Troubleshooting Guide for RNA Cleanup

Need some help with your RNA cleanups? We’re here to help. Our troubleshooting guide below outlines some of the most common pain points that scientists encounter during RNA cleanup. Still having trouble after reviewing this? Contact our technical support at any time.

Problem Cause Solution
Low RNA Yield Reagents added incorrectly Check protocol to ensure correct buffer reconstitution, order of addition of buffers and ethanol, and proper handling of column flow-through and eluents.
Insufficient mixing of reagents Ensure the ethanol is thoroughly mixed with RNA sample and RNA Cleanup Binding Buffer before applying the sample to the RNA Cleanup Column.
Incomplete elution during prep Ensure the nuclease-free water used for elution is delivered directly to the center of the column so that the matrix is completely saturated. Larger elution volumes, multiple elutions, and longer incubation times can increase yield of RNA, but will dilute the sample and may increase processing times. For typical RNA samples, the recommended elution volumes and incubation times should be sufficient.
High degree of RNA secondary structure Binding and elution of smaller RNAs (< 45 nt) can be affected by secondary structure of the RNA molecules. If poor yield of a small RNA is observed, we recommend diluting your sample with 2 volumes of ethanol instead of one volume in Step 2 of the protocol.
Purified RNA is Degraded RNase contamination In order to avoid RNase contamination during RNA cleanup, make sure to work on a clean lab bench, wear gloves and use disposable RNase-free pipet tips and microfuge tubes (not provided). Keep all kit components tightly sealed when not in use.
Improper storage of RNA Purified RNA should be used immediately in downstream applications or stored at -70°C.
Low A260/230  Ratios Residual guanidine salt carry-over   Ensure wash steps are carried out prior to eluting sample. Use care to ensure the tip of the column does not contact the flow-through. If unsure, repeat centrifugation. When reusing collection tubes, blot the rim of the tube on a Kimwipe prior to reattachment to the column to remove any residual wash buffer.
Low Performance of RNA in Downstream Steps Salt and/or ethanol carry-over Ethanol and salt remaining after the washes may inhibit downstream applications. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-centrifuge for 1 minute to ensure traces of salt and ethanol are not carried over in the eluted RNA.
DNA contamination DNA removal may be necessary for certain applications. Incubate RNA sample with DNase I (NEB #M0303) and cleanup RNA using the Monarch RNA Cleanup Protocol.