Against All Odds: RNA Extraction From Different Protocols Adapted to Formalin-fixed Paraffin-embedded Tissue

Abstract

For the preservation of tissue samples, formalin fixation followed by paraffin embedding (FFPE) has been the method of choice for decades, mainly because it maintains the morphologic characteristics of the original tissue particularly preserved, as well as its genetic material. FFPE cells can be used to perform molecular tests, such as conventional (c) or quantitative (q) reverse transcriptase polymerase chain reaction (RT-PCR), in retrospective investigations. However, extracting RNA from archived FFPE tissues is a challenging procedure, as it requires time and the use of complex extraction methods. As specific FFPE extraction methods are not always available in the laboratories, the objective of this study was to evaluate the performance of a method based on phenol-chloroform (PC) and 2 commercial methods for RNA extraction, adapting their protocols for FFPE tissues. For this study, a pool of FFPE tissues underwent RNA extraction by PC, QIAmp Viral RNA Mini, and RNeasy Mini Kit. Both the RT-cPCR and the RT-qPCR results were favorable, demonstrating the viability of the RNA. As these results expanded the alternatives for low-budget FFPE extraction, the choice of the ideal method to be used will depend on the availability of reagents and kits.

Differential expression of extracellular matrix‑related genes DCN, EPHA4, FN1, SPARC, SPON2 and SPP1 in colorectal carcinogenesis

“…RNA fragmentation in FFPE tissue samples has a size endpoint of ~80 nt and formalin modifications are partially reversed during isolation (56,57). Recent publications described that analysis of mRNAs from FFPE is reliable when performed appropriately (58,59). In the present study, only the samples that successfully passed initial quality control were further analysed, thus limiting the number of included samples.…”

Surveillance of hemorrhagic fever and/or neuroinvasive disease: challenges of diagnosis

“…This is consistent with prior studies reporting that conventional laboratory assays failed to detect a causative agent in approximately 40% of gastroenteritis 27 cases and 60% of encephalitis 28 cases. Part of our findings could be attributed to technical issues related to sample fixation [20][21] , but we also highlight the importance of better information availability when making the initial diagnosis, and having these data available to the reference laboratories (via online laboratory management systems). Lack of clinical data may limit implementing additional laboratory approaches.…”

“…Although the qPCR assay is able to provide information on the presence of infectious agents at the molecular level, being a very high specific analysis 18 , it is performed only on fresh frozen tissue in public surveillance laboratories. Despite its technical limitations 19,20 , FFPE analysis provides valuable support to immunohistochemical 21,22 and anatomopathological evidence. Cytological architecture is preserved and the samples can be used to perform molecular tests, such as conventional 23 or quantitative reverse transcriptase PCR 19,20 .…”

“…Despite its technical limitations 19,20 , FFPE analysis provides valuable support to immunohistochemical 21,22 and anatomopathological evidence. Cytological architecture is preserved and the samples can be used to perform molecular tests, such as conventional 23 or quantitative reverse transcriptase PCR 19,20 . It is important to consider, however, that although immunohistochemistry may reveal the presence of viral antigens within the tissue sample, its specificity may be limited due to the cross-reactivity observed in commercially available antibodies 20,[24][25][26] This reinforces the need to use molecular methods as a complementary tool for FFPE.…”

“…Cytological architecture is preserved and the samples can be used to perform molecular tests, such as conventional 23 or quantitative reverse transcriptase PCR 19,20 . It is important to consider, however, that although immunohistochemistry may reveal the presence of viral antigens within the tissue sample, its specificity may be limited due to the cross-reactivity observed in commercially available antibodies 20,[24][25][26] This reinforces the need to use molecular methods as a complementary tool for FFPE. Despite all efforts to identify an etiological agent, almost half of the cases investigated at the Pathology Center during the study did not receive a final laboratory diagnosis, thus relying solely on clinical and epidemiological information for a tentative conclusion 1 .…”