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  • 1. Pathway-Centric Tools and Technology™ Using Real-Time & End-Point PCR for Gene Expression Analysis and Array Data Verification SuperArray Bioscience Corporation George J. Quellhorst, Jr. Ph.D. Manager, Customer Education
  • 2. Pathway-Centric Tools and Technology™ Topics to be Covered Real-Time PCR Introduction, SYBR Green Detection Considerations for Success Examples of Dynamic Range & Specificity End-Point (Conventional) PCR Introduction, Considerations for Success More Quantitative with Internal Normalizer Dynamic Range, Control Systematic Variation Examples of Expression Profiling for both
  • 3. Pathway-Centric Tools and Technology™ Real-Time PCR Monitors amount of amplicon DURING reaction Amplicon amount DIRECTLY related to fluorescence Gene expression INVERSELY PROPORTIONAL to Ct VERY quantitative WIDE dynamic range and HIGH sensitivity DOES NOT require post-reaction processing REQUIRES dedicated equipment CAN BE expensive, but not necessarily
  • 4. Pathway-Centric Tools and Technology™ SYBR® Green Detection Fluoresces when bound to dsDNA Amplicon detected either during annealing step or at the end of the extension step Detects any dsDNA non-specifically Least expensive & easiest to use Applicable to most real-time systems
  • 5. Pathway-Centric Tools and Technology™ SYBR® Green Detection Melting Melting Annealing Extension
  • 6. Pathway-Centric Tools and Technology™ Considerations for Successful Real-Time PCR: Primer Design High Amplification Efficiency Short amplicon size (100 to 200 bp) Specificity Uniqueness in the genome – especially at 3’ end High sequence complexity No secondary structure or primer dimers Only one gene-specific amplicon detectable Chemical Properties Melting temperature / GC content
  • 7. Pathway-Centric Tools and Technology™ Considerations for Successful Real-Time PCR: Master Mix and Others HotStart enzyme Highly proficient enzyme = high amplification efficiency Minimizes amplification of non-specific priming events Further insures only one amplicon Wide Dynamic Range Five to six orders of magnitude Accommodation of SYBR® Green Detection Convenient: If lyophilized, just add water.
  • 8. Pathway-Centric Tools and Technology™ Five-Log Dynamic Range cDNA 0 10-5 10-4 10-3 10-2 10-1 1 Threshold cycle y = -13.73Lg(x) + 18.745 R2 = 0.9957 40 35 30 25 20 15 10 5 0 0.00001 cDNA 0 10-5 10-4 10-3 10-2 10-1 1** ** 1X cDNA = Product (1 µl) of 25-µl First Strand cDNA Synthesis using 0.15 µg Universal Reference RNA 0.0001 0.001 0.01 Dilution factor 0.1 1
  • 9. Pathway-Centric Tools and Technology™ High Specificity: Single PCR Products First Derivative Melting Curves BMP1 BMP2 BMP4 BMP6 BMP5 BMP3 BMP7 BMP15 Failed Primer Design Agarose Gel BMP1 BMP2 BMP3 BMP4 BMP5 BMP6 BMP7 BMP15 RT2 Real-Time™ Gene Expression Assay Kits for BMP family
  • 10. Pathway-Centric Tools and Technology™ Gene Expression Profiling with Real-Time PCR: Generating Standard Curve First Strand cDNA Synthesis Kit Serial Dilution of (Cat # C-01) XpressRef™ Human Universal Reference RNA (GA-004) RT2 Real-Time™ Gene Expression Assay Kit Threshold Cycle 30 25 TNFAIP3 Ct=-3.302Log(x)+16.612 20 15 GAPD Ct=-3.351Log(x)+13.4 10 5 0 0.001 0.01 0.1 Dilution factor 1
  • 11. Pathway-Centric Tools and Technology™ Gene Expression Profiling with Real-Time PCR: Generating Standard Curve First Strand cDNA Synthesis Kit (Cat # C-01) RNA from HeLa Cells Treated ± TNFα RT2 Real-Time™ Gene Expression Assay Kit TNFα untreated: Ct(TNFAIP3)=24.25, Ct(GAPD)=16.49 TNFα treated: Ct(TNFAIP3)=19.17, Ct(GAPD)=16.36 Threshold Cycle 30 (TNFAIP3/GAPD)treated 25 = (TNFAIP3/GAPD)untreated 20 0.17 / 0.14 0.0048 / 0.13 = 32.9 fold-change in TNFAIP3 expression 15 10 5 untreated 0 0.001 0.01 0.0048 0.1 Dilution Factor 0.17 0.13 0.14 1 TNFα treated TNFAIP3 GAPD Internal Normalizer
