Wikipedia talk:USEP/Courses/JHU MolBio Ogg 2012/Section 81/Group 81C/Rationale

Wiki Project: Article Pick Rationale (Group 81C)

Since the first documentation of RNA quantification via Northern blotting in 1977, this assay technique has been extensively used despite its shortcomings: (a)time-consuming, (b) requiring large amounts of RNA for detection, and (c) quantitative inaccuracy in the presence of low abundant RNA content (1)(2). With the discovery of reverse transcriptase during the study of viruses, the technique of reverse transcription polymerase chain reaction (RT-PCR) was developed to become the method of choice for RNA detection and quantification (3). Although the technique was more sensitive and reproducible compared to Northern blots, RT-PCR was not without problems of its own. The exponential growth of the reverse transcribed complementary DNA (cDNA) during the multiple cycles of PCR made the end point quantification less accurate due to the difficulty in maintaining linearity (4). To accurately detect and quantify the abundance of RNA in a sample, real-time RT-PCR (aka real-time qRT-PCR or real-time RT-qPCR*) was developed by a minor fluorescence-based modification to monitor the amplification of the products during each PCR cycle (5).

RT-PCR has risen to become the mainstream research tool for several reasons: (a) it does not require a post PCR processing ;(b) a wide range (>10^7 fold) of RNA abundance can be measured and (c) it provides both qualitative and quantitative data making real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels (6). Due to its simplicity, specificity and sensitivity, it is used in a wide range of applications from experiments as simple as quantification of yeast cells in wine to being used as a diagnostic tool for detecting bird flu (7)(8).

Our group chose the article on Reverse transcription polymerase chain reaction for several reason. First, upon reviewing the article it seemed under developed. Our group would like to expand on the history, protocol of RT-PCR, mechanism of RT-PCR, and application. Currently, the article states that RT-PCR is used in the diagnosis of genetic diseases. We would like to expand on diagnosis of genetic diseases such as Lesch–Nyhan syndrome. Lesch-Nyhan is a genetic disease that causes the malfunction of the HPRT1 gene, which clinically leads to fatal uric acid urinary stone and symptoms similar to gout (9). Furthermore, the article currently has no listed resources. We aim to add credible references to the article. This not only will give the article more credibility, but will provide links to additional resources in case readers want to further expand their knowledge.

• From what I can tell, real-time RT-PCR , real-time qRT-PCR, and real-time RT-qPCR were used interchangeably to represent quantitative RT-PCR being tracked in real time. Need to do more reading to make sure that the three names are synonymous.

Sources:

(1) Alwine JC, Kemp DJ, Stark GR (1977). "Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5350–4.

(2) Streit, S.; Michalski, C. W.; Erkan, M.; Kleef, J.; Friess, H. (2009). "Northern blot analysis for detection of RNA in pancreatic cancer cells and tissues". Nature Protocols 4 (1): 37–43.

(3) Bustin SA 2000 Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25 169–193.

(4) Shiao YH. A new reverse transcription-polymerase chain reaction method for accurate quantification. BMC Biotechnol. 2003; 3: 22.

(5) Lost source…

(6) Bustin, SA, Benes V, Nolan T, Pfaffl MW. Quantitative real-time RT-PCR – a perspective. J Mol Endocrinol June 1, 2005 34 597-601.

(7) Hierro N, Esteve-Zarzoso B, Gonzalez A, Mas A, Guillamon JM. Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine. Appl Environ Microbiol. 2006 November; 72(11): 7148–7155.

(8) Slomka MJ, Pavlidis T, Coward VJ, Voermans J, Koch G, Hanna A, Banks J, Brown IH. Validated real-time reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses.Influenza Other Respi. Viruses 2009 Jul;3: 151-164.

(9)Urbach A, Schuldiner M, Benvenisty N. Modeling for Lesch-Nyhan Disease by Gene Targeting in Human Embryonic Stem Cells. Stem Cells. 2004;22

635-641.

I found this article helpful/interesting also: http://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553