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cDNA Synthesis & Reverse Transcriptases

cDNA Synthesis

NEB offers several reagents for cDNA synthesis upstream of applications such as qPCR and qRT-PCR. For your convenience, reagents are available as kits or standalone products to suit your needs.

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 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.

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.

For help comparing the RTs and cDNA Synthesis reagents available, view our RT/cDNA Synthesis selection chart.

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Protocols for cDNA Synthesis & Reverse Transcriptases
    Publications related to cDNA Synthesis & Reverse Transcriptases
  1. 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, PubMedID: 25740998, DOI: 10.1128/JVI.00064-15
cDNA Synthesis Selection Chart
NEB offers several reagents for cDNA synthesis for use in applications including qPCR and qRT-PCR. For your convenience, reagents are available as kits or standalone products, depending on your needs.
RNA Polymerase Selection Chart
NEB offers RNA polymerases that can be used for in vitro synthesis of RNA for a wide variety of downstream applications. Additional polymerases are offered for the generation of untemplated homoribopolymeric tails for reverse transcription or labeling.
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This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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