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:
“Language is a process of free creation; its laws and principles are fixed, but the manner in which the principles of generation are used is free and infinitely varied. Even the interpretation and use of words involves a process of free creation.”
—Noam Chomsky (b. 1928)
“[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)
“Syntax is the study of the principles and processes by which sentences are constructed in particular languages. Syntactic investigation of a given language has as its goal the construction of a grammar that can be viewed as a device of some sort for producing the sentences of the language under analysis.”
—Noam Chomsky (b. 1928)