Reverse Transcription Reaction Setup—Seven Important Considerations

An important factor to the success of any experiment that begins with reverse transcription is the quality of the RNA template. Because RNA serves as the template in reverse transcription, it is critical to maintain the quality and integrity of the RNA during the isolation process. Special precautions should be exercised during extraction, processing, storage, and experimental use.

Prevent RNA degradation with laboratory best practices—wear gloves, pipette with aerosol barrier tips, use nuclease-free labware and reagents, decontaminate work surfaces, and use appropriate storage.

Carefully follow isolation workflows to stabilize RNA molecules, inhibit RNases, remove endogenous compounds and inhibitors, and maximize yield. Once purified, RNA should be stored at –80°C with minimal freeze-thaw cycles.

Once RNA is purified, there are several methods for assessing quality and quantity. A common approach is to measure absorbance across specific wavelengths with UV spectroscopy. RNA quantity can be determined from absorbance at 260 nm. 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. More accurate methods for determining quality and quantity of RNA are the Qubit RNA assay and the Qubit RNA IQ assay both using the Qubit Fluorometer. These assays use target-selective dyes that emit fluorescence only when bound to RNA making them more accurate than traditional UV absorbance methods. Common contaminants such as DNA, salts, free nucleotides, solvents, detergents, or proteins are well tolerated in Qubit assays.

Learn more: Qubit Fluorometer for RNA quantification and quality

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 using gel electrophoresis. 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 (Figure 1B) [1,2].

RNA integrity number (RIN) is a measure of the quality of RNA samples used in various molecular biology applications. A high RIN indicates high-quality RNA, while a low RIN suggests RNA degradation. Therefore, it is essential to ensure that the RNA samples used in experiments have a high RIN to obtain reliable and accurate results. To ensure high-quality RNA samples, researchers can take the following steps:

• Pay attention to sample collection and storage protocols. RNA samples should be collected and stored at –80°C or in liquid nitrogen to prevent RNA degradation. Additionally, samples should be processed as soon as possible after collection to prevent RNA degradation.
• Use appropriate equipment and reagents during RNA isolation. This includes using RNA-free, nuclease-free pipette tips, and avoiding the use of metal tools that can degrade RNA. Ensure that the RNA isolation process is performed carefully and consistently to prevent RNA degradation.
• Evaluate the RIN using an automated system to ensure the integrity of RNA samples.

If the RIN is low, take additional steps, such as:

• Re-extract the RNA to obtain higher quality samples.
• Troubleshoot the RNA isolation process to identify potential sources of RNA degradation, such as inadequate homogenization or prolonged exposure to RNases.
• Optimize the RNA isolation protocol by adjusting parameters such as temperature, time, and reagent concentrations.

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If trace amounts of genomic DNA (gDNA) are contaminating RNA preparations, the gDNA can cause issues such as high background and false positives. Almost all samples will have contaminating gDNA so using a DNase treatment is recommended, particularly for RT-PCR applications.

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, including 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.

Because DNase I may lead to RNA degradation, double-strand-specific DNases have been developed, such as ezDNase Enzyme, to eliminate contaminating gDNA without affecting the quality or quantity of RNA or single-stranded DNAs that may be pertinent to the reaction. They require only a simple inactivation at a relatively mild temperature (e.g., 55°C) without negative impacts. gDNA removal with ezDNase Enzyme takes 2 minutes at 37°C prior reverse transcription reactions. This streamlines the workflow (Figure 2).

Explore: ezDNase Enzyme for genomic DNA removal

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.

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Reverse transcriptases 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 (Table 2).

Learn more: Properties of reverse transcriptases

White paper:  SuperScript IV reverse transcriptase as a better alternative to AMV-based enzymes

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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, three basic primer types are available: oligo(dT) primers, random primers, and gene-specific primers (Figure 3).

