Reverse transcription─reaction setup

For molecular biology experiments, reverse transcription is primarily carried out to create complementary DNAs (cDNAs) representing tissue- or cell-specific RNA populations. For experiments to be successful there are critical considerations related to the template, reagents, and reaction conditions.

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Simplified Reverse Transcription

RNA template preparation

RNA serves as the template in reverse transcription. 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, inhibiting RNases, and maximizeing 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.

Inhibitors in Reverse Transcription Reaction

There are several methods for assessing RNA quality and quantity after purification. A common approach is UV spectroscopy to measure absorbance across specific wavelengths. RNA quantity can be determined from absorbance at 260 nm using the Beer-Lambert Law, and the presence or absence of specific contaminants may be inferred from ratios of absorbance at different wavelengths (Table 1). Note that UV absorbance is not specific to RNA alone, since all nucleic acids absorb UV at similar wavelengths. For more specific and sensitive analysis of RNA, fluorescence assays with dyes that emit signal only when specifically bound to target molecules may be considered.

What is a purity ratio?

Table 1. UV measurement guidelines for RNA analysis

Absorbance Indicates presence of: Target ratios
230 nm Organic compounds, sugars, urea, salts A260/A230 > 1.8
260 nm All nucleic acids A260 ≈ 0.1–1.0
270 nm Phenol A260/ A 270 > 1.0
280 nm Protein RNA: A260/A280 ≈ 2.0
DNA: A260/A280 ≈ 1.8
330 nm Light scattering A330 = 0

RNA integrity can be evaluated by the comparison of 28S and 18S ribosomal RNAs (rRNA) as a representation of total RNA. Total RNA is denatured and then resolved by size by gel electrophoresis, which is qualitative in nature. The ratio of intensities of 28S rRNA to 18S rRNA is then assessed, with a 2:1 ratio indicative of intact RNA (Figure 1A). A more quantitative method, developed by Agilent Technologies, combines microfluidics and a proprietary algorithm to assess RNA integrity. The method produces a digital readout, called the RNA Integrity Number, or RIN, where values ranging between 8 and 10 indicate high-quality RNA [1,2] (Figure 1B).

Figure 1. Analysis of RNA integrity by (A) gel electrophoresis and (B) microfluidics.

Genomic DNA removal

On occasion, 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.

DNase I is commonly added to the isolated RNA to eliminate gDNA. DNase I must be removed completely prior to RT-PCR, since any residual enzyme would degrade single-stranded DNA, such as primers and synthesized cDNA. Often, DNase I inactivation (e.g., treatment with EDTA and heat) or enzyme removal procedures results in RNA degradation or sample loss.

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 2).

gDNA removal procedures
Figure 2. 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.

Reverse transcriptase considerations

The class of enzymes known as reverse transcriptases synthesizes complementary DNA using RNA as template, but they can differ in functional activities and properties. Their properties impact their ability to reverse-transcribe long RNA transcripts, GC-rich RNA, RNA with significant secondary structures, and RNA of suboptimal quality.

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 is a 170-kDa heterodimer with an optimal reaction temperature range of 42–48°C. The AMV reverse transcriptase 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. MMLV reverse transcriptase is a 75-kDa enzyme with an optimal reaction temperature around 37°C. Although it 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 [3].

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 2) [4]. (Learn more: reverse transcriptase attributes).

Table 2. 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

Primer selection

To initiate reverse transcription, reverse transcriptases require a short DNA oligonucleotide called a primer to bind to its complementary sequences on the RNA template and serve as a starting point for synthesis of a new strand. Depending on the RNA template and the downstream applications, primers of three basic types are available: oligo(dT) primers, random primers, and gene-specific primers (Figure 3).

Common primers used in reverse transcription
Figure 3. Common primers used in reverse transcription.
  • Oligo(dT) primers consist of a stretch of 12–18 deoxythymidines that anneal to poly(A) tails of eukaryotic mRNAs, which make up only 1–5% of total RNA. These primers are the optimal choice for constructing cDNA libraries from eukaryotic mRNAs, full- length cDNA cloning, and 3′ rapid amplification of cDNA ends (3′ RACE). Because of their specificity for poly(A) tails, oligo(dT) primers are not suitable for degraded RNA, such as from formalin-fixed, paraffin-embedded (FFPE) samples, nor for RNAs that lack poly(A) tails, such as prokaryotic RNAs and microRNAs. Since cDNA synthesis starts at the 3′ poly(A) tail, oligo(dT) primers potentially can cause 3′ end bias. RNA with significant secondary structure may also disrupt full-length cDNA synthesis, resulting in under representation of the 5′ ends.

