Real-Time PCR Troubleshooting Mehmet Tevfik DORAK, MD PhD

Real-Time PCR 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

Linear vs Log View linear view log view

6.6 picogram template & C T > 40

Baseline Setting

Threshold Setting Easier to do in log-view

Threshold Setting Multiple Thresholds May be Necessary Easier to do in log-view

Baseline and Threshold Setting

High Background

High Background Insufficient quenching is one reason for high background.

High Background Spurious binding of the probe is another reason for high background. Redesign the primers and probe for increased specificity, and introduce LNAs or MGB to increase probe specificity.

High Background

Standard Curve

Low DRn/RFU Imperfect assay design Insufficient quenching High background Bad template quality (long probe, G at 5 end, incorrect choice of reporter and quencher pair, wrong primer-pair concentrations, probe has mismatches) Consider an alternative design Consider double-quenched probes

High efficiency?

Five C T difference rule SYBR green vs TaqMan Melting curve analysis

RNA quality

RNA quality: integrity (www)

RNA quality: purity (www)

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 2 nanogram is the optimal value. High template amount may increase background.

Optimal template amount

Optimal template amount (www)

Optimal template amount Use 1 to maximum 100 ng of cdna/dna per 20 μl reaction in qpcr/allelic discrimination assays, preferably no more than 2 ng cdna/dna per reaction Make sure the quantity used is within the dynamic range of the assay (www)

Reaction volume Manufacturers recommend 50 microl 20-25 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

Primer & probe concentration

cdna synthesis

cdna synthesis mrna in formalin fixed samples may have lost their poly-a tails.

cdna synthesis

Primer design

Probe design LNA and MGB increase probe Tm and allows shorter probes with higher specificity (and quenching).

Primer design

Amplicon length Amplicons should be as short as possible but not shorter than 75bp to be distinguished from primer-dimers and to allow primers and probe not to overlap To increase efficiency, amplicons should be smaller than 200bp Shorter amplicons allow probes to compete with the complementary strand of the amplicon resulting in faster and more efficient reactions with increased consistency of results Templates that contain more than four repeats of the same nucleotide should be avoided Templates should have a 50-60% GC content

Assay design

Primer concentrations may result in severe changes in Ct values and should remain constant in all experiments for the same assay (www)

Pipetting

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)

Good Assay ABI Understanding C T (www)

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

Controls

Controls Positive Signals in NTC: Most common cause is human error: Leave empty wells around NTC well and try again. If using SYBR Green, run melting analysis and see if the amplification is specific (no template increases the risk of primer-dimers). Switch to TaqMan assay if necessary. If contamination is due to PCR products, use dutp in the PCR master mix, and digestion with uracil-n-glycosylase. Late amplifications in NTC well may be tolerable if the lowest Ct value for the template is more than 5 Ct values away.

Replicate Variation Most common cause is pipetting error: Change hands, use better pipettes, use ROX dye for normalization. If using SYBR Green, run melting analysis and see if the amplification contains non-specific products. Switch to TaqMan assay if necessary. If the problem is cdna quality, try to use a mix of random primers and oligo(dt) for cdna synthesis (iscript or QuantoTect). Make sure the instrument is calibrated and there is no edge effect.

DDC T Assay Explanation of DDCt method (following slides): For the ease of understanding, in the first set of experiments normalizer Ct values were assumed to remain constant (equal mrna in each experiment, and an ideal normalizer). The difference of the Ct values obtained before (B) and after (A) a treatment or time translates into fold change in expression levels (0 = no change; -2 = increased and +2 = decreased expression). In reality, the normalizer Ct values do not remain constant due to changes in mrna quantity used in each assay but the calculations and interpretations remain the same. The use of normalizer makes sure that the results are adjusted for mrna quantity differences in each assay.

DCt 1 =10 DCt 2 =10 DCt 1 =10 DCt 2 =8 DCt 1 =10 DCt 2 =12 DDCt Method: Normalizer Ct values remain constant Ct 28 26 DDCt=0 DDCt=-2 DDCt= 2 24 16 0 (B) (A) (B) (A) (B) (A) Target (B) = before Normalizer (A) = after

DCt 1 =10 DCt 2 =10 DCt 1 =10 DCt 2 =8 DCt 1 =10 DCt 2 =12 DDCt Method: Normalizer Ct values vary Ct 28 DDCt=0 DDCt=-2 DDCt= 2 26 24 16 0 (B) (A) (B) (A) (B) (A) Target (B) = before Normalizer (A) = after

DDC T Assay

DDC T Assay: Validation

DDC T Assay

Dye selection

Annealing temperature optimization

Primer - probe optimization

Efficiency

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

Normalizer selection

Normalizer selection Stable endogenous controls do not yield DCt values greater than that of IPC and do not show much variation.

Normalizer selection

Normalizer selection Sabek et al. Transplantation 2002 (www)

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

GAPDH as a Normalizer The most commonly used but not the most suitable normalizer!

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)