Reverse transcriptases (RTs) are RNA-directed DNA polymerases that were first identified as part of the retroviral life cycle
. RTs catalyze the synthesis of a DNA copy (cDNA) of the target RNA molecules using a reverse transcription primer, dNTPs, and Mg2+ or Mn2+ as a cofactor. Scientists have since adapted these enzymes for use in a variety of in vitro applications including real-time and endpoint RT-PCR, labeled-cDNA probe generation and cDNA library construction. All RTs catalyze cDNA synthesis, but some enzymes are preferred over others for certain RNA targets and downstream applications. Choosing the best RT for your experiment is not difficult once you understand the basic enzymatic properties and important considerations such as the length of the target RNA, presence of complex RNA secondary structure, downstream application and an enzyme’s level of RNase H activity. This article summarizes many of these important considerations for the more popular reverse transcriptases.
Avian Myeloblastosis Virus Reverse Transcriptase
Avian myeloblastosis virus (AMV) reverse transcriptase is one of the most common RTs used in the lab. The 170kDa heterodimer requires 6–10mM Mg2+ or Mn2+ for activity, and reactions often include sodium pyrophosphate and spermidine to increase full-length cDNA production and decrease formation of hairpins during synthesis
. AMV RT is less sensitive to inhibition by strong RNA secondary structure than Moloney murine leukemia virus (M-MLV) RT
Optimal enzyme activity and maximum cDNA length occur at 42–48°C, but the reaction temperature can range from 25°C to 58°C
. The higher reaction temperature helps denature regions of strong RNA secondary structure, which can cause RTs to stall and limit cDNA size
. For this reason, AMV RT is often used to reverse transcribe RNAs with strong secondary structure. Like other RTs, AMV RT is compatible with gene-specific primers, oligo(dT)15 primers or random hexamers, although use of random hexamers requires a reduced reaction temperature of 37°C. Gene-specific RT primers with suitably high melting temperatures are recommended when the reaction temperature exceeds 42°C.
Although high reaction temperatures can effectively resolve regions of strong secondary structures, these temperatures are detrimental to RNA integrity. RNA is thermolabile and susceptible to metal-catalyzed degradation. Normally, hydrolysis occurs at a low frequency, but RNA hydrolysis becomes a concern under certain conditions (e.g., nonoptimal pH, high temperatures, the presence of divalent cations). Thus, cDNA synthesis—in particular cDNA synthesis of long RNAs—benefits from not exposing RNA to higher reaction temperatures. To minimize the amount of time that RNA spends at high temperatures, cDNA synthesis protocols using AMV and M-MLV RTs often incorporate an initial denaturation step, where the RNA and RT primer are combined, briefly heated to help denature any secondary structure then quickly cooled on ice to maintain the denatured state. The RT, reaction buffer and dNTPs are added, and the reaction is incubated at the desired temperature.
AMV RT possesses an intrinsic RNase H activity, which degrades the RNA strand of an RNA/DNA hybrid and can cleave the RNA template if the RT pauses during synthesis
. This reduces total cDNA yield and the percentage of full-length cDNA, limiting the usefulness of AMV RT to reverse transcribe RNAs longer than ~5kb.
Typical RT-PCR conditions include the use of up to 5µg of total RNA or up to 100ng of polyA+ mRNA, 20–30 units of enzyme and a 60-minute incubation at 42°C. AMV RT is more processive than M-MLV RT
, so fewer units are required to generate the same amount of cDNA; 25 units of AMV RT is equivalent to approximately 200 units of M-MLV RT. Prior to PCR, AMV must be inactivated because AMV RT, like M-MLV RT, can inhibit Taq DNA polymerase
. The enzyme can be inactivated by heating at 70–100°C, followed by a 5-minute incubation on ice. The reverse transcription reaction is often diluted prior to PCR or the volume of cDNA added to the PCR is limited because spermidine can inhibit PCR
. This limitation can negatively affect the ability to detect low-abundance RNAs.
AMV RT is recommended for one-step and two-step RT-PCR and RT-qPCR, reverse transcription of RNAs <5kb and primer extension, particularly if the template RNA has strong secondary structure.
Moloney Murine Leukemia Virus Reverse Transcriptase
Moloney murine leukemia virus RT is often used for reverse transcription of long RNAs (>5kb) due to the lower RNase H activity compared to AMV RT. However, the lower reaction temperature of 37°C can make reverse transcription of RNAs with strong secondary structure problematic. Variations of wildtype M-MLV RT exist and are popular due to their lack of RNase H activity and higher reaction temperatures. The most popular variant is the M-MLV RT RNase H– point mutant, which has a single amino acid substitution that dramatically reduces RNase H activity yet preserves full DNA polymerase activity
. The M-MLV RT RNase H– point mutant also is much more thermostable
. Whereas wildtype M-MLV RT has an optimal reaction temperature of 37°C, the M-MLV RT RNase H– point mutant can be used at temperatures of up to 55°C
, making the M-MLV RT RNase H– point mutant suitable for RNAs with strong secondary structure. However, in the absence of strong secondary structure, lower reaction temperatures will result in longer cDNA products.
Like many RTs, the M-MLV RT RNase H– point mutant is sensitive to inhibition by many common laboratory chemicals. M-MLV RT RNase H– point mutant is inhibited 50% by 5% (v/v) formamide, 17% (v/v) DMSO, 34% (v/v) glycerol, 15mM guanidine isothiocyanate, 1mM EDTA, 0.0025% SDS and 4µg/ml heparin. The enzyme is inhibited 70% by 0.5mM spermidine and 4mM sodium pyrophosphate, and >90% by 160mM guanidine hydrochloride, 70mM guanidine isothiocyanate, 2.5mM EDTA, 0.005% (w/v) SDS and 30µg/ml heparin
The lack of RNase H activity makes the M-MLV RT RNase H– point mutant the enzyme of choice for reverse transcribing long RNAs for cDNA library construction, cDNA probe generation and primer extension.
GoScript™ Reverse Transcriptase
GoScript™ Reverse Transcriptase uses M-MLV RT and state-of-the-art buffers designed specifically for efficient reverse transcription and qPCR of rare and abundant transcripts, even in the presence of inhibitors. Unlike other first-strand cDNA systems, there are no inhibitory effects when up to 20% of the RT reaction is added to a PCR as long as the final MgCl2 concentration is kept at an optimal level (1.5–5mM, depending on the template; optimization of MgCl2 concentration is strongly recommended). GoScript™ RT is active across a range of 25–55°C, with the greatest activity at 37–42°C.
GoScript™ Reverse Transcriptase is optimized for use in one-step and two-step RT-qPCR and reverse transcription of rare and abundant targets, and performs well in the presence of many common amplification inhibitors.