ReverseTranskriptasePCR

ReverseTranskriptasePCR, also known as reverse transcription polymerase chain reaction (RT-PCR), is a molecular biology technique used to detect and quantify RNA. It combines two enzymatic steps: first, reverse transcription, where an RNA template is converted into complementary DNA (cDNA) by a reverse transcriptase enzyme; and second, PCR, where specific DNA targets are amplified using DNA polymerase and sequence-specific primers.

Typical workflow begins with RNA isolation from a sample under RNase-free conditions. During reverse transcription, primers such as gene-specific primers, random hexamers, or oligo-dT primers guide synthesis of cDNA. In the subsequent PCR, the cDNA serves as template for amplification with forward and reverse primers, a DNA polymerase, nucleotides, and buffers. In one-step RT-PCR, reverse transcription and PCR occur in a single reaction mix; in two-step RT-PCR, cDNA synthesis and PCR are performed in separate vessels, allowing flexibility and multiplexing.

Real-time RT-PCR (qRT-PCR) monitors amplification with fluorescent dyes or probes, enabling quantitative measurement of RNA levels during the exponential phase of PCR. RT-PCR is widely used for measuring gene expression, detecting RNA viruses, and diagnostics in clinical microbiology, veterinary science, and research. It also supports multiplex assays and, in some formats, digital RT-PCR for absolute quantification.

Important considerations include maintaining RNase-free conditions, proper primer design, and appropriate controls to avoid contamination and false results. Limitations include sensitivity to RNA integrity, potential inhibitors, and reliance on reverse transcriptase efficiency.

RT-PCR has been a cornerstone technique since its development in the late 1980s and has evolved into numerous variants and workflows, with qRT-PCR and digital RT-PCR among the most common today.