Purified populations of PGCs were isolated as described above. RNA probes labeled with 32P were prepared and RNase protection assays performed as described previously and analyzed on 6% acrylamide-urea sequencing gels. A p-actin internal control (Ambion, Austin, TX) was included in each assay. The protected fragments used were the 277-base pair (bp) PvuII to SphI fragment of the extracellular domain of FGF receptor (FGFR)-1, the 326-bp EcoRV to EcoRI fragment in the extracellular domain of FGFR-2, the DdeI fragment in the extracellular domain of FgFr-3 , and the 387-bp EcoRV to BamHI fragment of FGFR-4. All fragments were subcloned in p-Bluescript SK or KS and transcribed with T7 or T3 polymerase (Stratagene, La Jolla, CA) as appropriate. buy cipro
Degenerate oligonucleotide analysis of FGFR expression by reverse transcription-polymerase chain reaction (RT-PCR) was performed as previously described with the following exceptions. All RNA preparations were treated with DNase I (Life Technologies) to eliminate genomic DNA contamination. Random-primed cDNA was prepared using Superscript II (Life Technologies) as suggested by the manufacturer. PCR was performed with Expand Taq (Boehringer-Mannheim, Indianapolis, IN) in the manufacturer’s supplied buffer supplemented with 10% glycerol.