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.
Read more about this topic: Reverse Transcription Polymerase Chain Reaction
Famous quotes containing the word principles:
“...at this stage in the advancement of women the best policy for them is not to talk much about the abstract principles of women’s rights but to do good work in any job they get, better work if possible than their male colleagues.”
—Virginia Crocheron Gildersleeve (1877–1965)
“The mode of founding a college is, commonly, to get up a subscription of dollars and cents, and then, following blindly the principles of a division of labor to its extreme,—a principle which should never be followed but with circumspection,—to call in a contractor who makes this a subject of speculation,... and for these oversights successive generations have to pay.”
—Henry David Thoreau (1817–1862)
“When great changes occur in history, when great principles are involved, as a rule the majority are wrong.”
—Eugene V. Debs (1855–1926)