The synthesis of DNA from an RNA template, via reverse transcription, results in complementary DNA (cDNA). cDNA can then serve as template in a variety of downstream applications for RNA studies such as gene expression; therefore, cDNA synthesis is the first step for many protocols in molecular biology. If you are new to cDNA synthesis or experience researcher wanting to optimize your protocol, consider these five critical steps to help you ensure your cDNA synthesis results in highest efficiency.

Step 1. Prepare sample

RNA serves as the template in cDNA synthesis. Total RNA is routinely used in cDNA synthesis for downstream applications such as RT-(q)PCR, whereas specific types of RNAs (e.g., messenger RNA (mRNA) and small RNAs such as miRNA) may be enriched for certain applications like cDNA library construction and miRNA profiling.

Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use. Best practices to prevent degradation of RNA include wearing gloves, pipetting with aerosol-barrier tips, using nuclease-free labware and reagents, and decontamination of work areas.

To isolate and purify RNA, a variety of strategies are available depending on the type of source materials (e.g., blood, tissues, cells, plants) and goals of the experiments. The main goals of isolation workflows are to stabilize RNA molecules, to inhibit RNases, and to maximize yield with proper storage and extraction methods. Optimal purification methods remove endogenous compounds, like complex polysaccharides and humic acid from plant tissues that interfere with enzyme activity; and common inhibitors of reverse transcriptases, such as salts, metal ions, ethanol, and phenol. Once purified, RNA should be stored at –80°C with minimal freeze-thaw cycles.

Troubleshooting tips

  1. Minimize the number of freeze-thaw cycles of RNA samples to prevent degradation.
  2. Store RNA in an EDTA-buffered solution to minimize nonspecific cleavage by nucleases that have metal ion cofactors.
  3. Use water that is certified nuclease-free or treated with DEPC (diethylpyrocarbonate) to ensure the absence of RNase.
  4. Assess the integrity of RNA by gel electrophoresis or microfluidics.

Step 2. Remove genomic DNA

Trace amounts of genomic DNA (gDNA) may be co-purified with RNA. Contaminating gDNA can interfere with reverse transcription and may lead to false positives, higher background, or lower detection in sensitive applications such as RT-qPCR.

The traditional method of gDNA removal is the addition of DNase I to preparations of isolated RNA. DNase I must be removed prior to cDNA synthesis since any residual enzyme would degrade single-stranded DNA. Unfortunately, RNA loss or damage can occur during DNase I inactivation treatment.

As an alternative to DNase I, double-strand–specific DNases are available to eliminate contaminating gDNA without affecting RNA or single-stranded DNAs. Their thermolabile property allows simple inactivation at a relatively mild temperature (e.g., 55°C) without negative impacts. Such double-strand–specific, thermolabile DNases can be incubated with RNA for 2 min at 37°C prior to reverse transcription reactions to streamline the workflow (Figure 1 ).

gDNA removal procedures
Figure 1. gDNA removal procedures: DNase I vs. Invitrogen ezDNase enzyme. Compared to DNase I, ezDNase enzyme offers a shorter workflow, simpler procedure, and less RNA damage. Inactivation of ezDNase enzyme prior to reverse transcription is optional since the enzyme does not cleave primers, ssRNA, or cDNA:RNA complexes.

Troubleshooting tips

  1. Trace amount of contaminants from RNA purification (e.g., SDS, EDTA) may inhibit DNase activities, therefore, re-precipitate the RNA with ethanol, wash the pellet with 75% ethanol, then dissolve in nuclease-free water.
  2. Select a gDNA removal protocol that has minimal impact on RNA integrity. If using ezDNase, increase treatment to 5 min incubation at 37oC.
  3. DNase is a highly sensitive enzyme; therefore, if you are using DNase treatment, the recommendation is to mix gently by pipetting up and down vs. vortexing.
  4. The most effective way to remove DNase is to perform phenol/chloroform extraction or use a spin column.

