Gene expression analysis of formalin-fixed paraffin-embedded (FFPE) material by quantitative real-time PCR is a valuable tool for the analysis of clinical samples. Several studies demonstrated high sample-to-sample expression variability of frequently used reference genes as GAPDH and ACTB in cancer samples. Alternative genes were suggested as the reference genes for the analysis of gene expression in cancer tissue. Accurate quantification requires simultaneous use of 2-4 independent reference genes for each assay. To simplify the use of several reference genes we analyzed a possibility to perform multiplex TaqMan PCR with three selected reference genes.
Several amplicons with size range 82-92 bp were selected out of previously validated reference genes MRPL19, TBP, HPRT1 on the basis of the minimal cross-hybridization of primers, probes and amplicons. The selected amplicons provided robust multiplex PCR with minimal cross-talk between probes when HEX labeled probe was used for MRPL 19, ROX for TBP and Cy5 for HPRT1 accordingly. RNA from 73 FFPE samples of breast cancer tissue was purified and relative expression of MRPL, TBP and HPRT1 genes was evaluated in multiplex qPCR assay. Expression for ER, PR and HER2 genes was measured using FAM-labeled probe in multiplex qPCR with the selected reference genes.
Maximal sample-to-sample variation of delta Ct for MRPL19/TBP; TBP/HPRT1 and MRPL19/HPRT1 was 0,95, 1,08 and 1,07 respectively. The mean value of Ct for MRPL19, TBP, and HPRT was used as a reference for the measurement of ER, PR and HER2/neu genes expression in 73 FFPE samples. Expression of ER, PR and HER2 in the majority of samples was in a good agreement with immunohistochemical data.
The suggested multiplex qPCR gene reference set provides accurate and reliable detection of gene expression in FFPE breast cancer samples. The work was supported by Russian RSF grant 15-14-10004.
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All authors have declared no conflicts of interest.