Synthesis of RNA and DNA Probes
A rat PAI-I cDNA cloned into a pBluescript vector and a 400-basepair (bp) £coRI fragment from rat tPA cDNA clone lambda 15 was subcloned into a pGEM-1 vector. Both vectors were linearized by HindIII and used as templates for probe synthesis using an in vitro transcription system (Promega-Biotech). A 400-bp tPA probe complementary to the 5′ region of rat tPA mRNA and a 376-bp PAI-I probe complementary to the 3′-untranslated region of PAI-I mRNA were obtained using the T7 and T3 promoters, respectively. The 32P-labeled antisense RNA probes were tested for specificity by hybridization to ovarian total RNA fractionated by formaldehyde agarose gel electrophoresis and blotted to nylon filters. The probes were found to be specific for tPA and PAI-I mRNA since they hybridized only to specific mRNA species with the size corresponding to tPA and PAI-I mRNA. A 250-nucleotide single-stranded p-actin DNA probe was prepared by primer extension as described. buy ampicillin
Preparation of Total RNA
Total RNA from granulosa cells was prepared using the NP-40 method. For hybridization analysis, total RNA was fractionated by agarose gel electrophoresis in the presence of formaldehyde and transferred to nitrocellulose filters or immobilized directly by using a slot blot filtration apparatus (Schleicher & Schuell). Dilutions of RNA from each sample were applied to nitrocellulose filters in triplicate for hybridization with PAI-I, tPA, and p-actin probes.