  • 12. Pathway-Centric Tools and Technology™ Gene Expression Profiling with Real-Time PCR: Quantification by ∆∆Ct** If the PCR efficiency is 1 (if the calibration curve slopes m equal -3.3), then the fold-change in gene expression = 2-∆∆Ct. Standard curves: TNFAIP3: Ct=-3.302Lg(x)+16.612 GAPD: Ct=-3.351Lg(x)+13.4 TNFα untreated: Ct(TNFAIP3)=24.25 TNFα treated: Ct(TNFAIP3)=19.17 Ct(GAPD)=16.49 Ct(GAPD)=16.36 ∆Ct (treated) = Ct (TNFAIP3) - Ct (GAPD) = 19.17 – 16.36 = 2.81 ∆Ct (untreated) = Ct (TNFAIP3) - Ct (GAPD) = 24.25 – 16.49 = 7.76 ∆∆Ct = ∆Ct (treated) - ∆Ct (untreated) = 2.81 – 7.76 = -4.95 The fold-change in TNFAIP3 expression = 2-∆∆Ct = 24.95 = 30.9 Error = |(30.9- 32.9)|/32.9 = 6% ** Use this method ONLY if the replication efficiencies for your gene of interest and the housekeeping gene are the same or similar.
  • 13. Pathway-Centric Tools and Technology™ Gene Expression Profiling with Real-Time PCR: Quantification by ∆∆Ct** If the PCR efficiency is less than 1, then the fold-change in gene expression = 10∆∆Ct/m. Standard curves: TNFAIP3: Ct=-3.302Lg(x)+16.612 GAPD: Ct=-3.351Lg(x)+13.4 Average slope m = ½ (- 3.302 - 3.351) = -3.327 ∆∆Ct = ∆Ct (treated) - ∆Ct (untreated) = -4.95 The fold-change in TNFAIP3 expression = 10∆∆Ct/m = 10-4.95/-3.327 = 30.8 Error = |(30.8- 32.9)|/32.9 = 6% ** Use this method ONLY if the replication efficiencies for your gene of interest and the housekeeping gene are the same or similar.
  • 14. Pathway-Centric Tools and Technology™ Real-Time PCR Summary: With proper primer design, SYBR Green detection applicable to all genes Gene-specific amplicons Five-fold dynamic range Simple determination of relative gene expression Useful for direct expression profiling and array data verification on gene-by-gene basis
  • 15. Pathway-Centric Tools and Technology™
  • 16. Pathway-Centric Tools and Technology™ RT2 Real-Time™ Gene Expression Assay Kits Available for any human, mouse, or rat gene Experimentally verified computer algorithm primer design Optimized for SYBR® Green detection Includes gene-specific primer pair & PCR master mix
  • 17. Pathway-Centric Tools and Technology™ End-Point (Conventional) PCR Determines amount of amplicon AFTER reaction Amplicon amount NOT DIRECTLY related to intensity NOT AS quantitative, but can be improved NARROWER dynamic range and LOWER sensitivity REQUIRES post-reaction processing DOES NOT require special equipment LESS expensive
  • 18. Pathway-Centric Tools and Technology™ Considerations for Successful End-Point PCR Primers Gene-specific amplicons As Wide a Dynamic Range as possible Two to three orders of magnitude Convenient Master Mix If lyophilized, just dissolve. Including gel loading dye saves a step. Mechanism to normalize results Internal Normalizer for housekeeping gene
  • 19. Pathway-Centric Tools and Technology™ GAPD Internal Normalizer Makes Conventional RT-PCR MORE quantitative. Detects the relative GAPD expression level in same tube as the gene of interest. Attenuates GAPD Signal. Band intensity does not saturate. Does not interfere with detection of the gene of interest. Places GAPD detection in same dynamic range as most genes. Relative Gene Expression = Target-to-GAPD Ratio Ratios compared between experimental conditions to control for systematic variables.