Step 3. Select reverse transcriptase

Most reverse transcriptases used in molecular biology are derived from the pol gene of avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (MMLV). The AMV reverse transcriptase was one of the first enzymes isolated for cDNA synthesis in the lab. The enzyme possesses strong RNase H activity that degrades RNA in RNA:cDNA hybrids, resulting in shorter cDNA fragments (<5 kb).

The MMLV reverse transcriptase became a popular alternative due to its monomeric structure, which allowed for simpler cloning and modifications to the recombinant enzyme. Although MMLV is less thermostable than AMV reverse transcriptase, MMLV reverse transcriptase is capable of synthesizing longer cDNA (<7 kb) at a higher efficiency, due to its lower RNase H activity.

To further improve cDNA synthesis, MMLV reverse transcriptase has been engineered for even lower RNase H activity (i.e., mutated RNase H domain, or RNaseH), higher thermostability (up to 55°C), and enhanced processivity (65 times higher). These attributes result in increased cDNA length and yield, higher sensitivity, improved resistance to inhibitors, and faster reaction times (Table 1).

Table 1. Common reverse transcriptases and their attributes.

  AMV reverse transcriptase MMLV reverse transcriptase Engineered MMLV reverse transcriptase
(e.g., Invitrogen SuperScript IV Reverse Transcriptase)
RNase H activity High Medium Low
Reaction temperature
(highest recommended)
42°C 37°C 55°C
Reaction time 60 min 60 min 10 min
Target length ≤5 kb ≤7 kb ≤12 kb
Relative yield
(with challenging or suboptimal RNA)
Medium Low High

Troubleshooting tips

  1. If starting with low RNA quantity, make sure to select a reverse transcription reagent that generates high linearity across a broad range of RNA inputs to ensure that low-abundance RNA can be quantified accurately.
  2. When using RNA that has been degraded or that contains residual salts and inhibitor, consider a reverse transcriptase that can work efficiently with degraded RNA or that is tolerant to salt, and carryover biological inhibitors and extraction reagents.

Step 4. Prepare reaction mix

In addition to enzyme and primers, the main reaction components for reverse transcription include RNA template (pre-treated to remove genomic DNA), buffer, dNTPs, DTT, RNase inhibitor, and RNase-free water (Figure 2).

Reverse transcription reaction with its main components
Figure 2. Reverse transcription reaction with its main components.

Troubleshooting tips

Component Key features

RNA template

Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use (see step 1):

  • Total RNA - routinely used in cDNA synthesis for downstream applications such as RT-(q)PCR
  • Messenger RNA (mRNA) and small RNAs such as miRNA) may be enriched for certain applications like cDNA library construction and miRNA profiling

Reaction buffer

  • Maintains a favorable pH and ionic strength for the reaction.
  • The supplied buffer may also contain additives to enhance the efficiency of reverse transcription.

dNTPs

  • Generally, should be at 0.5–1 mM each, preferably at equimolar concentrations
  • High-quality dNTPs, freshly diluted, are recommended to ensure proficient reverse transcription

DTT

  • Reducing agent, often included for optimal enzyme activity.
  • Reaction efficiencies may be compromised if DTT or other additives precipitate; hence, reaction components should be dissolved and well mixed.

RNase inhibitor

Often included in the reaction buffer or added to the reverse transcription reaction to prevent RNA degradation. They may be:

  • Co-purified during isolation
  • Introduced during reaction setup

A number of known RNases exist, and appropriate RNase inhibitors should be chosen based on their mode of actions and reaction requirements.

Water

Eliminate any RNases by using:

Contaminating RNases cannot be removed by simple filtration, and autoclaved water is not adequate because RNases are heat stable.

Troubleshooting tips

  1. qRT-PCR is a highly sensitive tool for analyzing RNA. As the PCR amplifies the target, errors are simultaneously amplified. Therefore, variability should be kept to a minimum whenever possible. Consider a "master mix", or mixture of the reaction reagents, should be used when setting up multiple reactions to minimize sample-to-sample and well-to-well variation and improve reproducibility
  2. Mix reagents properly to completely dissolve DTT and salts that may have precipitated.

