The Importance of Reference Gene Analysis of Formalin-Fixed, Paraffin-Embedded Samples from Sarcoma Patients - An Often Underestimated Problem

Objective: Reverse transcription quantitative real-time polymerase chain reaction is efficient for quantification of gene expression, but the choice of reference genes is of paramount importance as it is essential for correct interpretation of data. This is complicated by the fact that the materials often available are routinely collected formalin-fixed, paraffin-embedded (FFPE) samples in which the mRNA is known to be highly degraded. The purpose of this study was to investigate 22 potential reference genes in sarcoma FFPE samples and to study the variation in expression level within different samples taken from the same tumor and between different histologic types. Methods: Twenty-nine patients treated for sarcoma were enrolled. The samples encompassed 82 (FFPE) specimens. Extraction of total RNA from 7-mu m FFPE sections was performed using a fully automated, bead-base RNA isolation procedure, and 22 potential reference genes were analyzed by reverse transcription quantitative real-time polymerase chain reaction. The stability of the genes was analyzed by RealTime Statminer. The intrasamples variation and the interclass correlation coefficients were calculated. The linear regression model was used to calculate the degradation of the mRNA over time. Results: The quality of RNA was sufficient for analysis in 84% of the samples. Recommended reference genes differed with histologic types. However, PPIA, SF3A1, and MRPL19 were stably expressed regardless of the histologic type included. The variation in Cq value for samples from the same patients was similar to the variation between patients. It was possible to compensate for the time-dependent degradation of the mRNA when normalization was made using the selected reference genes. Conclusion: PPIA, SF3A1, and MRPL19 are suitable reference genes for normalization in gene expression studies of FFPE samples from sarcoma regardless of the histology.