Detection of human androgen receptor mRNA expression abnormalities by competitive PCR.
A competitive polymerase chain reaction (PCR) method for analysis of androgen receptor (AR) mRNA expression is described. The technique involves the use of an in vitro-transcribed RNA (cRNA) corresponding to a region of the AR mRNA transcript as a competitor in reverse transcription and PCR (RT-PCR) using total cellular RNA. The competitor RNA contains a site-directed mutation that produces a restriction fragment length polymorphism after RT-PCR and endonuclease digestion. We demonstrate that incorporation of the competitor RNA into RT-PCR reactions allows rapid semiquantitative determination of relative AR mRNA levels without the necessity of following PCR product formation kinetically; reaction products are assessed at the conclusion of the reaction sequence and without the use of radioactive probes or other specialized detection systems. We have used competitive PCR to demonstrate low levels of AR mRNA in an androgen-unresponsive human prostate cell line (PC3). In addition, we have also used this method to confirm that genital fibroblasts obtained from a subject with penoscrotal hypospadias (a non-intersex masculinization defect) that exhibit low levels of high-affinity androgen binding also exhibit abnormally low AR mRNA levels. These last results suggest that some non-intersex malformations of the urogenital tract are associated with abnormalities in the expression of the androgen receptor.
Edelstein
R A
RA
Urology Research Laboratory, Children's Hospital, Boston, MA 02115.
Carr
M C
MC
Caesar
R
R
Young
M
M
Atala
A
A
Freeman
M R
MR
D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D015536:Down-Regulation; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007021:Hypospadias / Q000235:genetics / Q000378:metabolism; D007223:Infant; D007231:Infant, Newborn; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012333:RNA, Messenger / Q000096:biosynthesis*; D011944:Receptors, Androgen / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012016:Reference Values; D012867:Skin / Q000378:metabolism; D015854:Up-Regulation
D017931:DNA Primers; D012333:RNA, Messenger; D011944:Receptors, Androgen; D004247:DNA