Wednesday August 15, 2007
There are a huge number of choices available for the purchase of cDNA synthesis kits and reagents. The majority of products achieve much the same goal – the production of cDNA from extracted RNA. The main differences between most of these kits are the type of enzyme used to perform the reverse transcription - avian myeloblastosis virus reverse transcriptase (AMV-RT) or Moloney murine leukaemia virus (MMLV-RT), and the additional reagents that make various kits useful for specific downstream applications such as standard PCR, qPCR (aka real-time PCR), end-cleanup and cloning (kits containing blunting enzymes and cloning vectors), size fractionation, cDNA labeling and microarrays (kits containing labeled NTPs), and so forth. Some kits even allow the production of cDNA directly from cells, rather than having to preform a separate RNA purification. Given that some kits are very specific in their use, it may be beneficial to purchase a basic kit that can be used for all applications (and these are often much less expensive) rather than buy many kits that can be used only for single applications. It should be noted that some kits are designed to produce cDNA from eukaryotic cells only (such as those that provide only poly-dT primers, rather than random hexamers which will work on any RNA sample). Many enzyme mixtures also contain proprietary ingredients that are designed to increase fidelity, reliability, thermostability as well as overall yields of transcription from difficult templates, such as those with high-GC content or strong secondary structures. Whether these mixtures can increase their performance in each specific application can only be assessed by trial and error, so it can be useful to obtain a sample from the manufacturer beforehand.
Scott Coutts
Research Scientist
Monash University
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