FAQ: Why do I have low cDNA yields?

There are several causes for low cDNA yields including sample integrity/purity and input copy numbers. To assess the most common causes:

1. Check the integrity of the RNA by denaturing agarose gel electrophoresis or BioAnalyzer^® evaluation.
2. RNA should have a minimum A₂₆₀/A₂₈₀ ratio of 1.8 or higher. Ethanol precipitation followed by a 70% ethanol wash can remove most contaminants such as EDTA and guanidinium that can carry over from upstream purification methods. Precipitation with lithium chloride can remove polysaccharides.
3. Extraction using phenol/chloroform extraction or ethanol can remove contaminant proteins such as proteases. 
4. Use sufficient amounts of input RNA, particularly for low abundance RNAs.