Low RNA Yield After Cleanup (RNA Cleanup)

Question

Accepted Answer

Recovered RNA concentration is significantly lower than expected based on input amount. Spectrophotometric measurement shows inadequate RNA quantity for downstream applications. Common causes: 1) Incorrect buffer reconstitution or order of reagent addition during protocol execution 2) Insufficient mixing of ethanol with RNA sample and RNA Cleanup Binding Buffer before column loading 3) Incomplete saturation of column matrix - elution water not delivered directly to center of column 4) High degree of RNA secondary structure affecting binding and elution of small RNAs (< 45 nt) 5) Premature discarding of flow-through or eluents during protocol steps Solutions: 1) Verify protocol adherence: confirm correct buffer reconstitution, reagent addition order, and proper handling of all flow-through and eluent fractions 2) Thoroughly mix ethanol with RNA sample and RNA Cleanup Binding Buffer by vortexing or pipetting before applying to column 3) Deliver nuclease-free elution water directly to the center of the column matrix; allow complete saturation before centrifugation 4) For small RNAs (< 45 nt) with suspected secondary structure, dilute sample with 2 volumes of ethanol instead of 1 volume in Step 2 5) Use larger elution volumes (note: will dilute sample), perform multiple elutions, or extend incubation time to 2-5 minutes on column before centrifugation