    Oligo(dT) primers may be modified to improve efficiency of reverse transcription. For instance, the length of oligo(dT) primers may be extended to 20 nucleotides or longer to enable their annealing in reverse transcription reactions at higher temperatures. In some cases, oligo(dT) primers may include degenerate bases like dN (dA, dT, dG, or dC) and dV (either dG, dA, or dC) at the 3′ end. This modification prevents poly(A) slippage and locks the priming site immediately upstream of the poly(A) tail. These primers are referred to as anchored oligo(dT).

  • Random primers are oligonucleotides with random base sequences. They are often six nucleotides long and are usually referred to as random hexamers, N6, or dN6. Due to their random binding (i.e., no template specificity), random primers can potentially anneal to any RNA species in the sample. Therefore, these primers may be considered for reverse transcription of RNAs without poly(A) tails (e.g., rRNA, tRNA, non-coding RNAs, small RNAs, prokaryotic mRNA), degraded RNA (e.g., from FFPE tissue), and RNA with known secondary structures (e.g., viral genomes).

    While random primers help improve cDNA synthesis for detection, they are not suitable for full-length reverse transcription of long RNA. Increasing the concentration of random hexamers in reverse transcription reactions improves cDNA yield but results in shorter cDNA fragments due to increased binding at multiple sites on the same template (Figure 4).

    Moreover, use of random primers only may not be ideal for some RT-PCR applications. For instance, overestimation of mRNA copy number is one concern [5]. A mixture of oligo(dT) and random primers is often used in two-step RT-PCR to achieve the benefits of each primer type. For microRNA (miRNA) expression assays, random hexamers are not suitable and special primers must be designed for reverse transcription of miRNA [6,7].

cDNA length and yield are impacted by primer choice and concentration
Figure 4. cDNA length and yield are impacted by primer choice and concentration. A 6.4-kb RNA with a poly(A) tail was reverse-transcribed into double-stranded (ds) cDNA using an oligo (dT) primer or random hexamers of varying concentrations. The results were analyzed by agarose gel electrophoresis. Increasing the concentration of random hexamers led to a higher concentration of short cDNAs relative to more discrete, longer products of transcription with oligo(dT) primers.
  • Gene-specific primers offer the most specific priming in reverse transcription. These primers are designed based on known sequences of the target RNA. Since the primers bind to specific RNA sequences, a new set of gene-specific primers is needed for each target RNA. As a result, more RNA is required for analysis of multiple target RNAs. Gene-specific primers are commonly used in one-step RT-PCR applications.

Table 3. Comparison of common reverse transcription primers.

  Oligo(dT) Random hexamers Oligo(dT) +
random hexamers
Gene-specific primer
Typical final concentration 2–5 µM 2–5 µM 1–2 µM each 0.5–1 µM
Pre-primer extension at 25°C    
Key benefits

Full-length reverse transcription of RNA with poly(A) tail

Reverse transcription of most RNA species, including degraded RNA

Combined benefits of oligo(dT) and random primers

Reverse transcription specific to the gene of interest

Main reaction components

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 5).

Reverse transcription reaction with its main components
Figure 5. Reverse transcription reaction with its main components.
  • The RNA template is prepared as described in the previous sections. Table 4 provides the recommended range of input RNA for reverse transcription reactions, and the optimal amount depends on the prevalence of target sequence and sensitivity of reverse transcriptase.

Table 4. Recommended ranges of input RNA amounts in reverse transcription reactions.

RNA template Recommended range
Total RNA 10 pg–5 μg
Poly(A) RNA 10 pg–0.5 μg
Specific RNA 0.01 pg–0.5 μg
  • The 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, a reducing reagent, is 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.
  • An RNase inhibitor is usually included in the reaction buffer or added to the reverse transcription reaction to prevent RNA degradation. RNases may have been co-purified during isolation or 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 used in reverse transcription reactions should be nuclease-free. Nuclease-free water from a commercial source, or water treated with DEPC (diethylpyrocarbonate) to eliminate any RNases, is best. Contaminating RNases cannot be removed by simple filtration, and autoclaved water is not adequate because RNases are heat stable.

