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 (3). AMV RT is less sensitive to inhibition by strong RNA secondary structure than Moloney murine leukemia virus (M-MLV) RT (4).
Optimal enzyme activity and maximum cDNA length occur at 42–48°C, but the reaction temperature can range from 25°C to 58°C (5). The higher reaction temperature helps denature regions of strong RNA secondary structure, which can cause RTs to stall and limit cDNA size (6–7). 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 (8). 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 (5–6), 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 (9). 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 (10). 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.