Quantitative measurement of human T-cell receptor V beta subfamilies by reverse transcription-polymerase chain reaction using synthetic internal mRNA standards.

Abstract:

:Quantitative determination of T-cell receptor (TCR) V beta expression is necessary to define the changes in TCR-V beta subfamily expression that occur during T-cell maturation and selection and to detect alterations of the TCR-V beta repertoire that may be associated with human diseases. Here we describe and validate a quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique to determine human TCR-V beta subfamily mRNA levels (as well as other mRNA species), based on the use of synthetic poly(A) mRNA internal standards that are coprocessed with native (sample) mRNA transcripts. The technique allows simultaneous reverse transcription of sample and standard mRNA and thus obviates errors arising during reverse transcription. In addition, the technique allows coamplification of several concentrations of standard mRNA (cDNA) with sample mRNA (cDNA) under conditions in which these mRNAs amplify with equal efficiency; thus, it avoids errors resulting from saturation of competition effects. Finally, the technique is sensitive to lower than 1.5-fold differences in input mRNA. To apply the technique, we also describe methods for the generation of poly(A) mRNA internal standards that can be used to quantitate TCR-V beta 2/6/7 and TCR-C beta mRNA.

journal_name

DNA Cell Biol

journal_title

DNA and cell biology

authors

Duchmann R,Strober W,James SP

doi

10.1089/dna.1993.12.217

subject

Has Abstract

pub_date

1993-04-01 00:00:00

pages

217-25

issue

3

eissn

1044-5498

issn

1557-7430

journal_volume

12

pub_type

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