cDNA Synthesis & Reverse Transcriptases

The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA). Reverse transcriptases (RTs) use an RNA template and a short primer complementary to the 3′ end of the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for applications such as PCR, qPCR, LAMP, or sequencing workflows.

NEB offers several RTs, available as standalone products or kits. ProtoScript^® II Reverse Transcriptase is a recombinant Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild-type M-MuLV. The enzyme is active up to 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.

The Luna RT featured in the Luna RT-qPCR and LunaScript RT Supermix products has been engineered to minimize RNase H activity. In the supermix products (NEB# E3010/M3010), the Luna RT has been paired with random primers for transcribing short RNA targets up to 3 kb. For longer targets, a primer-free version (NEB #E3025) allows for users to select their own primers for flexible cDNA synthesis.

Induro™ Reverse Transcriptase is a group II intron-encoded RT that exhibits high processivity, increased thermostability, and increased tolerance of inhibitors. It can support cDNA synthesis up to 20 kb.

The Template Switching RT Enzyme Mix features a unique RT and buffer system that enables efficient template switching. It has better specificity, sensitivity, and returns more sequencing reads than the Smart-Seq2 method for single cell RNA-seq. It also can be used in 5’ RACE and 2nd strand cDNA synthesis.

WarmStart^® RTx Reverse Transcriptase is an in silico designed RNA-directed DNA polymerase coupled with a reversibly-bound aptamer that inhibits enzyme activity below 40°C. It is ideal for use in isothermal amplification workflows and is featured in numerous LAMP mixes.

The use of engineered RTs improves the efficiency of full-length product formation, ensuring the copying of the 5′ end of the mRNA transcript is complete, and enabling the propagation and characterization of a faithful DNA copy of an RNA sequence. The use of the more thermostable RTs, where reactions are performed at higher temperatures, can be very helpful when dealing with RNA that contains high amounts of secondary structure.

Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and M-MuLV, Reverse Transcriptase are wild type retroviral RTs. M-MuLV RT lacks 3′ → 5′ exonuclease activity.