Determining the amount of mRNA is essential for accurate examination of genes expression. The basic PCR mechanism involves DNA replication as a nucleic acid pattern but not RNA. Thus, the first step is the synthesis of DNA named complementary deoxyribonucleic acids (cDNA) from an RNA template, via reverse transcription.
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an single-stranded RNA template. There are several types of RT enzymes: HIV-1, AMV, MMLV, and telomerase reverse transcriptase enzyme.
The cDNA can be synthesized using total RNA or purified mRNA.The required materials for this reaction are including buffer, dNTP, RNase-free water, reverse transcriptase enzyme, and RNA.
Three types of primers can be used in the cDNA constructionprocess:
- The desired gene Specific Primers
- Random hexamer Primers that can reproduce RNA types.
- Oligo (dt), which is specifically attached to the poly(A) tail at the end of the mRNA and replicate it.
The cDNA replication specifically occurs in the temperaturerange 37 to 50 °C, for each kit. At theend of the reaction, the enzyme inactivation was performed over a temperaturerange of 75 to 95 °C.