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cDNA Synthesis Protocol in Five Steps
We have compiled a list of five critical steps to help ensure your cDNA synthesis is as efficient as possible. cDNA synthesis, is the process of creating complementary DNA (cDNA) from an RNA template through reverse transcription. It is a crucial first step in many molecular biology protocols. From gene expression studies to a variety of downstream applications, cDNA is a valuable tool in RNA research. Follow these tips to get the most out of your cDNA synthesis.
What is cDNA?
cDNA is complementary DNA. 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.
cDNA synthesis protocol
Prepare sample RNA for cDNA synthesis
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 RNA (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, inhibiting RNases, and maximizing yield with proper storage and extraction methods. Optimal purification methods remove common inhibitors that interfere with activity of reverse transcriptases. Such inhibitors include both endogenous compounds from biological sample material, or inhibitory carryover compounds from RNA isolation reagents, like salts, metal ions, ethanol, and phenol. Once purified, RNA should be stored at –80°C with minimal freeze-thaw cycles.
Explore: RNA isolation
Remove genomic DNA preparing sample for cDNA synthesis
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. However, DNase I must be removed prior to cDNA synthesis since any residual enzyme would degrade single-stranded DNA, compromising RT-PCR results. Unfortunately, DNase I inactivation methods can often result in RNA loss or damage.
As an alternative to DNase I, double-strand–specific DNases, such as Invitrogen ezDNase Enzyme, 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 offer shorter protocols and require only 2 minutes at 37°C prior to reverse transcription reactions to complete gDNA digestion (Figure 1).
Click image to enlarge
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.
Select reverse transcriptase for improved cDNA yield
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. These attributes can 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 | ≤14 kb |
| Relative yield (with challenging or suboptimal RNA) | Medium | Low | High |
Explore: SuperScript IV reverse transcriptase
Prepare reaction mix for cDNA synthesis
In addition to enzyme, the main reaction components for cDNA synthesis include RNA template (pre-treated to remove genomic DNA), buffer, dNTPs, DTT, RNase inhibitor, nuclease-free water, and primers (Figure 2).
Figure 2. Reverse transcription reaction with its main components.
Table 2. Reaction components for cDNA synthesis
| Component | Key features |
|---|---|
RNA template | Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use (see step 1):
|
Reaction buffer |
|
dNTPs |
|
DTT |
|
RNase inhibitor | Often included in the reaction buffer or added to the reverse transcription reaction to prevent RNA degradation by RNases. RNase inhibitors may be:
A number of known RNases exist, and appropriate RNase inhibitors should be chosen based on their mode of actions and reaction requirements. |
Water | Use DEPC-treated or nuclease-free water from a commercial source to minimize the risk of contaminating RNases. Contaminating RNases cannot be removed by simple filtration, and autoclaved water is not adequate because RNases are heat stable. |
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.
If RNA is GC-rich or is known to contain secondary structures, optional denaturation step can be performed by heating RNA-primer mix at 65°C for 5 minutes and then chilling on ice for 1 minute.
If using random hexamers, we recommend incubating the reverse transcription reaction at room temperature (~25 °C) for 10 minutes to anneal and extend the primers.
DNA polymerization is a critical step—in this step, reaction temperature and duration may vary depending on reverse transcriptase used. Reverse transcriptases differ by thermostability, which in turn determines the highest optimal polymerization temperature. 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). Thus, reactions at higher temperature can result in an increase in cDNA yield, length, and representation.
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 minutes to synthesize cDNA. In contrast, an engineered reverse transcriptase with high processivity may take as little as ten minutes to complete cDNA synthesis.
Learn more: Reverse transcription troubleshooting guide
Explore: Five step workflows
Prepare sample RNA for cDNA synthesis
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 RNA (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, inhibiting RNases, and maximizing yield with proper storage and extraction methods. Optimal purification methods remove common inhibitors that interfere with activity of reverse transcriptases. Such inhibitors include both endogenous compounds from biological sample material, or inhibitory carryover compounds from RNA isolation reagents, like salts, metal ions, ethanol, and phenol. Once purified, RNA should be stored at –80°C with minimal freeze-thaw cycles.
Explore: RNA isolation
Remove genomic DNA preparing sample for cDNA synthesis
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. However, DNase I must be removed prior to cDNA synthesis since any residual enzyme would degrade single-stranded DNA, compromising RT-PCR results. Unfortunately, DNase I inactivation methods can often result in RNA loss or damage.
As an alternative to DNase I, double-strand–specific DNases, such as Invitrogen ezDNase Enzyme, 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 offer shorter protocols and require only 2 minutes at 37°C prior to reverse transcription reactions to complete gDNA digestion (Figure 1).
Click image to enlarge
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.
Select reverse transcriptase for improved cDNA yield
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. These attributes can 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 | ≤14 kb |
| Relative yield (with challenging or suboptimal RNA) | Medium | Low | High |
Explore: SuperScript IV reverse transcriptase
Prepare reaction mix for cDNA synthesis
In addition to enzyme, the main reaction components for cDNA synthesis include RNA template (pre-treated to remove genomic DNA), buffer, dNTPs, DTT, RNase inhibitor, nuclease-free water, and primers (Figure 2).
Figure 2. Reverse transcription reaction with its main components.
Table 2. Reaction components for cDNA synthesis
| Component | Key features |
|---|---|
RNA template | Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use (see step 1):
|
Reaction buffer |
|
dNTPs |
|
DTT |
|
RNase inhibitor | Often included in the reaction buffer or added to the reverse transcription reaction to prevent RNA degradation by RNases. RNase inhibitors may be:
A number of known RNases exist, and appropriate RNase inhibitors should be chosen based on their mode of actions and reaction requirements. |
Water | Use DEPC-treated or nuclease-free water from a commercial source to minimize the risk of contaminating RNases. Contaminating RNases cannot be removed by simple filtration, and autoclaved water is not adequate because RNases are heat stable. |
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.
If RNA is GC-rich or is known to contain secondary structures, optional denaturation step can be performed by heating RNA-primer mix at 65°C for 5 minutes and then chilling on ice for 1 minute.
If using random hexamers, we recommend incubating the reverse transcription reaction at room temperature (~25 °C) for 10 minutes to anneal and extend the primers.
DNA polymerization is a critical step—in this step, reaction temperature and duration may vary depending on reverse transcriptase used. Reverse transcriptases differ by thermostability, which in turn determines the highest optimal polymerization temperature. 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). Thus, reactions at higher temperature can result in an increase in cDNA yield, length, and representation.
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 minutes to synthesize cDNA. In contrast, an engineered reverse transcriptase with high processivity may take as little as ten minutes to complete cDNA synthesis.
Learn more: Reverse transcription troubleshooting guide
Explore: Five step workflows
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