There are several possibilities:

1. 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).
2. Phenol/chloroform extraction and ethanol extraction can remove contaminant proteins such as proteases (2).
3. Some target RNA may contain strong pauses for RT; Use random priming instead of d(T)₂₃VN.
4. Use sufficient amount of RNA.