Robinson, Murray O. and Simon, Melvin I. (1991) Determining transcript number using the polymerase chain reaction: Pgk-2, mP2, and PGK-2 transgene mRNA levels during spermatogenesis. Nucleic Acids Research, 19 (7). pp. 1557-1562. ISSN 0305-1048. http://resolver.caltech.edu/CaltechAUTHORS:20120420-071323728
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We describe a technique that uses reverse transcription and the polymerase chain reaction (per) to rapidly quantitate numbers of specific mRNA transcripts from nanogram quantities of total cellular RNA. Linearity of input molecules to output signal was maintained by limiting the cycle number and the amount of input RNA and by minimizing the number of manipulations. Absolute levels of specific transcripts were determined by the inclusion of a separate standard curve composed of serially diluted in vitro transcribed RNA run alongside the experimental samples. This allowed rapid quantitation of many samples simultaneously. We applied this technique to measuring the expression of phosphoglycerate kinase 2 (Pgk-2) transgenes in the mouse testis during development. A human PGK-2 transgene, a PGK-2/CAT transgene, and the endogenous mPgk-2 gene all displayed similar patterns and levels of expression, consistent with the conclusion that peak RNA accumulation occurs in pachytene spermatocytes. Mouse protamine 2 (mP2) is expressed at a level approximately tenfold higher than Pgk-2 and displays a different pattern of expression consistent with initiation of transcription occuring in haploid round spermatids.
|Additional Information:||© 1991 Oxford University Press. Received December 11, 1990; Revised and Accepted February 27, 1991. We would like to thank Andrew Pakula and Tom Wilkie for critical review of the manuscript. This work was supported by N.I.H. grant AG 07687.|
|Official Citation:||Murray O. Robinson and Melvin I. Simon Determining transcript number using the polymerase chain reaction: Pgk-2, mP2, and PGK-2 transgene mRNA levels during spermatogenesis Nucl. Acids Res. (1991) 19(7): 1557-1562 doi:10.1093/nar/19.7.1557|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Ruth Sustaita|
|Deposited On:||20 Apr 2012 15:35|
|Last Modified:||26 Dec 2012 15:05|
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