Plasmid

                                                      I-with UDG   I-without UDG        Q-with UDG        Q-without UDG




                                                                                                      20 cycles,
                                                                                               ~1,000,000 fold difference




                        0          5         10       15           20              25                30             35    40   45   50
                                                                            Cycle Number




                                           Not all UDG kits are built equal!
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                               Which Detection system?




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                                                            Detection chemistry?

 • dsDNA specific stains
    – Ethidium bromide (used in first experiments)
    – SYBR Green I (most widely used today)

 • Probe-Based Systems (unlabeled primers plus probes)
    – TaqMan and TaqMan MGB probe system (5’ nuclease assay)
    – Dual Probe System, uses FRET between probes hybridized side by
      side to the amplicon
    – Molecular Beacon probe (hairpin probe, quencher and fluorophore
      are separated when hybridized to the amplicon)
    – Epoch probe (second structure probe, quencher and fluorophore are
      separated when hybridized to the amplicon)

 • Primer/oligo labeled systems
    – Amplifluor: hairpin primer with fluorophore and quencher
    – LUX™: primer with one fluorophore and no quencher, proprietary to
      Invitrogen


                                          Decision, Decision…
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                                                                      Detection chemistry?




        SYBR Green I®
                                          TaqMan®




                                                    Q




                                                        Amplifluor™             LUX™
                 Quantiprobe®



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                                                     Which detection system?




 - Monoplexing                                      - Multiplexing
 - Cost saving                                      - High Specificity
 - Fast initial screening                           - High Sensitivity

                                                         Probe based
                Sybr Green I®                                or
                                                        LUX™ primers



                           A detection system for every applications!
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                                                                                 LUXTM (Light Upon eXtension)




                                                                                                   LUX™


The fluorescent intensity is modulated due to the proximity of the fluorophore to specific primary & secondary structures of the
oligonucleotide. The design rules have been incorporated into the LUX Designer™. The labeled primer exhibits low fluorescence
but incorporation during PCR produces a large increase in fluorescence


                                            Light Upon eXtension!
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                                                           Quenching effect




• The fluorogenic LUX primer has an attached fluorophore at the 3’end and a
    short sequence tail on the 5’ end that is complementary to the 3’end of the

    primer. The resulting hairpin secondary structure provides optimal
    quenching of the attached fluorophore.


• The quenching of fluorescence in duplex (ds DNA) is provided by the
    terminal dG-dC or dC-dG base pair when the dye is attached to a thymidine
    / cytosine within three nucleotides from the 3΄-end.




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                                                                  Competitive Audit Data


     • Certified LUX primers were compared to TaqMan® Gene
       Expression Assays from ABI and QuantiTect Gene Expression Assays
       from QIAGEN using Platinum Super-Mix UDG (1X ROX).
             – Standard curve generated from ten-fold serial dilutions of ORF clone (107 –102
               copies).
ABI 7700

        LUX IL6                           TaqMan                      Quantitect
                                            IL6                          IL6




               PCR Efficiency: 92%          PCR Efficiency: 93%           PCR Efficiency: 94%
                    R2:0.999                     R2:0.998                      R2:0.996




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                                                           Comparison of LUX™ to TaqMan




                                          LUX™ sensitivity advantages
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                                                                     Melting curve analysis


     Method:
1.    Real-time PCR
2.    After amplification: 60°C to 95°C (slow ramp)
3.    Fluorescence signal is recorded continuously during the slow temperature ramp
4.    Melting curve : Fluorescence signal vs. Temperature
5.    Melting curve is converted to melting peaks by plotting –dF/dT vs Temperature




                                           LUX™ Built-in control feature!
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                                                         Advantages LUX™ versus TaqMan®
                                                                                  0.2




                                                                                   Norm.Fluoro.
               Specificity – Melting Curve                                                                Sample 1
                                                                                                          Sample 2


               Fast control of the PCR product specificity
                                                                                                          Sample 3
                                                                                 0.15
                                                                                                          Positive Control

               without:                                                                                   NTC
                                                                                                          NTC

               - opening the tube and
               - running a gel
                                                                                  0.1




                                                                                 0.05
0.8
 -dF/dT




                  Sample 1
                  Sample 2
                  Sample 3                                                            0
                                                                                                  0   5         10           15   20   25   30   35   40   45   Cycle   50
0.6               Positive Control
                  NTC
                  NTC




0.4


                                                                                 Provides accurate real time assessment of
                                                                                 quality:
                                                                                 - eliminates risk of false positives
0.2




                                                                                 - reduces risk of contamination
  0
          60         65              70   75   80   85   90   Temperature   95




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                                                         Advantages LUX™ versus TaqMan®


                                                                          Alexa
                                                                           546
                        Excitation        Emission
                        (nm)              (nm)              All
FAM                     492               517            detectors
JOE                     520               548
TET                     521               536
HEX                     533               550
                                                                          TET
Alexa Fluor 546        554               570
Data from Molecular Probes spectra




Triplex amplification using standard protocol:

- 10,000 copies of Gene X are amplified using a LUX primer set           FAM
labeled with Alexa Fluor 546
- 1,000 copies of Gene Z are amplified using a LUX primer set
labeled with TET
- 100 copies of Gene Y are amplified using a LUX primer set labeled
with FAM


                                                     Easy multiplexing!
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                                               Advantages LUX™ versus TaqMan®

   Simple Primer Design with the new D-LUX™ Designer




                                    www.invitrogen.com/dluxdesigner
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                                                  3 Choices to access LUX™ primers


                                           Scientific project




  D-LUX™ Designer
     Self Service                                                   EvoQuest™ LUX™
                                                                      Custom Service



                                          Certified LUX™ primers
                                                    - HSKG
                                                 -Virus /Bacteria
                                                 - Human genes




                                                Flexibility!
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                               Other convenient formats?




