cDNA synthesis

To study gene expression levels, the first step is to extract RNAs from cells and then synthesize cDNA (complementary DNA) molecules using the enzyme reverse transcriptase. The reverse transcriptase enzyme can synthesize DNA molecules from RNA. cDNA is a strand of DNA obtained from mature mRNA. After synthesizing cDNA, quantitative analysis of the expression level of the target gene can be performed through Real-Time PCR. Various primers can be used in the production of cDNA, including oligo-dT, specific primers, and random hexamers.

The oligo-dT primer is used to generate complementary DNA from mRNA. Oligo-dT is complementary to the polyadenylated tail of mRNA, as it is rich in thymine, and binds to its 3' end. Random hexamers can be employed in synthesizing complementary DNA from other extracted RNAs, as their binding occurs randomly to different RNA sections. When working with RNA types such as microRNA, specific primers are used. The stages of cDNA synthesis are illustrated in the figure below.

The steps for synthesizing cDNA are as follows: first, the RNA-containing samples are placed into tubes. The aqueous solvent in which RNA is dissolved must be free of RNases. Next, primers are added to the tubes. The samples then need to be placed in a thermal cycler at 65 degrees Celsius for five minutes, allowing the RNA strands to fully denature. After this step, the samples are briefly placed on ice. A specified amount of master mix is then added to each tube, and finally, the samples undergo thermal cycling.

In the first cycle, at 55 degrees Celsius, the primers and then the enzymes bind, initiating the synthesis process. In the second cycle, at 85 degrees Celsius, the enzymes are inactivated, and in the third cycle, the samples are cooled. The samples are then transferred to -20 degrees Celsius for storage.