Molecular analysis of different classes of RNA molecules from formalin-fixed paraffin-embedded autoptic tissues: a pilot study

Abstract

For a long time, it has been thought that fresh and frozen tissues are the only possible source of biological material useful to extract nucleic acids suitable for downstream molecular analysis. Recently, for forensic purpose such as personal identification, also fixed tissues have been used to recover DNA molecules, whereas RNA extracted from such material is still considered too degraded for gene expression studies. In the present pilot study, we evaluated the possibility to use forensic formalin-fixed paraffin-embedded (FFPE) samples, collected at autopsy at different postmortem intervals (PMI) from four individuals, to perform advanced molecular analyses. In particular, we performed qualitative and quantitative analyses of total RNAs extracted from different FFPE tissues and put expression profiles in relation with the organ type and the duration of PMI. Different classes of RNA molecular targets were studied by real-time quantitative RT-PCR. We report molecular evidence that small RNAs are the only RNA molecules still detectable in all the FFPE autoptic tissues. In particular, microRNAs (miRNAs) represent a consistent, stable, and well-preserved molecular target detectable even from tissue sources displaying signs of ongoing putrefaction at autopsy. In this pilot study, we show that miRNAs could represent a highly sensitive and potentially useful forensic marker. Amplification of specific miRNAs using paraffin-embedded blocks could facilitate retrospective molecular analysis using specific forensic-archived tissues chosen as most suitable according to PMI, and this approach would address molecular evidence in forensic cases in which fresh or frozen material is no longer available.

Reliability of miRNA Analysis from Fixed and Paraffin-Embedded Tissues

“…In particular, this trend is even more evident for miR-30d-5p because of its lower expression level in ovarian cancers. The highest fragmentation of the RNAs in Bouin’s fixative is in line with the fixative composition, which has a 9–10% concentration of formaldehyde, 5% glacial acetic acid, and 0.9% of picric acid [19], and it is in agreement also with results reported by other authors [3,4,20,21,22]. Only Gloghini and colleagues have demonstrated the detection of 921 base stretches by RT-PCR from both formalin- and Bouin’s-fixed tissues, but the sample fixation time was reduced to 5 hours [23].…”

“…This fixation method has been used for specific purposes because it better preserves some morphological details, such as nuclear conformation [3]. In the past decades, several studies have already shown that Bouin’s-fixed samples are amenable for nucleic acid and protein analyses, but only to a certain point, as the fixative has resulted to be more detrimental compared to 10% buffered formalin mixture in the preservation of biomolecules [1,3,4]. Currently, no investigation has been carried out on the analysis performance with respect to miRNAs, despite the publication of two samples in a report [4].…”

Expanding the Utilization of Formalin-Fixed, Paraffin-Embedded Archives: Feasibility of miR-Seq for Disease Exploration and Biomarker Development from Biopsies with Clear Cell Renal Cell Carcinoma

“…These archives remain an underused resource, as NGS of FFPE biopsies was previously thought to yield results of insufficient quality. However, results comparable to those of FF samples have been obtained, even using highly degraded FFPE samples [ 28 , 29 , 30 , 31 ] Several investigations of the miRNA profile of ccRCC have been performed [ 7 , 32 ], as have studies on FFPE specimens [ 14 , 33 ]. However, to the best of our knowledge only the work of Weng et al has investigated the miRNA profile of ccRCC with NGS of samples derived from FFPE [ 28 ] Although novel, the work of Weng et al included only three cases of ccRCC and their findings, therefore, require further investigation and validation.…”

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process

“…However, human postmortem tissue shows a high degree of biological variance [ 23 , 24 ] which may influence the results obtained by quantitative gene expression analysis. Indeed, it is well known that both antemortem (fever, hypoxia-ischemia, and acidosis) and postmortem ( postmortem interval—PMI, brain or cerebrospinal fluid pH changes) factors can influence the production of gene transcripts [ 25 – 29 ].…”