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:
“Ah, I fancy it is just the same with most of what you call your “emancipation.” You have read yourself into a number of new ideas and opinions. You have got a sort of smattering of recent discoveries in various fields—discoveries that seem to overthrow certain principles which have hitherto been held impregnable and unassailable. But all this has only been a matter of intellect, Miss West—superficial acquisition. It has not passed into your blood.”
—Henrik Ibsen (1828–1906)
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—Catherine E. Beecher (1800–1878)
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—Ralph Waldo Emerson (1803–1882)