Reverse transcription generates complementary DNA (cDNA) from RNA, and the cDNA can then serve as template in a variety of downstream applications for RNA studies. Therefore, it is important to recognize and prevent potential issues with cDNA synthesis to maintain the validity of experimental results. The troubleshooting tips provided here pertain to reverse transcription in most common applications, with emphasis on (quantitative) reverse transcription PCR, or RT-(q)PCR.

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Possible cause Recommendations
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 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
  • Review the recommendations on reverse transcription primer types for specific RNA templates. For example, use random primers, instead of the oligo(dT) primer, for bacterial RNA or RNA lacking a poly(A) tail, as well as for potentially degraded RNA.
  • With a gene-specific primer, ensure the primer’s sequence is complementary to the 3’ end of the target.
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.
Possible cause Recommendation
Contamination with genomic DNA (gDNA)
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.
Possible cause Recommendation
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.
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.
Possible cause Recommendation
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.
Possible cause Recommendation
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.