Quantitation of messenger RNA by competitive RT-PCR: a simplified read out assay.
A competitive RT-PCR method that permits reliable quantification of minute amounts of reverse-transcribed mouse lymph node mRNA is described. Using this technique, an absolute number of cDNA copies ranging from 10(3) to 10(5) can be determined, with a precision superior to 25%. The standard templates described in the present study permit the quantitation of beta-actin, IFN gamma, IL2, IL3, IL4, IL10, IL12 (p40 subunit), TGF beta 1, inducible nitric oxide synthase, ELAM-1, VCAM-1, and ICAM-1 mouse mRNA. The expression of a particular transcript is normalized to an arbitrary number of actin transcripts. The standard templates and wild-type cDNA have nearly identical sequences, but they can be distinguished by unique restriction sites. Known amounts of these standard templates, are co-amplified with serial dilutions of the cDNA derived from the mRNA of interest. Oligonucleotide primer pairs possessing 3' octamers found infrequently in the mouse genome (< or = 0.26 x 10(-6)) are used to amplify sequences, chosen to contain no GC stretches longer than 8 (PCRare software) (Griffais et al., 1991). Samples of each PCR product are digested separately with restriction endonucleases unique either for the wild-type or the standard amplicon. The quantitation of the test product and the standard product is easily carried out following their electrophoresis in an ethidium bromide-stained agarose gel.
Colle
J H
JH
Unité d'Immunophysiologie Cellulaire, Institut Pasteur, Paris, France. jhcolle@pasteur.fr
Falanga
P B
PB
Singer
M
M
Hevin
B
B
Milon
G
G
D000199:Actins / Q000235:genetics; D000818:Animals; D004247:DNA / Q000032:analysis; D007371:Interferon-gamma / Q000235:genetics; D015847:Interleukin-4 / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*
D000199:Actins; D012333:RNA, Messenger; D015847:Interleukin-4; D007371:Interferon-gamma; D004247:DNA