Technique / Molecular Biology / RNA transcriptional and post-transcriptional regulation

General reverse transcription troubleshooting guide

General reverse transcription troubleshooting guide

Low cDNA yield:

1. Check reverse transcription temperature. Different reverse transcriptase may require different reaction temperature, ranging from 37c to 65c.

2. RNA template is degraded or concentration is wrong. Use RNase inhibitor in the reaction mix. Measure RNA concentration accurately to avoid too high or too low RNA concentration.

3. dNTP is degraded. Use fresh dNTP mix and store dNTP stock in -20c freezer.

4. Primer is degraded or concentration is wrong. Use random primer or gene-specific primer.

5. High degree of RNA secondary structure within the template RNA. Try increase the reaction time or use an reverse transcriptase with higher reaction temperature. Use DMSO or GC-rich reagent to overcome the difficult RNA secondary structure.

Last update 06-May-2006, Rating of 1 votes.