Step 5. Perform cDNA synthesis

Reverse transcription reactions involve three main steps: primer annealing, DNA polymerization, and enzyme deactivation. The temperature and duration of these steps vary by primer choice, target RNA, and reverse transcriptase used.

The critical step is during DNA polymerization. In this step, reaction temperature and duration may vary according to the primer choice and reverse transcriptase used. If using random hexamers, then we recommend incubating the reverse transcription reaction at room temperature (~25 °C) for 10 min after enzyme addition to extend the primers.

Among reverse transcriptases there are differences in thermostability, which in turn determines the highest optimal polymerization temperature for each. Using a thermostable reverse transcriptase allows, a higher reaction temperature (e.g., 50°C), to help denature RNA with high GC content or secondary structures without impacting enzyme activity (Figure 3). With such enzymes, high-temperature incubation can result in an increase in cDNA yield, length, and representation.

Effect of thermostability and its impact on reverse transcriptase activity
Figure 3. Effect of thermostability and its impact on reverse transcriptase activity. Samples containing RNA of varying lengths were reverse-transcribed, using oligo(dT) primers and radiolabelled dNTPs. Reaction products were resolved by gel electrophoresis and visualized by autoradiography. The thermostable reverse transcriptase produces high cDNA yields even above 50°C.

Polymerization time depends on a reverse transcriptase’s processivity, which refers to the number of nucleotides incorporated in a single binding event. For instance, wild-type MMLV reverse transcriptase with low processivity often requires >60 min to synthesize cDNA. In contrast, an engineered reverse transcriptase with high processivity may take as little as 10 min to synthesize a 9 kb cDNA.

Troubleshooting tips

  1. If RNA sample contains high GC content or secondary structure, then minimize the formation of hairpin sequences by performing reverse transcription at a higher temperature (e.g., 50oC). Consider using a highly thermostable reverse transcriptase that withstands elevated reaction temperatures.
  2. Problematic primer design can be minimized by performing revere transcription at an elevated temperature to help increase the specificity of primer binding, and use a thermostable reverse transcriptase.

Step 1. Prepare sample

RNA serves as the template in cDNA synthesis. Total RNA is routinely used in cDNA synthesis for downstream applications such as RT-(q)PCR, whereas specific types of RNAs (e.g., messenger RNA (mRNA) and small RNAs such as miRNA) may be enriched for certain applications like cDNA library construction and miRNA profiling.

Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use. Best practices to prevent degradation of RNA include wearing gloves, pipetting with aerosol-barrier tips, using nuclease-free labware and reagents, and decontamination of work areas.

To isolate and purify RNA, a variety of strategies are available depending on the type of source materials (e.g., blood, tissues, cells, plants) and goals of the experiments. The main goals of isolation workflows are to stabilize RNA molecules, to inhibit RNases, and to maximize yield with proper storage and extraction methods. Optimal purification methods remove endogenous compounds, like complex polysaccharides and humic acid from plant tissues that interfere with enzyme activity; and common inhibitors of reverse transcriptases, such as salts, metal ions, ethanol, and phenol. Once purified, RNA should be stored at –80°C with minimal freeze-thaw cycles.

Troubleshooting tips

  1. Minimize the number of freeze-thaw cycles of RNA samples to prevent degradation.
  2. Store RNA in an EDTA-buffered solution to minimize nonspecific cleavage by nucleases that have metal ion cofactors.
  3. Use water that is certified nuclease-free or treated with DEPC (diethylpyrocarbonate) to ensure the absence of RNase.
  4. Assess the integrity of RNA by gel electrophoresis or microfluidics.

Step 2. Remove genomic DNA

Trace amounts of genomic DNA (gDNA) may be co-purified with RNA. Contaminating gDNA can interfere with reverse transcription and may lead to false positives, higher background, or lower detection in sensitive applications such as RT-qPCR.

