Optimization of the method of RNA isolation from paraffin blocks to assess gene expression in breast cancer

Abstract

Molecular oncology increasingly needs the assessment of tumor gene expression profile (transcriptome), most commonly by determination of RNA-based molecular markers employing the technique of quantitative real-time polymerase chain reaction (Q-PCR). However, as all are methods based on RNA, to date, the experience in Q-PCR is mostly limited to freshly collected material frozen at -80 degrees C, i.e. showing no signs of RNA degradation. The aim of the present study was to implement into practice a method of RNA isolation from formalin-fixed and paraffin-embedded (FFPE) breast carcinoma samples collected during routine surgical and histopathological procedure, to further employ it in expression analysis by Q-PCR. The RNA isolation kit RNeasy FFPE (QIAGEN) was used. It was demonstrated that in samples subjected to DNAse digestion, the mean concentration of the obtained RNA was low (46 ng/microl), while during the isolation performed using solely gDNA Eliminator columns, the authors obtained RNA with an almost fourfold higher concentration value. A comparison was made between isolation effectiveness using varying amounts of input material. It was noted that isolation efficacy was lower when three sections were employed (the concentration value of 178 ng/microl) as compared to 5-8 sections (279 and 302 ng/microl, respectively). RNA quality assessment was also performed employing the method of capillary electrophoresis by the "lab-on-a-chip" technology of Agilent Bioanalyzer 2100. Freshly prepared material yielded in single cases samples containing RNA18S and RNA28S populations, while in samples isolated from archival paraffin blocks, the obtained RNA showed more considerable degradation, thus, was of lesser quality. In the analysis of 20 samples from the second collected series, the majority of samples were characterized by the RNA Integrity Number (RIN) values in the range of 2-2.5, still indicative of a substantial degree of RNA degradation. The mean isolation effectiveness in the second series was 885 ng/microl. In 10 of 20 blocks isolated, we succeeded in obtaining sufficient RNA concentration, above 500 ng/microl. It was also noted that the storage time did not affect the amount of RNA obtained from a block: while isolating RNA from freshly prepared blocks, we achieved similar concentrations as when analyzing the archival material.

Conclusions: the key in preserving RNA quality in paraffin blocks is the timing of material collection and fixing. Routine paraffin blocks allow for obtaining RNA for molecular studies, yet with features of considerable degradation.