Each target sequence was assayed in a separate set of PCR reactions consisting of standards (n = 5) and cDNA samples (n = 6-8 per group) assayed in triplicate. Hot-start PCR was performed using 0.4 ^M of each primer, 0.2 mM deoxyribonucleotides, 0.7 ^Ci [32P]deoxycytidine triphosphate (3000 Ci/mmol), 2.5 U AmpliTaq Gold, 3% dimeth-ylsulfoxide, 2.5 mM MgCl2 (LIF reactions) or 2 mM MgCl2 (cyclophilin reactions), and 10 ^l cDNA in 25 ^l AmpliTaq Gold PCR buffer. The PCR profile consisted of an initial incubation (11 min, 96°C) to denature the cDNA and activate the polymerase; 34-40 cycles of denaturing (3 min, 94°C), annealing (20 sec; 60°C for LIF, 55°C for cyclophilin), and extending (20 sec plus 2 sec/cycle, 72°C) the products; and a final extension (6 min, 72°C). PCR products were separated on a 5% native polyacrylamide gel, dried, and quantified using a GS-525 Molecular Imager (Bio-Rad Laboratories, Hercules, CA). After comparison of sample signal intensity to its respective standard curve, LIF expression was normalized to that of cyclophilin. ventolin 100 mcg
Southern Blot Hybridization
PCR products were separated by electrophoresis on 1.2% agarose gels and transferred to Nytran Plus membranes by downward alkaline blotting. Random-primer labeling of gene-specific subclones was performed using the RadPrime DNA Labeling System (Gibco BRL, Gaithersburg, MD) and 50 ^Ci [32P]dCTP Hybridizations were performed in double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), 0.1% SDS, with 109 dpm/^g of probe at 65°C for 16 h. Membranes were washed under high stringency (0.1-strength SSC, 0.1% SDS, 55°C) and analyzed with a GS-525 Molecular Imager.