It is important to ensure that the RNA is purified prior to use. It should be suspended in nuclease-free water free from EDTA and salts.
Heating the RNA at 65°C for 5 minutes prior to setting up the reaction removes secondary structures on the 5´ end of the transcript. Extend time to 10 minutes for RNA with known highly structured 5’ ends.
SAM is unstable at pH 7–8, 37°C. Hence, it should be diluted just prior to starting the reaction.
We strongly recommend wearing gloves, using nuclease-free tubes and reagents, and thoroughly cleaning pipettes and bench surfaces to avoid RNase contamination.