  • 20. Pathway-Centric Tools and Technology™ GAPD Internal Normalizer Corrects for Differences in Template Loading First Strand cDNA Synthesis Kit XpressRef™ Human (Cat # C-01) Universal Reference Total RNA (GA-004) Standard GAPD Primers Human MX1 and IRF7 RT2 End-Point™ Kits Standard GAPD Primers cDNA 1 µL 2 µL 1 µL 2 µL GAPD or Internal Normalizer (436 bp) MX1 (222 bp) 0.58 0.50 N/A N/A Target/GAPDH IRF7 (163 bp)
  • 21. Pathway-Centric Tools and Technology™ GAPD Internal Normalizer Corrects for Differences in Template Loading First Strand cDNA Synthesis Kit XpressRef™ Human (Cat # C-01) Universal Reference Total RNA (GA-004) Standard GAPD Primers cDNA 1 µL 2 µL 1 µL 2 µL Standard or Internal Normalizer GAPD Primers Human MX1 and IRF7 RT2 End-Point™ Kits GAPD Internal Normalizer cDNA 1 µL 2 µL 1 µL 2 µL GAPD or Internal Normalizer (436 bp) MX1 (222 bp) 0.58 0.50 N/A N/A Target/GAPDH 1.1 1.1 0.40 0.41 Target/GAPDH IRF7 (163 bp)
  • 22. Pathway-Centric Tools and Technology™ Dynamic Range of End-Point RT-PCR Using Internal Normalizer XpressRef™ Human Universal Reference Total RNA (GA-004) cDNA cDNA First Strand cDNA Synthesis Kit (Cat # C-01) Spike with Human PARP-1 plasmid cDNA RT2 End-Point™ Kit Human PARP1 1µL 1µL PARP-1 2x106 106 1µL 1µL 1µL 5x105 2.5x105 105 1µL 1µL 6x104 3x104 1µL 0 copies PARP-1 Human GAPD Internal Normalizer
  • 23. Pathway-Centric Tools and Technology™ Dynamic Range of End-Point RT-PCR Using Internal Normalizer - continued Human PARP1:GAPD Ratio y = 2E-06x R2 = 0.9826 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 100000 200000 300000 400000 Copies of Target 500000 600000
  • 24. Pathway-Centric Tools and Technology™ Expression Profiling with End-Point PCR and Internal Normalizer First Strand cDNA Synthesis Kit RNA from HT-29 Cells (Cat # C-01) Treated ± IFNα (10 ng/ml 3h 37C) MX1 IFN-α + 1000bp 750bp 500bp IRF7 MX1 (414 bp) 250bp GAPD Internal Normalizer (226 bp) Human MX1 and IRF7 RT2 End-Point™ Kits IFN-α + 1000bp 750bp 500bp 250bp Untreated IFNα Fold Increase MX1 / GAPD 0.61 2.44 4.0 IRF7 / GAPD 0.17 0.79 4.6 IRF7 (315 bp) GAPD Internal Normalizer (226 bp)
  • 25. Pathway-Centric Tools and Technology™ Expression Profiling with RT2 End-Point™ Gene Expression Assay Kits First Strand cDNA Synthesis Kit RNA from HeLa Cells (Cat # C-01) Treated with TNFα For various times 15.0 TNFα treated 30min 1hr 2hr 3hr 4hr GAPD TNFAIP3 Target/GAPD 0.41 0.98 1.5 1.3 1.4 1.1 GAPD NFKBIA Target/GAPD 0.13 0.77 1.5 1.5 0.81 1.0 GAPD IL6 Target/GAPD 0.30 RT2 End-Point™ Kits 2.3 2.3 1.2 0.90 1.0 Induction level untreated Human TNFAIP3, NFKBIA, IL6 TNFAIP3 NFKBIA IL6 12.5 10.0 7.5 5.0 2.5 0.0 0 1 2 3 Induction time (hrs) 4
  • 26. Pathway-Centric Tools and Technology™ End-Point PCR Summary With use of an Internal Normalizer, Provides adequate dynamic ranges Controls for systematic tube-to-tube variation Simple determination of relative gene expression Useful for direct expression profiling and array data verification on gene-by-gene basis Provides quantitative RT-PCR to researchers without access to real-time equipment
  • 27. Pathway-Centric Tools and Technology™ RT2 End-Point™ Gene Expression Assay Kits Includes enough reagents for 24 reactions: Primer pair designed with experimentally-verified computer algorithm HotStart “Sweet ” PCR master mix Unique GAPD Internal Normalizer Band of correct size from universal RNA source Any gene in the human, mouse, or rat genome
  • 28. Pathway-Centric Tools and Technology™ Using Real-Time & End-Point PCR for Gene Expression Analysis and Array Data Verification SuperArray Bioscience Corporation George J. Quellhorst, Jr. Ph.D. Manager, Customer Education