Decorative artful nucleic acids

Reverse transcription is a powerful tool for creating complementary DNA (cDNA) from RNA, which can then be used in a variety of downstream applications for studying RNA. It is crucial to recognize and prevent potential issues with cDNA synthesis in order to maintain the reliability of experimental results.

These troubleshooting tips are relevant for most common reverse transcription applications, with a focus on quantitative reverse transcription PCR (RT-(q)PCR). Follow these tips to ensure successful reverse transcription and get the most out of your RNA studies.

Learn how to recover from these reverse transcription issues:

Low or no amplification in RT-(q)PCR

Low or no amplification in RT-(q)PCR

Possible causeRecommendations
Poor RNA integrity
Low RNA purity
  • Follow RNA purification protocols designed for specific sources, such as tissue, blood, and plants.
  • Assess RNA purity by UV spectroscopy, reading the absorbance across a range of wavelengths.
  • Review RNA extraction procedures. Avoid exceeding the recommended quantities of source materials, to allow efficient sample lysis and to minimize inhibitor carryover. Ensure that wash steps are properly carried out to remove impurities and inhibitors.
  • Dilute input RNA in nuclease-free water to reduce the concentration of potential inhibitors, if necessary.
  • Repurify RNA samples to remove residual salts and inhibitors, if necessary.
  • Consider a reverse transcriptase that is resistant to inhibition by salts, carryover biological inhibitors, and extraction reagents.
High GC content and/or secondary structures
  • Denature secondary structures by heating RNA at 65°C for ~5 min, then chilling rapidly on ice, prior to reverse transcription.
  • Minimize the formation of hairpin sequences by performing reverse transcription at a higher temperature (e.g., 50°C).
  • Use a highly thermostable reverse transcriptase that withstands elevated reaction temperatures.
Low RNA quantity
Suboptimal reverse transcriptase
  • To increase cDNA yields, choose a high-performance reverse transcriptase that has better sensitivity, processivity, and resistance to inhibitors. High-performance reverse transcriptases are well-suited for challenging RNA such as in degraded or inhibitor-containing samples.
  • Use reverse transcriptases with high processivity and improved thermostability for short reaction times and high reaction temperatures, respectively.
Suboptimal time and temperature of reverse transcription
Incorrect primer design
Reaction component quality (or stability)
  • Follow manufacturer recommendations for storage and use of the reagents.
  • Ensure that reagents used are fresh and designed for the selected reverse transcriptase.
  • Mix reagents properly to completely dissolve DTT and salts that may have precipitated.
Nonspecific amplification in RT-(q)PCR

Nonspecific amplification in RT-(q)PCR

Possible causeRecommendation
Contamination with genomic DNA (gDNA)
  • Check gDNA contamination by PCR, using a control reaction without reverse transcriptase (a minus-RT or no-RT control).
  • Treat RNA samples with a DNase prior to reverse transcription. Consider a gDNA removal procedure that minimizes nonspecific degradation of RNA and does not require harsh DNase inactivation.
Problematic primer design
  • If a gene-specific primer is used, review the recommendations for primer design. Verify that the binding site of the primer is specific to the gene of interest.
  • Perform reverse transcription at an elevated temperature to help increase the specificity of primer binding, and use a thermostable reverse transcriptase.
  • In (q)PCR setup, choose PCR primers that span exon-exon junctions to enable specific amplification of cDNA.
Truncated cDNA

Truncated cDNA

Possible causeRecommendation
Poor RNA integrity
Presence of reverse transcriptase inhibitors
  • Follow RNA purification protocols designed for specific sources, such as tissue, blood, and plants.
  • Assess RNA purity by UV spectroscopy, reading the absorbance across a range of wavelengths.
  • Review RNA extraction procedures. Avoid exceeding the recommended quantities of source materials, to minimize inhibitor carryover. Ensure that wash steps are properly carried out to remove inhibitors.
  • Repurify RNA samples to remove residual salts and inhibitors, if necessary.
  • Dilute input RNA in nuclease-free water to reduce inhibitor concentration, if necessary.
  • Consider a reverse transcriptase that is resistant to inhibition by salts, carryover biological inhibitors, and extraction reagents.
High GC content and/or secondary structures
  • Denature secondary structures by heating RNA at 65°C for ~5 min, then chilling rapidly on ice, prior to reverse transcription.
  • Minimize the formation of hairpin sequences by performing reverse transcription at a higher temperature (e.g., 50°C).
  • Use a highly thermostable reverse transcriptase that withstands elevated reaction temperatures.
Problematic primers
  • If a gene-specific primer is used, verify that the binding site of the primer is unique to the gene of interest.
  • Use an oligo(dT) primer for synthesis of full-length cDNA when possible.
  • Consider random primers, when working with potentially degraded RNA, for the most efficient reverse transcription.
  • When random primers are used, optimize primer concentrations to obtain long cDNA fragments while maintaining a high yield.
Suboptimal reverse transcriptase
  • Select a reverse transcriptase that is capable of synthesizing long cDNA. This type of reverse transcriptase often exhibits low RNase H activity, increased processivity, and high resistance to inhibitors.
Poor representation (low coverage) in a cDNA pool

