Reverse Transcription Troubleshooting | Thermo Fisher Scientific

Reverse Transcription Troubleshooting Guide

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.

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	Check the protocol(s) for recommended amounts of input RNA. Confirm RNA quantity by UV spectroscopy. Consider an alternative, fluorescence-based quantitation method for higher accuracy and specificity. Select a genomic DNA removal protocol that has minimal impact on RNA integrity during inactivation/removal of the DNase used. Use a reverse transcriptase with high efficiency and sensitivity to reverse-transcribe low-abundance RNA. Select reverse transcription reagents that generate high linearity across a broad range of RNA inputs to ensure that low-abundance RNA can be quantified accurately.
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	Make sure reaction time and temperature are appropriate for the selected reverse transcriptase.
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.

Nonspecific amplification in RT-(q)PCR

Possible cause	Recommendation
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 during inactivation/removal of the DNase used.
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

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.

Poor representation (low coverage) in a cDNA pool

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	Optimize reaction time and temperature for the selected reverse transcriptase.
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

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.

For more troubleshooting tips, please visit our Reverse Transcription and RACE Support Center, cDNA Libraries and cDNA Library Construction, and Next-Generation Sequencing Support Center, or contact our technical support team.

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Reverse Transcription Troubleshooting Guide

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