The reverse transcription linked polymerase chain reaction (RT-PCR) is a powerful technique for detecting mRNAs of low abundance while enabling distinction between homologous mRNAs such as family members and between alternative splice variants. We utilized this technique for quantitative analysis of expression of nine fibroblast growth factor (FGF) and four FGF receptor (FGFR) family genes in mouse brain during development and adulthood. The primer sets and reaction conditions for each family member were optimized for efficient amplification, and the amplified products were detected by hybridization with specific probes to ensure specificity. To achieve quantitative measurement, serial concentrations of the cloned cDNAs were simultaneously amplified and the results were used to titrate the amount of mRNA in the samples. Since FGF family has been recently recognized to be important in various functions of central nervous system and the protocol described here is directly applicable for a variety of small tissue samples, this protocol is very helpful in understanding the involvement of FGF family in various physiological phenomena.
Animals Brain