Real-Time PCR: Practical Issues and Troubleshooting Mehmet Tevfik DORAK, MD PhD
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1 Real-Time PCR: Practical Issues and Troubleshooting Mehmet Tevfik DORAK, MD PhD Dept of Environmental & Occupational Health Robert Stempel College of Public Health and Social Work Florida International University Miami, Florida USA MOBGAM, Istanbul, Turkey June 3, 2011
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11 Linear vs Log View linear view log view
12 Linear vs Log View linear view log view
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15 6.6 picogram template & C T > 40
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18 Baseline Setting
19 Baseline Setting
20 Baseline Setting
21 Baseline Setting
22 Baseline Setting
23 Baseline Setting
24 Threshold Setting Easier to do in log-view
25 Threshold Setting Multiple Thresholds May be Necessary Easier to do in log-view
26 Baseline and Threshold Setting
27 High Background
28 Standard Curve
29 Low DRn/RFU Imperfect assay design Incorrect quencher Bad template quality (long probe, G at 5 end, incorrect choice of reporter and quencher pair, wrong primer-pair concentrations, probe has mismatches)
30 High efficiency? Efficiency > 110% (slope <-3.1) is due to: Pipetting errors Wrong threshold setting (not in the log-linear phase) Primer-dimers in SYBR green assays Probe degradation in TaqMan assays High variability at low concentrations PCR inhibition by reverse transcriptase
31 High efficiency?
32 Five C T difference rule SYBR green vs TaqMan Melting curve analysis
33 RNA quality
34 RNA quality: integrity (www)
35 RNA quality: purity (www)
36 RNA quantity As long as you are staying within the dynamic range of the assay established by initial validation, use any amount, but no less than 10 picogram and no more than 1 microgram. Usually 1 to 100 nanogram is sufficient. High template amount may increase background.
37 (www)
38 Optimal template amount
39 Optimal template amount (www)
40 Reaction volume Manufacturers recommend 50 microl 25 microl is fine 10 microl is generally fine 5 microl may be fine (for allelic discrimination) but not recommended for qpcr Aim for 3-5 microl template volume in the reaction Aim for duplicates unless using so little template (around 10 picogram or Ct > 35); then you need to use triplicates
41 cdna synthesis mrna in formalin fixed samples may have lost their poly-a tails.
42 cdna synthesis
43 Primer design
44 Primer design
45 Assay design
46 Primer concentrations may result in severe changes in CT values and should remain constant in all experiments for the same assay (www)
47 Pipetting
48 Good Assay Good efficiency, good sensitivity and good predictive power. Albumin (ALB) gene dosage by real-time PCR Laurendeau et al. Clin Chem 1999 (www)
49 Good Assay ABI Understanding C T (www)
50 Good Assay Optimized for: High signal intensity: High RFU / DRn Low background (noise) Low Ct values Maximum Ct = 40 for lowest template amount in dilution series
51 Controls
52 Controls
53 Controls
54 DDC T Assay
55 DDC T Assay: Validation
56 DDC T Assay: Validation
57 DDC T Assay
58 Dye selection
59 Normalizer selection
60 Normalizer selection
61 Normalizer selection Stable endogenous controls do not yield DCt values greater than that of IPC and do not show much variation.
62 Normalizer selection
63 Normalizer selection Sabek et al. Transplantation 2002 (www)
64 18S as a Normalizer Most abundant RNA: may need singleplex runs using diluted samples or competimers (Ambion); not suitable for rare target transcripts Forces separate baseline settings in some instruments Not mrna Does not have 3 poly-a tail Ct value should be smaller than 22 for valid results
65 GAPDH as a Normalizer The most unstable and inconsistent normalizer! Just don t use it! A case of majority not always being right!
66 Interpretation Any assessment of the biological consequences of variable mrna levels must include additional information regarding regulatory RNAs, protein levels and protein activity Weak Correlation Between mrna and Protein Levels in Eukaryotes A total of 150 signature genes showed significant changes at either the protein and/or the mrna level in two bovine bone marrow derived cell lines. 113 signature genes (76%) exhibited changes for mrnas and their cognate proteins in the same direction (1st and 3rd quadrants), only 29 of them changed significantly at both mrna and protein levels and were thus dubbed correlated genes (red). In contrast, 67 genes showed significant changes at the mrna but not the protein level (green), whereas 52 genes showed significant changes at the protein but not the mrna level (blue). Another two genes showed opposite expression patterns of mrna and protein (brown). The correlation coefficient between mrna and protein is 0.64 for the signature genes and 0.59 for all the genes examined. Tian, 2004 (www) (www)
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Contents.
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