The traditional method of gDNA removal is the addition of DNase I to preparations of isolated RNA. DNase I must be removed prior to cDNA synthesis since any residual enzyme would degrade single-stranded DNA. Unfortunately, RNA loss or damage can occur during DNase I inactivation treatment.

As an alternative to DNase I, double-strand–specific DNases are available to eliminate contaminating gDNA without affecting RNA or single-stranded DNAs. Their thermolabile property allows simple inactivation at a relatively mild temperature (e.g., 55°C) without negative impacts. Such double-strand–specific, thermolabile DNases can be incubated with RNA for 2 min at 37°C prior to reverse transcription reactions to streamline the workflow (Figure 1 ).

gDNA removal procedures
Figure 1. gDNA removal procedures: DNase I vs. Invitrogen ezDNase enzyme. Compared to DNase I, ezDNase enzyme offers a shorter workflow, simpler procedure, and less RNA damage. Inactivation of ezDNase enzyme prior to reverse transcription is optional since the enzyme does not cleave primers, ssRNA, or cDNA:RNA complexes.

Troubleshooting tips

  1. Trace amount of contaminants from RNA purification (e.g., SDS, EDTA) may inhibit DNase activities, therefore, re-precipitate the RNA with ethanol, wash the pellet with 75% ethanol, then dissolve in nuclease-free water.
  2. Select a gDNA removal protocol that has minimal impact on RNA integrity. If using ezDNase, increase treatment to 5 min incubation at 37oC.
  3. DNase is a highly sensitive enzyme; therefore, if you are using DNase treatment, the recommendation is to mix gently by pipetting up and down vs. vortexing.
  4. The most effective way to remove DNase is to perform phenol/chloroform extraction or use a spin column.

Step 3. Select reverse transcriptase

Most reverse transcriptases used in molecular biology are derived from the pol gene of avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (MMLV). The AMV reverse transcriptase was one of the first enzymes isolated for cDNA synthesis in the lab. The enzyme possesses strong RNase H activity that degrades RNA in RNA:cDNA hybrids, resulting in shorter cDNA fragments (<5 kb).

The MMLV reverse transcriptase became a popular alternative due to its monomeric structure, which allowed for simpler cloning and modifications to the recombinant enzyme. Although MMLV is less thermostable than AMV reverse transcriptase, MMLV reverse transcriptase is capable of synthesizing longer cDNA (<7 kb) at a higher efficiency, due to its lower RNase H activity.

To further improve cDNA synthesis, MMLV reverse transcriptase has been engineered for even lower RNase H activity (i.e., mutated RNase H domain, or RNaseH), higher thermostability (up to 55°C), and enhanced processivity (65 times higher). These attributes result in increased cDNA length and yield, higher sensitivity, improved resistance to inhibitors, and faster reaction times (Table 1).

Table 1. Common reverse transcriptases and their attributes.

  AMV reverse transcriptase MMLV reverse transcriptase Engineered MMLV reverse transcriptase
(e.g., Invitrogen SuperScript IV Reverse Transcriptase)
RNase H activity High Medium Low
Reaction temperature
(highest recommended)
42°C 37°C 55°C
Reaction time 60 min 60 min 10 min
Target length ≤5 kb ≤7 kb ≤12 kb
Relative yield
(with challenging or suboptimal RNA)
Medium Low High

Troubleshooting tips

  1. If starting with low RNA quantity, make sure to select a reverse transcription reagent that generates high linearity across a broad range of RNA inputs to ensure that low-abundance RNA can be quantified accurately.
  2. When using RNA that has been degraded or that contains residual salts and inhibitor, consider a reverse transcriptase that can work efficiently with degraded RNA or that is tolerant to salt, and carryover biological inhibitors and extraction reagents.

Step 4. Prepare reaction mix

In addition to enzyme and primers, the main reaction components for reverse transcription include RNA template (pre-treated to remove genomic DNA), buffer, dNTPs, DTT, RNase inhibitor, and RNase-free water (Figure 2).

