Troubleshooting Reverse-Transcription PCR (RT-PCR)
- Inhibition of PCR by Reverse Transcriptase:
- Purify products of RT by common cDNA purification kits or any method that removes the RT. RT is a known and relatively strong inhibitor of PCR and especially in qPCR causes problems.
- Dilution (10...200 fold) might help as well.
- Ref: L.N. Sellner et al. "Reverse transcriptase inhibits Taq polymerase activity"
Nucleic Acids Research, 1992, Vol. 20, No. 7 1487-1490
- Inhibition of RT and PCR by melanin:
- Melanin (from skin samples) copurifies with RNA prepared with TRIZOL. Use RNA clean-up columns (e.g. from the Sigma RNA purification kit) to remove the melanin. If your RNA sample is not colorless you will experience problems in RT-PCR.
- Inhibition of PCR by heparin:
- Heparin is a naturally occurring polysaccharide found primarily in the liver, lung and artery walls. It is known to inhibit enzyme reactions relating to nucleic acids. Addition of heparinase (around 0.5 unit) into the reaction mixture before PCR decreases the inhibition by heparin.