Reaction temperature and time considerations

Reverse transcription reactions involve three main steps: primer annealing, DNA polymerization, and enzyme deactivation (Figure 6). The temperature and duration of these steps vary by primer choice, target RNA, and reverse transcriptase used.

Three main steps of cDNA synthesis
Figure 6. Three main steps of cDNA synthesis.
  • Primer annealing: The primer is mixed with the RNA template, heated to 65°C for 5 min, then incubated on ice for at least 1 min. This helps ensure that the RNA is single-stranded and that the primer anneals to the target efficiently. After annealing, the reverse transcriptase and necessary components (e.g., buffer, dNTPs, RNase inhibitor) are added.
  • DNA polymerization: In this step, reaction temperature and duration may vary according to the primer choice and reverse transcriptase used. With an oligo (dT) primer (Tm ~35–50°C), the reaction may be incubated directly at the optimal temperature of the reverse transcriptase (37–50°C). Random hexamers typically have lower Tm (~10–15°C) due to their shorter length. Therefore, when random hexamers are used (solely or in combination with oligo(dT)), 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 7). With such enzymes, high-temperature incubation can result in an increase in cDNA yield, length, and representation.

    Polymerization time depends on 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 (learn more about reverse transcriptase processivity).
Effect of thermostability and its impact on reverse transcriptase activity
Figure 7. 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.
Reverse transcription in 10 minutes
  • Enzyme deactivation: The final step in reverse transcription reactions is to deactivate the reverse transcriptase. The deactivation temperature may be in the range of 70–85°C, depending upon the thermostability of the enzyme. Deactivation is usually carried out for a period of 5–15 minutes, with a higher temperature requiring a shorter time.

First-strand and second-strand cDNA synthesis

Synthesis of cDNA from an RNA template, as described in the previous section, generates a cDNA:RNA hybrid. This process is referred to as first-strand cDNA synthesis. If RNase H activity is present (as in wild-type AMV and MMLV reverse transcriptases), the RNA of the cDNA:RNA hybrid is cleaved during first-strand synthesis. The first-strand cDNA (with or without the RNA annealed to it) may be used directly in some applications such as RT-PCR, where a thermostable DNA polymerase (e.g., Taq DNA polymerase) replicates the complementary strand of cDNA.

In cDNA library construction and sequencing, the first-strand cDNA is used as a template to generate double-stranded cDNA representing the RNA targets. This process is known as second-strand cDNA synthesis. In the second-strand cDNA synthesis, reverse transcriptases with minimal RNase H activity are recommended in order to maximize the length and yield of cDNA.

Synthesis of double-stranded cDNA often employs a different DNA polymerase (e.g., T7 DNA polymerase, DNA polymerase I, Taq DNA polymerase) to produce the complementary strand of the first cDNA strand. Additional enzymes may be included in the double-stranded cDNA synthesis, such as those listed below for a modified method of Gubler and Hoffman [8] (Figure 8).

Double-stranded cDNA synthesis by the Gubler-Hoffman procedure
Figure 8. Double-stranded cDNA synthesis by the Gubler-Hoffman procedure.
  • E. coli RNase H nicks the RNA strands of cDNA:RNA complexes, providing 3′-OH priming sites for DNA synthesis.
  • E. coli DNA polymerase I extends the nicked RNA strands by 5′ 3′ polymerase activity and replaces the RNA strand in the direction of synthesis by 5′ 3′ exonuclease activity, in a process known as nick translation.
  • E. coli DNA ligase seals the nicks between the newly synthesized cDNA segments. (T4 DNA ligase should not be used as a substitute, as it can ligate blunt-end double-stranded cDNA fragments and form chimeric structures.)
  • T4 DNA polymerase blunts the termini of the double-stranded cDNA (optional in the final step).

In conclusion, the success of reverse transcription is highly dependent upon reaction components and conditions. Reactions should be carried out appropriately to fit with downstream applications of interest.

References