The process of cDNA synthesis involves several steps:
Isolation of mRNA: The first step is to isolate mRNA from the tissue of interest. This is often done using techniques such as poly-A tail capture, which takes advantage of the polyadenylated tail found on most eukaryotic mRNA molecules.
Reverse Transcription: The isolated mRNA is then used as a template for reverse transcription. The enzyme reverse transcriptase synthesizes a complementary DNA strand by adding nucleotides that are complementary to the mRNA sequence.
Second Strand Synthesis: Once the first strand of cDNA is synthesized, the RNA template is often degraded, and a second strand of DNA is synthesized to create a double-stranded cDNA molecule. This can be achieved using DNA polymerase.
