Alternative splice variant mRNA expression
Expression of ASV mRNA was analyzed using RT-PCR and quantitative RT-PCR. Human normal RNA and tumor RNA (Total RNA Master Panel II) was purchased from Clontech and was DNase treated. First-strand cDNA was synthesized using the ABI High Fidelity Kit (Applied Biosystems, Foster City, CA, USA). For PCR amplification, the primers were designed using Oligo 6 (Molecular Biology Insights, Inc., Cascade, CO, USA). The condition for PCR amplification of FGFR4 and FGFR4-ASV was 30 cycles of 95°C for 45 seconds, 60°C for 50 seconds, and 72°C for 1 minute. The reaction was terminated with an elongation step of 72°C for 10 minutes.
For quantitative RT-PCR, gene-specific primers and probes were designed and assayed for specificity and efficiency using a human universal RNA sample. Quantitative RT-PCR was performed using an ABI 7900 HT sequence detection system (Applied Biosystems, Foster City, CA, USA) and TaqMan® chemistries. cDNA was amplified in triplicate wells for both the normal and variant gene on the same plate. Cycle threshold values were determined and the average cycle threshold values were calculated and analyzed using The Institute for Genomic Research, TIGR Multiexperiment Viewer hierarchical clustering module [45].