Reverse transcription reaction with its main components
Figure 2. Reverse transcription reaction with its main components.

Troubleshooting tips

Component Key features

RNA template

Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use (see step 1):

  • Total RNA - routinely used in cDNA synthesis for downstream applications such as RT-(q)PCR
  • Messenger RNA (mRNA) and small RNAs such as miRNA) may be enriched for certain applications like cDNA library construction and miRNA profiling

Reaction buffer

  • Maintains a favorable pH and ionic strength for the reaction.
  • The supplied buffer may also contain additives to enhance the efficiency of reverse transcription.

dNTPs

  • Generally, should be at 0.5–1 mM each, preferably at equimolar concentrations
  • High-quality dNTPs, freshly diluted, are recommended to ensure proficient reverse transcription

DTT

  • Reducing agent, often included for optimal enzyme activity.
  • Reaction efficiencies may be compromised if DTT or other additives precipitate; hence, reaction components should be dissolved and well mixed.

RNase inhibitor

Often included in the reaction buffer or added to the reverse transcription reaction to prevent RNA degradation. They may be:

  • Co-purified during isolation
  • Introduced during reaction setup

A number of known RNases exist, and appropriate RNase inhibitors should be chosen based on their mode of actions and reaction requirements.

Water

Eliminate any RNases by using:

Contaminating RNases cannot be removed by simple filtration, and autoclaved water is not adequate because RNases are heat stable.

Troubleshooting tips

  1. qRT-PCR is a highly sensitive tool for analyzing RNA. As the PCR amplifies the target, errors are simultaneously amplified. Therefore, variability should be kept to a minimum whenever possible. Consider a "master mix", or mixture of the reaction reagents, should be used when setting up multiple reactions to minimize sample-to-sample and well-to-well variation and improve reproducibility
  2. Mix reagents properly to completely dissolve DTT and salts that may have precipitated.

Step 5. Perform cDNA synthesis

Reverse transcription reactions involve three main steps: primer annealing, DNA polymerization, and enzyme deactivation. The temperature and duration of these steps vary by primer choice, target RNA, and reverse transcriptase used.

The critical step is during DNA polymerization. In this step, reaction temperature and duration may vary according to the primer choice and reverse transcriptase used. If using random hexamers, then we recommend incubating the reverse transcription reaction at room temperature (~25 °C) for 10 min after enzyme addition to extend the primers.

Among reverse transcriptases there are differences in thermostability, which in turn determines the highest optimal polymerization temperature for each. Using a thermostable reverse transcriptase allows, a higher reaction temperature (e.g., 50°C), to help denature RNA with high GC content or secondary structures without impacting enzyme activity (Figure 3). With such enzymes, high-temperature incubation can result in an increase in cDNA yield, length, and representation.

Effect of thermostability and its impact on reverse transcriptase activity
Figure 3. Effect of thermostability and its impact on reverse transcriptase activity. Samples containing RNA of varying lengths were reverse-transcribed, using oligo(dT) primers and radiolabelled dNTPs. Reaction products were resolved by gel electrophoresis and visualized by autoradiography. The thermostable reverse transcriptase produces high cDNA yields even above 50°C.

Polymerization time depends on a reverse transcriptase’s processivity, which refers to the number of nucleotides incorporated in a single binding event. For instance, wild-type MMLV reverse transcriptase with low processivity often requires >60 min to synthesize cDNA. In contrast, an engineered reverse transcriptase with high processivity may take as little as 10 min to synthesize a 9 kb cDNA.

Troubleshooting tips

  1. If RNA sample contains high GC content or secondary structure, then minimize the formation of hairpin sequences by performing reverse transcription at a higher temperature (e.g., 50oC). Consider using a highly thermostable reverse transcriptase that withstands elevated reaction temperatures.
  2. Problematic primer design can be minimized by performing revere transcription at an elevated temperature to help increase the specificity of primer binding, and use a thermostable reverse transcriptase.

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