Quantitative measurement of human T-cell receptor V beta subfamilies by reverse transcription-polymerase chain reaction using synthetic internal mRNA standards.
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.
Duchmann
R
R
Mucosal Immunity Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
Strober
W
W
James
S P
SP
D000942:Antigens, Bacterial / Q000276:immunology; D001427:Bacterial Toxins; D001483:Base Sequence; D004768:Enterotoxins / Q000276:immunology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D011061:Poly A / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000138:chemical synthesis; D012194:RNA-Directed DNA Polymerase; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D018089:Superantigens; D013601:T-Lymphocytes / Q000276:immunology
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004768:Enterotoxins; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta; D018089:Superantigens; C030107:enterotoxin F, Staphylococcal; D011061:Poly A; D012194:RNA-Directed DNA Polymerase