Quantification of androgen receptor mRNA in tissues by competitive co-amplification of a template in reverse transcription-polymerase chain reaction.
We describe a polymerase chain reaction (PCR)-based method for the quantification of androgen receptor (AR) mRNA in tissues. The amount of PCR products depends on the exponential amplification of the initial cDNA copy number; therefore minor differences in the efficiency of amplification may dramatically influence the final product yield. To overcome these tube-to-tube differences in reaction efficiency, an internal control AR cRNA was reverse transcribed along with the target mRNA using the same primers. This standard was obtained by deleting a 38 bp fragment from an amplified bovine AR sequence, which was then subcloned and transcribed into cRNA. Known dilutions of the competitor cRNA were spiked into a series of RT-PCR reaction tubes containing equal amounts of the target mRNA. Following RT-PCR, the co-amplified specimens obtained were separated by gel electrophoresis and quantified by densitometric analysis of ethidium bromide stain. We applied this method to quantify the AR-mRNA in skeletal muscle of castrated as well as from intact male cattle. The applicability of the quantification system for AR-mRNA described herein was demonstrated for other species, e.g. man.
Malucelli
A
A
Institut für Physiologie, Forschungszentrum für Milch und LebensmittelWeihenstephan, Technische Universität München, Freising, Germany.
Sauerwein
H
H
Pfaffl
M W
MW
Meyer
H H
HH
D000728:Androgens / Q000378:metabolism; D000818:Animals; D002417:Cattle; D017931:DNA Primers; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D011944:Receptors, Androgen / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D000728:Androgens; D017931:DNA Primers; D012333:RNA, Messenger; D011944:Receptors, Androgen