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cDNA Synthesis

cDNA Synthesis describes the generation of complementary DNA (cDNA) from an RNA template by reverse transcription. Reverse transcriptases (RTs) use an RNA template and a primer complementary to the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is required.

Many RTs are available from commercial suppliers. Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase are RTs that are commonly used in molecular biology workflows. M-MuLV Reverse Transcriptase lacks 3´ → 5´ exonuclease activity. ProtoScript® II Reverse Transcriptase is a recombinant 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. WarmStart RTx Reverse Transcriptase is a unique, in silico-designed RNA-directed DNA polymerase coupled with a reversibly-bound aptamer that inhibits enzyme activity below 40°C. This enzyme can synthesize a complementary DNA strand initiating from a primer using RNA (cDNA synthesis) or single-stranded DNA as a template. RTx is a robust enzyme for RNA detection in amplification reactions and is particularly well suited for use in LAMP (loop-mediated isothermal amplification). The Template Switching RT Enzyme Mix features an RT that adds a few non-templated nucleotides after it reaches the 5′ end of the RNA template, enabling efficient template switching activity in a RT reaction. The Luna Reverse Transcriptase featured in Luna RT-qPCR/qPCR products possesses higher thermostability than many other RTs, allowing an optimal reaction temperature of 55°C. Induro Reverse Transcriptase is a group II intron-encoded RT that exhibits high processivity, increased thermostability, and increased tolerance of inhibitors in the synthesis of cDNA from RNA. It is an ideal enzyme for challenging cDNA synthesis from long transcripts, RNAs with strong secondary structures, and RNA samples with inhibitors.

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Protocols for cDNA Synthesis
    Publications related to cDNA Synthesis
    • Terry Fei Fan Ng, Nikola O Kondov, Xutao Deng, Alison Van Eenennaam, Holly L Neibergs, Eric Delwart (2015) A metagenomics and case-control study to identify viruses associated with bovine respiratory disease. J Virol; 89, 5340-9. PubMedID: 25740998, DOI: 10.1128/JVI.00064-15
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