Extraction of DNA and RNA from Formalin-fixed Paraffin-embedded Tissue Specimens

Background

In recent years, next-generation sequencing (NGS) has provided seamless benefits in the fields of medical diagnostics, oncology, and precision medicine (Kerick et al., 2011). While NGS has enabled better therapeutic approaches, the identification of a particular mutation causing a disease and the course of action for treatment remedies needs substantial validation (Arreaza et al., 2016). An ideal and sensitive approach is to validate the candidate genes as biomarkers using RT-qPCR, for which a good quality starting material in the form of biomolecules (DNA/RNA) is required. However, the extraction procedure using samples from various collection and preservation methods has been a great challenge, especially formalin-fixed paraffin-embedded (FFPE) tissue blocks (Heydt et al., 2014). FFPE tissue blocks are one of the most common methods of tissue preservation since they allow preservation of cellular components and architecture (Donczo and Guttman, 2018; Groelz et al., 2018), can provide specimen quickly, and are essential in the histopathology lab for diagnosis. The disadvantage of this method is that longer exposure to formalin may cause crosslinking of proteins and nucleic acids, which can result in potential DNA/RNA damage (Williams et al., 1999; Do and Dobrovic, 2015, Atanesyan, 2017). Previously, FFPE samples were not considered a good source for molecular analysis, since nucleic acids are extensively modified in this process (Blow et al., 2007; Bass et al., 2014). Steps such as preparation, fixation, embedding, and storage can affect DNA/RNA quality, which can further influence later stage diagnostics and research (Nam et al., 2014; Turashvili et al., 2012). In the recent past, efforts have been made to isolate smaller quantities/aliquots of DNA/RNA from FFPE samples using appropriate proteinase digestion and buffers (Sah et al., 2013, Sarnecka et al., 2019). The purified biomolecules could be used for further downstream processes such as polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-qPCR), microarray, and next-generation sequencing analyses (Taga et al., 2013; Ludyga et al., 2012; Gupta et al., 2020). Although commercial kits are available for FFPE extraction, there is a need for a better protocol describing extraction procedures for biomolecules (DNA/RNA) from FFPE tissue specimens. This protocol is mainly based on the deparaffinization of FFPE blocks and proteolytic digestion with proteinase K/protease. We have further optimized this protocol for DNA and RNA extraction using a QIAamp FFPE DNA extraction kit and the TRIzol method, respectively.

Part I: Protocol for DNA isolation