Evaluation of RNA quality from formalin fixed and paraffin embedded samples: Applications and limitations.

摘要

RNA molecules isolated from FFPE samples are highly fragmented and modified, and generally deemed unsuitable for downstream gene expression profiling. With the development of molecular biology, there has been growing interest in profiling archival FFPE samples. Successful profiling of transcripts from FFPE samples would greatly expand tissue sources for large scale gene expression studies; also it would pave the way for future applications on the type of tissue readily available in the clinical setting. So far, there is a lack of systemic studies evaluating the quality of RNA isolated from routinely processed FFPE samples, and it has remained difficult to assess how well FFPE-derived RNA mirrors the status of RNA isolated before fixation. In this project, the similarity of miRNA and mRNA profiles between matched frozen and FFPE lymphoid hyperplasia tissues (N=7 for miRNA comparison, N=4 for mRNA comparison) were evaluated. We found consistently good correlation (mean of Pearson coefficient=0.939, mean of Spearman coefficient=0.905, mean of Kendall tau=0.744) between matched frozen and FFPE-derived miRNA profiles, suggesting FFPE samples may retain miRNA expression information quite well. This has major positive implications for research using FFPE samples, as miRNA profiling becomes more prominent in bioprofiling studies. On the contrary, mRNA isolated from FFPE samples showed less correlation (Spearman coefficient less than 0.75) with its frozen counterpart on the Agilent microarray platform. With a post extraction heat treatment aimed at reversing base modifications and cross linking structures, obvious global mRNA quality improvement was observed in cases where samples appeared to be heavily cross linked, but was less effective and even detrimental in cases where cross linking was less prominent. This research suggests that the extent of cross linking may be critical in terms of determining whether a particular FFPE tissue will become a useful source of mRNA for global profiling studies.