Competitive reverse transcriptase-polymerase chain reaction using a synthetic internal RNA standard to quantitate transcripts for leukocyte-derived hormones.
Leukocytes synthesize a variety of hormones that were once thought to be unique products of endocrine tissues. Understanding the regulation of leukocyte-derived hormone synthesis requires an accurate means for measuring steady-state expression of specific mRNA transcripts. Here we describe a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) technique to accurately quantitate macrophage-derived insulin-like growth factor-I (IGF-I) mRNA, and demonstrate the utility of this approach for measuring expression of leukocyte-derived hormone transcripts. A riboprobe was constructed to generate approximately 1 kb of synthetic competitor IGF-I RNA (exons 1 and 3-6) that differed from cellular IGF-I RNA by insertion of 122 bp of beta-actin RNA. One set of oligonucleotide primers could thus be used to simultaneously reverse transcribe and amplify both 144 bp of cellular (exons 3 and 4) and 266 bp of competitor IGF-I RNA. Densitometric scanning of the PAGE-separated PCR products revealed that the ratio of competitor to cellular amplified DNA bore a linear relationship (r2 > or = 0.98) to the amount of competitor RNA for both rat liver and splenocytes. However, rat liver contained 104 x 10(6) IGF-I molecules per microgram of total cellular RNA compared to only 2 x 10(6) IGF-I molecules for splenocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Animal Sciences, University of Illinois, Urbana 61801, USA.
Q
Q
Liu
S
S
Arkins
A
A
Biragyn
C
C
Minshall
P
P
Parnet
R
R
Dantzer
K W
KW
Kelley
D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis*; D007962:Leukocytes / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014158:Transcription, Genetic / Q000235:genetics
D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D012194:RNA-Directed DNA Polymerase