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:
“[E]very thing is useful which contributes to fix us in the principles and practice of virtue.”
—Thomas Jefferson (1743–1826)
“[The sceptic] must acknowledge, if he will acknowledge any thing, that all human life must perish, were his principles to prevail. All discourse, all action would immediately cease, and men remain in a total lethargy, till the necessities of nature, unsatisfied, put an end to their miserable existence.”
—David Hume (1711–1776)
“It must appear impossible, that theism could, from reasoning, have been the primary religion of human race, and have afterwards, by its corruption, given birth to polytheism and to all the various superstitions of the heathen world. Reason, when obvious, prevents these corruptions: When abstruse, it keeps the principles entirely from the knowledge of the vulgar, who are alone liable to corrupt any principle or opinion.
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—David Hume (1711–1776)