There are several causes for low cDNA yields including sample integrity/purity and input copy numbers. To assess the most common causes:
- Check the integrity of the RNA by denaturing agarose gel electrophoresis or BioAnalyzer® evaluation (Agilent®).
- 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.
- Extraction using phenol/chloroform extraction or ethanol can remove contaminant proteins such as proteases.
- Use sufficient amounts of input RNA, particularly for low abundance RNAs.