cDNA Synthesis Outline & Application
cDNA synthesis is the process of synthesizing DNA from messenger RNAs (mRNAs). The resultant complementary DNA (cDNA) constitutes the starting material for molecular cloning, hybridization probes and the creation of clone libraries, but is also commonly used for transcriptome analysis in qPCR or microarray experiments. It is a necessary initial step for PCR of an RNA sample.
For research purposes, cDNA is typically synthesized from purified mRNA using an in-vitro reverse transcriptase reaction. There are plenty of kits available on the market, called either cDNA synthesis or reverse transcription kits, supplying all the reagents needed to make your cDNA synthesis easy and cost-effective. However, there are a few factors to consider when choosing the right product.
The starting templates are generally:
Typically, cDNA synthesis kits synthesize either first-strand or double-stranded cDNA:
If you plan to use cDNA clones only for transcriptome analysis, you will need a first-strand synthesis kit
Double-stranded synthesis is required for cloning applications
It is also essential to be aware of the sensitivity of your kit, particularly for quantitative applications. Kits able to amplify minute differences in mRNA expression can be the key to successful experiments. Similarly, thermostable enzymes allow for the use of higher temperatures that can disentangle complex RNA secondary structures, resulting in a more representative amplification and more precise quantification.
Last but not least, the kit should fit your experimental workflow. In this regards, sample processing speed might be worth considering, alongside with suitability for automatic processing or buffer compatibility with your downstream applications.
Check out our cDNA Synthesis Troubleshoot for further guidance.