Reverse Transcription Polymerase Chain Reaction - Principles

Principles

In RT-PCR, the RNA template is first converted into a complementary DNA using a reverse transcriptase. The cDNA is then used as a template for exponential amplification using PCR. RT-PCR is currently the most sensitive method of RNA detection available. The use of RT-PCR for the detection of RNA transcript has revolutionalized the study of gene expression in the following important ways:

• Made it theoretically possible to detect the transcripts of practically any gene
• Enabled sample amplification and eliminated the need for abundant starting material that one faces when using northern blot analysis
• Provided tolerance for RNA degradation as long as the RNA spanning the primer is intact

Despite its major advantages, RT-PCR is not without drawbacks. The extreme sensitivity of the technique can be a double edged sword since even the slightest DNA contamination can lead to undesirable results. Additionally, planning and design of quantification studies can be technically challenging due to the existence of numerous sources of variation including template concentration and amplification efficiency.