Poor representation (low coverage) in a cDNA pool

Possible causeRecommendation
Poor RNA enrichment
  • Use  RNA isolation methods that are efficient yet minimally biased in depleting unwanted RNA (e.g., ribosomal RNA) and enriching RNA of interest (e.g., poly(A)-tailed RNA).
Poor RNA integrity
  • Assess the integrity of RNA prior to cDNA synthesis by gel electrophoresis or microfluidics.
  • Minimize the number of freeze-thaw cycles of RNA samples to prevent degradation.
  • Avoid RNase contamination by following laboratory best practices.
  • Include an RNase inhibitor in reverse transcription setup.
  • Use water that is certified nuclease-free or treated with DEPC (diethylpyrocarbonate) to ensure the absence of RNases.
  • Store RNA in an EDTA-buffered solution (0.1 mM EDTA, or 10 mM Tris + 1 mM EDTA) to minimize nonspecific cleavage by nucleases that have metal ion cofactors.
  • Select a genomic DNA removal protocol that has minimal impact on RNA integrity during inactivation/removal of the DNase used.
  • Consider a reverse transcriptase that can work efficiently with degraded RNA samples.
Low RNA purity
  • Follow RNA purification protocols designed for specific sources, such as tissues, blood, and plants.
  • Assess RNA purity by UV spectroscopy, reading the absorbance across a range of wavelengths.
  • Review RNA extraction procedures. Avoid exceeding the recommended quantities of source materials, to allow efficient sample lysis and to minimize inhibitor carryover. Ensure that wash steps are properly carried out to remove impurities and inhibitors.
  • Repurify RNA samples to remove residual salts and inhibitors, if necessary.
  • Consider a reverse transcriptase that is resistant to inhibition by salts, carryover biological inhibitors, and extraction reagents.
High GC content and/or secondary structures
  • Denature secondary structures by heating RNA at 65°C for ~5 min, then chilling rapidly on ice, prior to reverse transcription.
  • Minimize the formation of hairpin sequences by performing reverse transcription at a higher temperature (e.g., 50°C).
  • Use a highly thermostable reverse transcriptase that withstands elevated reaction temperatures.
Problematic primers
  • Optimize primer mix and concentrations (e.g., oligo(dT) and random hexamers) to decrease bias and increase detection of different targets.
  • Choose random primers for potentially degraded RNA templates, to ensure proper coverage.
Suboptimal time and temperature of reverse transcription
Suboptimal reverse transcriptase
  • Select a high-performance reverse transcriptase that has better sensitivity, processivity, and thermostability. These enzyme properties enable detection of low-abundance RNAs, long transcripts, degraded samples, and inhibitor-containing templates.
  • Use reverse transcriptases with high processivity and improved thermostability for short reaction times and high reaction temperatures, respectively.
Sequence error in cDNA

Sequence error in cDNA

Possible causeRecommendation
Suboptimal reverse transcriptase
  • Check the error rate or fidelity of the reverse transcriptase used. Also, verify the reliability of sequencing results from high-quality reads, paired-end sequencing, and sample replicates.
Genomic DNA (gDNA) contamination
  • Check gDNA contamination by PCR, using a control reaction without reverse transcriptase (a minus-RT or no-RT control).
  • Treat RNA samples with a DNase prior to reverse transcription. Select a gDNA removal procedure that minimizes nonspecific degradation of RNA during inactivation/removal of the DNase used.
  • If PCR-amplified cDNA is sequenced, choose PCR primers that span exon–exon junctions to enable specific amplification of cDNA.
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For Research Use Only. Not for use in diagnostic procedures.