Wistar rat (60–90 day old) tissues were obtained commercially (Zivic Laboratories Inc, Pittsburgh, PA, USA). Tissues were placed in RNA
Later (Ambion, Inc Austin TX, USA) solution overnight at 4°C to allow penetration and fixation. The tissues were shipped on dry ice. Upon arrival, tissues were immediately stored at -70°C. Each tissue was homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and total RNA was extracted from the following tissues: caput, corpus, cauda, testis, seminal vesicle, prostate, spleen, heart, lung, liver and kidney from a single adult male and ovary, uterus, mammary gland and cervix from a single adult female. Total RNA (2 μg) was reverse transcribed using 50 U Stratascript (Stratagene, La Jolla, CA, USA) and 0.5 μg of oligodT (Invitrogen) according to the manufacturer's instructions. 2 μl of the resultant cDNA was amplified by PCR using gene specific primers (Table
1). The number of cycles to amplify each cDNA in the linear range was determined by preliminary PCR under the following conditions: 94°C for 1 min followed by 25–35 cycles at 94°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, and with a final round of extension at 72°C for 10 min.
Defb21-36 were amplified for 32 cycles and
Gapdh for 28 cycles. PCR amplified gene products were analyzed by electrophoresis on 2 % agarose gels. Identity of major amplicons was determined by sequencing at the UNC-CH Genome Analysis Facility using ABI PRISM model 377 DNA sequencer (PE Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (
Gapdh) expression was used as the internal control. To study the androgen regulation of
Defb transcripts, epididymides from sham operated, castrated and testosterone supplemented Sprague-Dawley rats (n = 5 in each group) were obtained. Testosterone supplementation was supplied by a 20 mg dihydrotestosterone pellet implanted subcutaneously immediately after castration. All the animals were sacrificed 14 days after castration. Epididymides were stabilized in RNA
Later solution and stored at -70°C till further use. All procedures were performed in accordance with the Guiding Principles in the Care and Use of Animals established by the National Institute of Health and approved by the Institutional Committee on the use of Animals in Research and Education. For studies on the developmental regulation of defensins, epididymides from 10–60 day old Wistar rats, one rat for each age, were obtained commercially (Zivic Laboratories).
Table 1
Gene specific primer sequences for rat β-defensins
Defb21
|
Forward – 5' ATA CCT GGA TCT ACT GTC CTA CCT 3'
Reverse – 5' TTA TGT GTC CAT CCG TGA AGT C 3'
|
Defb24
| Forward – 5' GTC ATC ACC TTC ACC CCG GGA 3' Reverse – 5' CAG CTT CTC TGG AAG TCT GTG CAT 3' |
Defb27
| Forward – 5' CAC GAG GAA CAC CCT GGA TTT CC 3' Reverse – 5' TGC CTA GGT CC ACCT TCG TTT CTG 3' |
Defb30
| Forward – 5' GAG TGA CTT TCC TTT CCT CAG 3' Reverse – 5' TCA GAA TTC CCA GAG GAA CCC TGG A 3' |
Defb36
| Forward – 5' TTG GGC CTT CTC CCA CCA TGA AGC 3' Reverse – 5' TGC ATC GTC TGG GCT TCC GGC TT 3' |