Top : Molecular Biology : RNA : Reverse Transcription (RT) & cDNA Synthesis

Summary: Reverse Transcription (RT reaction) is a process in which single-stranded RNA is reverse transcribed into complementary DNA (cDNA) by using total cellular RNA or poly(A) RNA, a reverse transcriptase enzyme, a primer, dNTPs and an RNase inhibitor. The resulting cDNA can be used in RT-PCR reaction. RT reaction is also called first strand cDNA synthesis. Three types of primers can be used for RT reaction: oligo (dT) primers, random (hexamer) primers and gene specific primers with each having its pros and cons. For a RT reaction, 1-2 micrograms of RNA is typically used. Steps are: 1) RNA is first incubated with a primer at 70 degree to denature RNA secondary structure and then quickly chill on ice to let the primer anneal to the RNA. 2) Other components of RT are added to the reaction including dNTPs, RNase inhibitor, reverse transcriptase and RT buffer. 3) RT reaction is extended at 42 degree for 1 hr. 4) Heat the reaction at 70 degree to inactivate the enzyme. 5) Sometimes removal of the template RNA by treating the RT reaction with RNase H is necessary before using the reaction in RT-PCR.
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