cDNA synthesis

cDNA (complementary DNA) is complementary to mRNA and is synthesized by the enzyme reverse transcriptase (RT)from the mRNA template. Because it is based from mRNA, cDNA only includes the expressed sections of a gene, compared to genomic DNA which contains the entire gene sequence.

When investigating protein-coding regions of the genome, working with mRNA presents some challenges. Most importantly, RNA is easily degraded due to ubiquitous RNases. Therefore, reverse transcription from mRNA to cDNA creates a more stable and reliable sample to analyze this section the genome, for example by PCR. Most importantly, PCR-based techniques like sequencing and qPCR involve the use of DNA polymerases, which can use cDNA but not mRNA as a substrate.

The primer used for cDNA synthesis from mRNAs is usually an oligo(dT) primer, which is able to anneal to the polyA tail of mRNA molecules. Random hexamer primers are also sometimes used, depending on the sequence of the hexamer, which would bind randomly to any RNA in the sample depending on the hexamer's sequence. After cDNA synthesis a DNA-RNA hybrid is formed. Finally, the RNA is digested by RNaseH giving a single-stranded cDNA molecule that can be directly used in any PCR-based technique. If a double-stranded cDNA is required, the resulting single-stranded DNA can fold back on itself and the 3' end binds to a complementary sequence on the same strand. The resulting double-stranded DNA serves as primer for DNA polymerase that produces the complementary strand.

When reverse transcribing mRNA isolated from a tissue sample, all mRNA molecules are transcribed. Therefore, very abundant copies of mRNAs (due to high gene expression) will lead to very abundant copies of cDNA. Likewise genes that are not expressed, thus with little to no mRNA copies transcribed, will not be represented in the cDNA sample.