Poor RNA Performance in Downstream Applications (RNA Cleanup)

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Accepted Answer

Purified RNA fails to perform as expected in RT-PCR, RT-qPCR, RNA-seq library preparation, or other downstream assays despite acceptable concentration and A260/280 ratio. Common causes: 1) Salt carryover from wash buffer inhibiting reverse transcriptase or other enzyme activity 2) Ethanol carryover from wash steps interfering with downstream enzymatic reactions 3) Column tip contacted flow-through during final centrifugation, introducing contaminants into eluate 4) DNA contamination in RNA preparation affecting RNA-specific applications or quantification accuracy Solutions: 1) Ensure column tip does not contact flow-through at any step; if uncertain, re-centrifuge for 1 minute to remove traces of salt and ethanol 2) After final wash, perform an additional dry-spin: centrifuge empty column for 1-2 minutes to evaporate residual ethanol 3) Use fresh collection tube for elution step to eliminate any possibility of contaminant carryover 4) For DNA-sensitive applications, treat RNA sample with DNase I (NEB #M0303) following manufacturer's protocol, then perform RNA cleanup using the Monarch RNA Cleanup Protocol 5) Verify A260/230 and A260/280 ratios are within acceptable ranges (1.8-2.0 and ~2.0 respectively) before proceeding to downstream applications