Transcripts may have different and not yet annotated 3' ends, which might be mistaken for internal priming events of the oligo(dT) primer, when in fact those are true 3' ends. Artificial spike-in transcripts such as the SIRVs or the ERCC spike-in transcripts only have one defined 3' end, this which provides the only true measure to determine internal priming.
If true internal priming is detected, this could be a result of mis-priming during reverse transcription, for instance if the temperature before or during reverse transcription was too low. As outlined in the general section of the User Guide, unless explicitly mentioned, all steps should be carried out at room temperature between 20 °C and 25 °C. In particular, for First Strand cDNA Synthesis (p.11): Do not cool the FS2 / E1 mastermix (step 3) and have the RNA / FS1 samples at 42 °C when adding the FS2 / E1 mastermix (step 4) to avoid mishybridization. Mix properly by pipetting. Do not forget to shortly spin down the samples at room temperature before and after adding the FS2 / E1 mastermix.
ATTENTION: Centrifugation should not be carried out at 4 °C, always spin down at room temperature! Raising the reaction temperature up to 50 °C for reverse transcription can be used for additional prevention of mis-priming.
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