Reverse Transcription PCR

Reverse transcription (RT)-PCR is a technique used to amplify RNA targets. Because DNA polymerase requires a double-stranded DNA template, RNA must be transcribed into complementary (c) DNA prior to PCR by the enzyme reverse transcriptase. The cDNA then serves as the template for the first PCR temperature cycle (Fig. 11.4). The combined use of RT and PCR to amplify RNA targets was first described in 1987. Early studies coined the terms RT-PCR, RNA-PCR, RNA phenotyping, and message amplification phenotyping. Reviews describing the numerous applications of RT-PCR are available (Larrik, 1992). RT-PCR is

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

_mRNA(or total RNA)

Reverse Transcription down stream primer

Reverse transcriptase

PCR Cycle 1

RNA degradation or denature first strand cDNA (complementary DNA) upstream primer downstream primer Taq DNA polymerase

PCR Cycle 1

Figure 11.4. Amplification of RNA.

Figure 11.4. Amplification of RNA.

an important technique in the diagnosis of infectious and genetic diseases and is the key procedure used to detect and quantify RNA as a measure of gene expression.

Two reverse transcriptase enzymes commonly used are Moloney murine leukemia virus (M-MuLV) reverse transcriptase and avian myeloblastosis virus (AMV) reverse transcriptase. Both enzymes have the same fundamental activities but differ in some characteristics, including temperature and pH optima. In addition to M-MuLV and AMV, other variants of this enzyme are available for use in the molecular diagnostic laboratory. These enzymes are available in preoptimized RT-PCR kits.

In vitro reverse transcription is primer directed. A single primer is used to generate cDNA and can be one of the primers used in the subsequent PCR reaction (sequence-specific) or a random oligonucleotide. Specificity is not required of reverse transcription. Random oligonucleotides are convenient in that one RT kit or reaction can be used for all RNA targets.

Traditionally, the RT step has been performed in a separate tube containing only components necessary for reverse transcription. After RT, an aliquot is removed, added to a PCR reaction tube, and subjected to amplification. Drawbacks of the separate tube method include inconvenience and cross-contamination risk. More recently, single-tube RT-PCR assays, either two-enzyme or single-enzyme, have been described.

RT-PCR is often used interchangeably to describe reverse transcription PCR and real-time PCR. To avoid confusion, "real-time" will not be abbreviated in this chapter.

Was this article helpful?

0 0

Post a comment