- Check the integrity of the RNA by denaturing agarose gel electrophoresis (2).
RNA should have a minimum A260/A280 ratio of 1.7 or higher. Ethanol precipitation followed by a 70% ethanol wash can remove most contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides (2).
- Phenol/chloroform extraction and ethanol extraction can remove contaminant proteins such as proteases (2).
- Some target RNA may contain strong pauses for RT; Use random priming instead of d(T)23VN.
- Use sufficient amount of RNA.