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 primers of 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) sample. Oligo(dT) primers are also not suited 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 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. 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 hexamer 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 hexamer primers only may not be ideal for some RT-PCR applications. For instance, overestimation of mRNA copy number is one concern [3]. A mixture of oligo(dT) and random RNA primers is often used in two-step RT-PCR to achieve the benefits of each primer type. For microRNA (miRNA) analysis, random hexamers are not suitable and special primers must be designed for reverse transcription of miRNA [4,5].

Gene specific primers offer the most specific priming in reverse transcription. These primers must be 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.

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In addition to enzyme and primers, the main reaction components for reverse transcription include the RNA template (pre-treated to remove genomic DNA), buffer, dNTPs, DTT, RNase inhibitor, and RNase-free water (Figure 5).

• RNA template—serves as the template in reverse transcription. Table 4 shows the range of RNA amount needed in reverse transcription reactions. The optimal amount depends on the prevalence of target sequence and sensitivity of reverse transcriptase.
• Reaction buffer—a buffer providing a favorable pH and ionic strength for the reaction which may also contain additives to improve the efficiency of reverse transcription.
• dNTPs—nucleotides, preferably at equimolar concentrations (0.5–1 mM each). High-quality and freshly diluted dNTPs are recommended for reverse transcription.
• DTT—a reducing reagent that is often included to optimize enzyme activity.
• RNase inhibitor—prevents RNA degradation by RNase.
• Water—Nuclease-free water from a commercial source, or water treated with DEPC (diethylpyrocarbonate) to eliminate any RNases, is recommended.

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

Denaturation and annealing: In the first step of the reverse transcription reaction setup, the template RNA is mixed with selected primers and dNTPs. If the RNA template is GC-rich or is known to contain secondary structures, the mix may be incubated at 65°C for 5 minutes and chilled on ice for 1 minute before proceeding to the next step. This helps ensure that the RNA is single-stranded and that the primer anneals to the target efficiently. If random hexamer primers are used, which have lower melting temperature, additional annealing step at room temperature for up to 10 minutes is recommended after enzyme addition to extend the primers and stabilize template: primer duplexes.

DNA polymerization: Once the primer is annealed to the RNA template, the reverse transcriptase extends the primer by adding complementary nucleotides in a 5' to 3' direction to synthesize cDNA. The reaction temperature and duration for DNA polymerization depend on the processivity and thermostability of the reverse transcriptase used. The duration of polymerization varies with the processivity of the RT, with wild-type MMLV reverse transcriptase often requiring 60 minutes to synthesize cDNA, while an engineered RT with increased processivity may take as little as 10 minutes to complete full-length cDNA synthesis. Optimal reaction temperature depends on the thermostability of the reverse transcriptase and ranges from 37°C to 55°C. Using a more thermostable reverse transcriptase allows for a higher reaction temperature (e.g., 50°C), which can help denature RNA with high GC content or secondary structures without impacting the enzyme activity. As a result, improved cDNA yield, length and representation is achieved.

Enzyme deactivation: Finally, to stop the reverse transcription reaction, the enzyme is typically inactivated by heating the reaction mixture to a temperature of 70–85°C for 5 to 15 minutes, depending on the thermostability of the enzyme.

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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 to maximize the length and yield of cDNA. Synthesis of double-stranded cDNA often employs a different 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 (Figure 8) [6].

• 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)

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1. Schroeder A, Mueller O, Stocker S et al. (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 7:3.
2. Mueller O, Lightfoot S, Schroeder A (2016) RNA Integrity Number (RIN) – Standardization of RNA Quality Control. Agilent Technologies Publication 5989-1165EN.
3. Zhang J, Byrne CD (1999) Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR. Biochem J 337 (Pt 2):231–241.
4. Chen C, Ridzon DA, Broomer AJ et al. (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179.
5. Kramer MF (2011) Stem-loop RT-qPCR for miRNAs. Curr Protoc Mol Biol Unit 15.10.
6. Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25(2-3):263–269.