Troubleshooting Guide for Total RNA Extraction & Purification

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

PROBLEM CAUSE SOLUTION
Clogged column Insufficient sample disruption or homogenization
  • Increase time of sample digestion or homogenization

  • Centrifuge sample after Proteinase K digestion or homogenization to pellet debris and use only supernatant for next steps

  • Use larger volume of DNA/RNA Protection Reagent (NEB #T2011) and/or RNA Lysis Buffer (NEB #T2012) for sample disruption and homogenization. See sample specific protocol in the Product Manual.
Too much sample
Low yield Incomplete elution
  • After addition of Nuclease-free Water (NEB #B1500) to column matrix, incubate 5-10 min at room temperature and then centrifuge to elute

  • Perform a second elution (note: this will dilute sample)
Sample is degraded
  • Store input sample at -80°C prior to use

  • Use Monarch DNA/RNA Protection Reagent (NEB #T2011) to maintain RNA integrity during storage
Insufficient disruption or homogenization
  • Increase time of sample digestion or homogenization

  • Centrifuge sample after Proteinase K digestion or homogenization to pellet debris and use only supernatant for next steps

  • Use larger volume of DNA/RNA Reagent (NEB #T2011) and/or RNA Lysis Buffer (NEB #T2012) for sample disruption and homogenization. See sample specific protocol in the Product Manual.

  • For Proteinase K treated samples, doubling Proteinase K (from 5% to 10%) may lead to an increase in RNA yield
Too much sample
RNA degradation Starting material not handled/stored properly
  • Store input sample at -80°C prior to use. Degradation of RNA may occur if sample is not flash frozen or protected by a preservation reagent. Use Monarch DNA/RNA Protection Reagent (NEB #T2011) to maintain RNA integrity during storage.
Deviation from the stated protocol may expose RNA to unwanted RNase activities
RNase contamination of eluted materials or kit buffers may have occurred
Low OD ratios Low A260/280 values indicate residual protein in the purified sample
  • Ensure the Proteinase K step was utilized for the recommended time. Ensure samples have no debris prior to addition of ethanol and loading onto RNA Purification Column.
Low A260/230 values indicate residual guanidine salts have been carried over during elution
  • 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 after the final wash. If unsure, please repeat centrifugation. When reusing collection tubes, blot rim of tube on a Kimwipe prior to reattachment to the column to remove any residual wash buffer.
DNA contamination Genomic DNA not removed by column
Too much sample
Low performance of RNA in downstream steps Salt and/or ethanol carryover has occurred
  • Use care to ensure the tip of the RNA Purification Column does not contact the flow-through after the final wash. If unsure, please repeat centrifugation

  • Be sure to spin the RNA Purification Column for 2 minutes following the final wash with RNA Wash Buffer

  • When reusing collection tubes, blot rim of tube on a Kimwipe prior to reattachment to the column to remove any residual wash buffer

  • Add additional wash step and/or extend spin time for final wash
Unusual Spectrophotometric readings RNA concentration is too low for spectrophotometric analysis
Silica fines in eluate
  • Re-spin eluted samples and pipet aliquot from the top of the liquid to ensure the A260/230 is unaffected by possible elution of silica particles