CDNA Synthesis

For PCR, only the first cDNA strand needs to be synthesized, since the second strand is copied in the first cycle of the PCR reaction. Many companies sell first-strand synthesis kits, and these are recommended. This reaction uses the enzyme reverse transcriptase, which is an RNA-dependent DNA poly-merase, i.e., it copies RNA into DNA. The two most commonly used enzymes are avian myeloblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia virus (M-MuLV) reverse transcriptase. These enzymes have slightly different activities, but both function adequately. It should also be noted that AMV is used at 42°C, whereas M-MuLV works best at 37°C. The RNA extraction procedure outlined above yields a sample of total RNA and, therefore in addition to including mRNA, it also contains rRNA and tRNA. To copy mRNA preferentially in the reverse transcription reaction, the primer that is used is an oligo dT oligomer. This will bind specifically to the poly (A) tail of mRNA molecules and ensure that they are replicated in preference to the other RNAs in the sample. The other reagents include the appropriate buffer for the reverse transcriptase enzyme, deoxynucleotide triphosphates from which the DNA will be synthesized, RNase inhibitor, and water to make up the volume. The procedure is carried out according to the instructions of the manufacturer of the cDNA kit. In a first-strand synthesis reaction, I would use between 2 and 5 pL of the RNA extracted as above, depending on the amount of starting material.

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