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C.A. Suarez-Quian1 *, R. Smith2 , C. Noguchi2
Dept Cell Biol; Georgetown Uni Med Center, Washington, D,C. 1
LCB, NIDDK, Bethesda, MD 2
Development and maintenance of male reproductive tissues are dependent on normal expression levels of the AR gene, although what this level should be is not clear. Herein, we report development of a highly sensitive and reproducible assay to measure levels of AR gene transcripts in testis (T), epididymis (E), prostate (P) and seminal vesicles (SV) from adult mice. Total RNA was prepared from tissues by conventional small scale procedure, including a digestion step with RNAse-free DNAse I (Promega) to eliminate genomic DNA. First strand cDNA was synthesized using oligo dT and modified M-MLV reverse transcriptase (Life Technologies) from 1 mg of the respective tissue RNAs. cDNAs were amplified by PCR using mouse specific primers spanning the border of exons 5-6 and rendered a product of 87bp. Differences (judged by EtBr staining of product separated in agarose gels) in the amount of product produced between the four tissues examined, however, could not be discerned. The Q-PCR step was performed in an ABI 7700 Sequence Detection System using Sybr green I (Molecular Probes) as the reporter. The threshold cycle (Ct) for each tissue was plotted against a standard curve generated from a mouse AR containing plasmid (M Danielson, GU) that was similarly processed. A no substrate reaction served as control. AR expression levels, expressed as amol/mg total RNA in the different tissues were as follows: T = 0.29 ± 0.003; E = 25.00 ± 0.097; P = 7.32 ± 0.028; and SV = 06.21 ± 0.046. This approximate 3.5 fold greater level of AR transcripts present in E versus P and SV was unexpected, given the prior results of AR immunoperoxidase IHC. In conclusion, we demonstrate the application of using real time, Q-PCR to accurately determine AR gene expression in distinct reproductive tissues. Work was funded in part by NIH grant HD 42384 to CASQ
This abstract is being presented on Tuesday, August 3 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.