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                                                                                 Cells Direct kit

                             Cell Lysis        No RNA isolation step required.


                                               DNase treatment eliminates genomic DNA so you can be
  All                  DNase Treatment         confident that results are due to cDNA amplification.
done
 in 1
tube!                                          The cDNA synthesis is performed by SuperScript™ III
                        cDNA Synthesis
                                               RNase H- RT.



                           Application:
                             qPCR

                                      From cells to cDNA in ONE TUBE!
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                                                                  Cells Direct kit




                              From 1 cell to 10,000 cells without NAP!
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                                                                                      RTS kits

                                                    Vialing   Optimized PCR SuperMix

        Proprietary Polymer Mix




                                                         Temperature-controlled Lyophilization

                               Packaging

                              Shelf-life = 1 year



        Complete Cycle within 3 days


                                          Room Temperature Stable
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                                                                                                                                         RTS kits
                                                                              Standard Curve RTS one step
                                                            40                                                y = -3.548(x) + 37.1
                                                                                                                   R2 = 0.998
                                                            35




                                          Cycle Threshold
                                                            30


                                                            25

                                                            20

                                                            15

                                                       10
                                                       1.00E+00    1.00E+01    1.00E+02   1.00E+03   1.00E+04     1.00E+05    1.00E+06
                                                                                     Starting Concentration




                                                                 Standard Curve aqueous (SuperScript III one step)
                                                            40                                                y = -3.505(x) + 36.8
                                                                                                                   R2 = 0.999
                                                            35
                                          Cycle Threshold




                                                            30

                                                            25

                                                            20

                                                            15

                                                       10
                                                       1.00E+00    1.00E+01    1.00E+02   1.00E+03   1.00E+04     1.00E+05    1.00E+06
                                                                                     Starting Concentration



               RTS and regular mixes have equal performances
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                       What mix for what instrument?




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                                                 Invitrogen qPCR Reagent Selection Guide

                           SYBR Green Detection                                Fluorescent Probes/Primers
                                                                                 (LUX™, TaqMan®, etc.)
                 ABI 7000,               Roche              Any            ABI 7000,           Roche              Any
                7700, 7900            LightCycler       Instrument        7700, 7900        LightCycler       Instrument
   DNA         Platinum SYBR          Platinum SYBR     Platinum SYBR       Platinum          Platinum          Platinum
    or          Green qPCR             Green qPCR        Green qPCR        Quantitative      Quantitative      Quantitative
  cDNA         SuperMix-UDG           SuperMix-UDG      SuperMix-UDG      PCR SuperMix-     PCR SuperMix-     PCR SuperMix-
                   w/ROX                   w/BSA                           UDG w/ROX            UDG               UDG


   RNA,        SuperScript III        SuperScript III   SuperScript III   SuperScript III   SuperScript III   SuperScript III
    1-         Platinum SYBR          Platinum SYBR     Platinum SYBR     Platinum One-     Platinum One-     Platinum One-
   Step          Green One-             Green One-        Green One-      Step qRT-PCR      Step qRT-PCR      Step qRT-PCR
               Step qRT-PCR           Step qRT-PCR      Step qRT-PCR        Kit w/ROX             Kit               Kit
                 Kit w/ROX               Kit w/BSA            Kit


   RNA,        SuperScript III        SuperScript III   SuperScript III   SuperScript III   SuperScript III   SuperScript III
    2-         Platinum SYBR          Platinum SYBR     Platinum SYBR     Platinum Two-     Platinum Two-     Platinum Two-
   Step          Green Two-             Green Two-        Green Two-      Step qRT-PCR      Step qRT-PCR      Step qRT-PCR
               Step qRT-PCR           Step qRT-PCR      Step qRT-PCR        Kit w/ROX             Kit               Kit
                 Kit w/ROX               Kit w/BSA            Kit




                                          A solution for each problem!
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                                          SYBR Green LC qPCR versus Roche qPCR




       HPRT primers were used with plasmid standards from 1x107 to 1x101 copies. Invitrogen
       SYBR Green LC (blue) quantified all 7 logs with a slope of –3.644 and an R-value of -1.00.
       No template controls (NTC’s) were negative with the SYBR LC kit.
       Roche’s FastStart DNA Master Plus (green) quantified 5 of 7 logs with a slope of –3.700 and
       an R-value of -1.00. The NTCs along with the last two dilutions showed contamination.



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                       What method of quantitation?




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                                                             What Method and When




                            http://www.gene-quantification.de/strategy.html
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                                          Absolute quantitation




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                                          Relative Quantitation: ΔΔCt Method



   CtGOIs – Ctnorms = ΔCtSample

   CtGOIc – Ctnormc = ΔCtCalibrator

  GOI = Gene of Interest
  Norm = Normalizer (Housekeeping) gene



  ΔCtSample – ΔCtCalibrator = ΔΔCt

  Fold Induction = 2- ΔΔCt




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                                    Where to find more info?




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                                                         Invitrogen qPCR Central




                                    www.invitrogen.com/qpcr
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                                                         Invitrogen qPCR Central




                                    www.invitrogen.com/qpcr
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                                          Invitrogen multidisplinary qPCR group




     •     R&D qPCR group in Carlsbad, CA
     •     Enzymologists in Carlsbad, CA
     •     Chemists from Molecular Probes in Eugene, OR
     •     Custom Primer manufacturing facility in Frederick, MA
     •     EvoQuest service group in Carlsbad, CA
     •     And more…




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                                             The end


                                          www.invitrogen.com/qpcr
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