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Role of Fibroblast Growth Factors: MATERIALS AND METHODS(4)

MATERIALS AND METHODS(4)

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.