Troubleshooting Guide: RNA Extraction for Sequencing

Extracting and purifying intact RNA from cells is a crucial step in many molecular biology experiments. This includes tasks like constructing cDNA libraries, conducting northern hybridization, measuring transcript levels, and performing techniques like RNA sequencing, quantitative PCR, RT-PCR, and in vitro translation.

Based on our practical experiences in service projects and consultations, we've put together some practical tips for RNA isolation and purification. These insights are designed to help you overcome common challenges and achieve successful results in your RNA-related work.

Furthermore, A Guide to RNA Extraction, RNA Purification and Isolation might also be a helpful article for you. It aims to provide a clear and comprehensive understanding of the RNA processing workflow, supporting your research efforts.

RNA Sequencing Sample Submission and Preparation Guidelines and A Beginner's Guide to RNA Sequencing may be useful articles for your RNA sequencing projects.

1. Incomplete Solubilization of RNA Products

Causes:

• Too long drying time.
• Excessive amount of product.
• Excessive impurities.

Solutions:

• Control the drying time after washing with 75% ethanol to avoid over-drying.
• Prolong the dissolution time or heat at 55~60°C for 23 minutes.
• Increase the amount of ddH20, extend the dissolution time, or heat at 55~60°C for 23 minutes.
• After the sample is fully lysed, centrifuge the supernatant before subsequent operations.

2. RNA Degradation

Causes:

• Presence of RNase contamination.
• Improper sample storage or too long sample storage time.
• Repeated freezing and thawing of samples.
• Electrophoresis reasons.

Solutions:

• Ensure that all centrifuge tubes, tips, and related solutions are free of RNase contamination.
• Wear a mask and clean disposable gloves, and operate in a separate clean area.
• Use fresh samples or samples frozen in liquid ammonia at -85~-65°C.
• Store samples in separate packages to avoid degradation due to repeated freezing and thawing.
• Quickly add samples taken out from liquid nitrogen to the lysis solution and mix well.
• Pre-treat electrophoresis tanks with 3% hydrogen peroxide or RNase and nucleic acid removers before electrophoresis.
• Rinse with RNase-free ddH20 and prepare electrophoresis buffer with RNase-free ddH20.
• Replace the Loading Buffer with a new one before electrophoresis.

3. Downstream Inhibition or Low Purity

Causes:

• Protein contamination.
• Polysaccharide contamination.
• Fat contamination.
• Salt residue.

Solutions:

• Decrease the sample starting volume; increase the volume of single-phase lysis reagent.
• Decrease the starting volume of the sample; increase the step of sample processin.
• Increase the number of 75% ethanol rinses.
• Less shaking at the end of centrifugation to reduce the amount of supernatant aspirated.

4. Genomic DNA Contamination

Causes:

• Add isopropanol and centrifuge without seeing the precipitate.
• High sample input.

Solutions:

• Reduce the starting sample volume.
• Increase the volume of single-phase lysis reagent.
• When lysing samples, add an appropriate amount of HAc.
• Use reverse transcription reagents containing genome removal modules.
• When designing primers, use trans-intron primers to avoid genomic DNA participation in the amplification reaction.

5. Loss of Precipitate

Causes:

• After low sample input or addition of isopropanol, leave the sample at 28°C for 10~30 minutes and then centrifuge after the RNA content is low; add 1 μL of glycogen (20 mg/mL) for co-precipitation.
• Loss of precipitate.
• The sample's metabolites are dispersed on the walls of the centrifuge tube.

Solutions:

• When discarding the supernatant, use the method of aspiration rather than decanting to avoid losing the precipitate.
• Precipitate will be dispersed on the wall of the centrifuge tube; when discarding the supernatant, aspirate slowly along the surface of the liquid.

6. No RNA Precipitation

Causes:

• Incomplete Homogenization: Genomic DNA molecules are large and sticky. Incomplete homogenization results in flocculent agglutination of deformed proteins and genomic DNA, hindering effective RNA release.
• Incomplete Precipitation: When dealing with small tissue or cell quantities, proportional reduction of TRIzol volume is essential during extraction. Failure to do so can prevent RNA precipitation due to excessive dilution.

Solutions:

• Optimize homogenization conditions to overcome challenges posed by large and sticky genomic DNA.
• Adjust TRIzol volume proportionally for small tissue or cell quantities to prevent excessive dilution.

7. Low Extraction Rate

Causes:

• Too Much Sample: Excessive sample amounts lead to incomplete homogenization. Increased DNA and protein proportion per unit volume prevents effective RNA release into the supernatant. Difficulties in phase separation and reduced RNA precipitation efficiency.
• Too Little Reagent: TRIzol extraction relies on acidic phenol extraction. In acidic pH, DNA precipitates at the phenol-solution interface, while RNA stays in the aqueous phase. Inadequate TRIzol volume, leading to neutral pH, causes DNA to dissolve in the aqueous phase. Please refer to TRIzol RNA Extraction Protocol.
• Incomplete Dissolution of RNA: Inadequate dissolution time or improper conditions hinder RNA solubility.
• RNAase Contamination: Exogenous or endogenous RNAase contamination can lead to RNA degradation.
• Mixing of Phases: Aspirating the aqueous phase while inadvertently mixing with the organic phase.
• Short Lysis Time: Homogenized samples were lysed for less than 5 minutes at room temperature.

Solutions:

• Adjust sample amounts to ensure effective homogenization and prevent incomplete RNA release.
• Ensure sufficient TRIzol volume for acidic phenol extraction, maintaining the proper pH for RNA precipitation.
• Enhance RNA solubility by extending the dissolution time.
• Implement stringent measures to prevent RNAase contamination.
• Exercise caution to prevent mixing of phases during aspiration.
• Increase the sample lysis time to over 5 minutes for effective RNA extraction.