In addition to progesterone concentrations, PGFM was also measured in all plasma samples collected during the experiment. The samples were assayed by Professor Hans Kindahl, Swedish University of Agricultural Sciences, according to the method described by Granstram and Kindahl. The limit of detection was 8-10 pg/ml for 0.5 ml of plasma. buy flovent inhaler
Extraction of mRNA and RT-PCR
RNA was isolated from CL using the guanidium thio-cyanate method. Concentrations were estimated by absorbance at 260 nm, and the samples were stored in diethyl pyrocarbonate-treated H2O at -80°C (1-^g aliquots) until required. The A260/A280 ratio for all RNA samples was > 1.6. First strand cDNA synthesis was carried out using a modification of the method described previously. Briefly, total RNA (1 ^g) was reverse transcribed in RTase buffer (Tris-HCl [250 mM, pH 8.3], KCl [375 mM], MgCl2 [15 mM]; Gibco BRL, Life Technologies, Paisley, Scotland, UK), dNTP mix (0.5 ^M; Pharmacia Biotech, UK), random hexamers (50 ^M; Pharmacia Biotech), RNasin (4 U; Pro-mega UK Ltd., Southampton, UK), and Superscript II reverse transcriptase (13.5 U; Gibco BRL) at 37°C for 60 min. The reverse transcriptase reaction medium was diluted to 100 ^l after the addition of 8 ^l 10-strength PCR buffer and stored at – 20°C. The PCR reaction was carried out using 6 ^l of the diluted reverse transcriptase reaction (equivalent to 0.06 ^g of the original total RNA) in 12 ^l dH2O, 1 ^l 10-strength PCR buffer, 100-200 pmol each of 5′ and 3′ primers, and 1 U Taq DNA polymerase (Gibco BRL) in a total volume of 20 ^l.