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mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts by Gregory J Hurteau; Simon D Spivack is a scholarly article available to read on EtoBox.

What is mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts about?

Reverse transcription-polymerase chain reaction (RT-PCR) has become the method of choice for detection of mRNA transcripts, including those of low abundance obtained from small precious samples of human tissue. A major confounding problem for standard reverse-transcription-priming strategies is the presence of contaminating genomic DNA (gDNA) carried over from the original "RNA" extract into the RT and PCR steps. The contaminating gDNA contains a processed pseudogene sequence-which lacks introns but contains a poly(A) tail-for commonly studied internal reference genes beta-actin and GAPDH, and target genes GSTM1, GSTP1, and others. These pseudogene sequences therefore confound standard-design "RNA-specific" PCR primer pairs which rely, for cDNA versus gDNA specificity, on the pair-spanning introns, or one of the individual primer oligos spanning an exon/exon splice site, because these features are lacking in processed pseudogene sequences. The result is false RT-PCR positives for these "housekeeper" genes in total RNA extracts; the gDNA processed pseudogene is mistaken for mRNA gene transcript. A universal RT primer has been designed that targets the poly(A) tail of mRNA and adds a

Author

Gregory J Hurteau; Simon D Spivack

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2002

Language

EN