Gonadotropin-Independent Regulation of Steroidogenesis: MATERIALS AND METHODS(6)

7 Nov
2012

MATERIALS AND METHODS(6)RNA Extraction

Total RNA was isolated from the fetal pituitaries of animals aged E16.5 and older by the CsCl/guanidium isothio-cyanate method as described previously . Because of difficulties in identifying the fetal pituitary at E15.5, total RNA was isolated from the whole fetal head at this age. buy levaquin online

Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)

For detection of the LH|3-subunit mRNA, the RT and PCR reactions were performed sequentially in the same tube . Two micrograms of total RNA were reverse-transcribed by using a AMV-reverse transcriptase (Promega, Madison, WI) and oligonucleotide (oligo) pi as primer, corresponding to nucleotides 400 to 381 (5′-GTCACAGGTCAT TGGTTGAG-3′) of the rat LH0 cDNA sequence. The reverse-transcribed cDNA then was amplified using PCR oligo (32 corresponding to the rat LH(3 cDNA sequence of 154 to 173 (5′ -CTTCACC ACC AGC ATCTGTG-3′) at the 5′-end, and oligo pi. Fifty microliters of the RT-PCR mixture contained 1 nM of each oligo primer, 200 |xM deoxy (d)NTPs, 1.5 mM MgCl2, 20 U RNasin (Promega), 12.5 U AMV-reverse transcriptase, and 2.5 U Taq-DNA polymerase (Promega). The reaction was started at 50°C for 10 min (RT), followed by a period of 3 min at 97°C, and then run for 32 PCR cycles (96°C for 1 min, 57°C for 1 min, 72°C for 2 min, and final extension for 10 min at 72°C). To exclude potential genomic DNA contamination of the PCR reactions or cross-contamination of pituitary RNA in the other RT-PCR reactions, samples were run on PCR by omitting the reverse transcription step or by using RNA from liver and water as control samples. Each of these control samples yielded negative reactions.

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