There are several causes for low cDNA yield. Here are some suggestions to improve the yield:
- Check the integrity of the RNA by denaturing agarose gel electrophoresis or BioAnalyzer (Agilent). Intact RNA of high purity is essential for full-length cDNA synthesis.
- RNA should have a minimum A260/A280 ratio of 1.7 or a RIN number greater than 8. Ethanol precipitation followed by a 70% ethanol wash can remove contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides.
- An RNA purification step using RNA extraction kits or phenol/chloroform extraction can remove contaminant proteins such as proteases.
- Increase the amount of RNA used, especially for low abundant RNA.