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Molecular Pathology

PCR thermocycling machines

Reverse Transcriptase PCR and Real-Time Reverse Transcriptase PCR

 

Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)

Revised: 22 September 2012, last major update July 2010

Copyright: (c) 2008-2010, PathologyOutlines.com, Inc.

 

General

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● In 1977, RNA levels were first routinely quantified via Northern blotting

● Northern blotting uses denaturing gel electrophoresis blotting with labeled DNA probes, extensive washing steps, followed by multiple film exposures to insure that an exposure within the linear range of the film is achieved

● Northern blotting is relatively complex and time consuming and requires a large amount of RNA, making the examination of many different transcripts difficult

● Additionally, Northern blotting is poor at detecting low abundance RNA species

           

● With the discovery of reverse transcriptase, which converts RNA to DNA, PCR could be used to amplify very low levels of RNAs (reverse transcriptase PCR or RT-PCR)

● RNA could be converted by reverse transcriptase into a cDNA in one step, and then PCR could amplify the cDNA

● With minor modifications, this technique can be made real-time, allowing the comparison of the relative abundance of different RNA species

● Compared to Northern blotting, RT-PCR has several advantages: (a) it requires little post PCR processing, unlike the cumbersome multi-steps of Northern blotting;(b) it can analyze a wide range (>107 fold) of difference in RNA quantities, unlike Northern blotting; and (c) the assay is far more quantitative than Northern blotting, allowing more accurate measurements of RNA species amounts

 

Other advantages of RT-PCR

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● There are many diseases in which RNA analysis is preferable to DNA analysis

● In chronic myelogenous leukemia (CML), analysis of the BCR-ABL fusion gene transcript is fairly straight forward with RT-PCR, since designing oligonucleotide primers which can span the region where the two genes are fused in not difficult

● However, analysis of the same fusion region in DNA would require often impossibly long PCR reactions, as the breakpoint regions within the DNA sequence can be great distances from each other and also occur at different point within the BCR gene

● Thus DNA analysis by PCR would require the design of many different primers and probes to cover the different breakpoints in the chronic myelogenous leukemia the BCR-ABL gene fusion gene

● RT-PCR requires far fewer, making the analysis far simpler

 

● Often the meaning of the abbreviation “RT-PCR” is ambiguous and can mean either reverse transcriptase-PCR or real-time-PCR; when writing about these techniques, you should clearly define exactly what technique is being discussed

 

● An essential step in RT-PCR is the priming of the reverse transcriptase reaction; this is usually done by one of three methods:

 

Random Primers:

● Usually this method uses random heximer primers

● All or most of the RNA templates within a sample are primed and amplified at multiple origins

● Advantages: (a) the random primer mix can prime any RNA mix; (b) there is no need to design specific probes; (c) works well for applications not requiring exact calculations of the abundance of a specific RNA

● Disadvantages: (a) it preferentially amplifies ribosomal RNA, distorting the actual abundance of less common RNAs in the subsequent PCR reactions (the distortion of actual RNA abundance can be as high as 19-fold); (b) random primers give a narrower linear range in PCR amplification curves than do reactions primed by sequence-specific primers

 

Oligo-dT Priming:

● This priming method is more specific than the random method and is best used when one wants obtain a faithful representation of the mRNA pool and when one has a limited amount of mRNA to be amplified

● Disadvantages include that it does not work if (a)  the mRNA to be amplified is fragmented; (b) the mRNA has extensive secondary structure that may inhibit PCR; (c) the later DNA amplification 5’ priming site is very far away from the end of the oligo-dT priming site, such as in mRNAs with very long untranslated 3’ sequences; and (d) the mRNA has multiple splice variants, which differ at the 5’ mRNA end and may not bind a specific 5’ primer

 

Target-Specific Primers:

● Target-specific primers give the most specific cDNA, providing the greatest sensitivity for quantitative assays

● The main problem with this method is that it requires specific RNA amplifying primers and separate reactions for each target and therefore cannot be used on the same RNA sample; thus, it is a poor way to make cDNA when one has limited RNA

● Multiplex priming is possible, but requires very careful primer design

 

End of Molecular Pathology > Polymerase Chain Reaction > Reverse Transcriptase PCR and Real-Time Reverse Transcriptase PCR

 

 

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