Quality Assessment And Utilization Of Nucleic Acids Extracted From Immunostained Formalin Fixed Paraffin Embedded (FFPE) Slides In Molecular Cancer Biology
The conventional Formalin Fixed Paraffin Embedded (FFPE) technique is the gold standard for preserving histomorphology of the tissue, while cryopreservation is the gold standard for biomolecules’ preservation. Once the tissue is stained, the slides are routinely archived along with FFPE blocks for a long time in biobanks/hospitals. However, and despite the huge need for fully annotated biospecimens, the use of already-fixed and stained biospecimens to extract DNA and RNA is not a common practice worldwide. This study focuses on the feasibility of extracting DNA and RNA from immunohistochemistry (IHC) stained slides to assess its quality and usefulness for further downstream molecular analysis such as methylation, gene expression, and miRNA validation at the genetic level. With the shortage in high-quality and fully annotated biospecimens, we believe that this technique, once optimized, will improve our best practices associated with the use of biospecimens in biorepositories and medical research. FFPE IHC slides from colorectal carcinoma (CRC) consented patients were made and stored in the Centre of Excellence in Genomic Medicine Research Biobank, King Abdulaziz University, Saudi Arabia. The study workflow consisted successively of removing the slide cover-slip by immersion in Acetone, Benzene, Tetrahydrofuran (THF) with different temperature (room temp. vs. 37˚C) and immersion for 5 seconds in liquid Nitrogen (LN). Only pipelines with removal time less than 24 hours were selected for the study. After cover slip removal, a soft crude dissection of the IHC-stained tissue was performed followed by DNA and RNA extraction using the QIAamp DNA/RNA FFPE tissue kit according to manufacturer's instructions. After DNA and RNA quality assessment, a number ofdownstream molecular analyses was performedto validate the quality of the extracted DNA and RNA.A high-quality DNA and RNA was obtained that allowed successful downstream molecular analysis of CRC. Reused DNA was utilized to evaluate both themethylation profile of HIC1gene as well as the mutational status of the selected proto-oncogene KRAS andCTNNB1. For RNA, it was used in microRNA (miRNA) expression of miR-124a since it has been previously shown to be implicated to function as a tumor suppressor. This study suggests a promising technique to reuse DNA and RNA from IHC stained slides to harvest suitable DNA and RNA concentration with good integrity as starting biomolecules for further downstream molecular applications for diagnostic and/or research purposes.