Quantitative analysis of lncRNA from human FF and FFPE brain speimens

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Both Fresh frozen (FF) and formalin‐fixed paraffin‐embedded (FFPE) brain tissues are important resources for molecular medical study. However, long non‐coding RNA (lncRNA) analysis is restricted in FFPE samples due to its high‐level degradation. To compare the quality of lncRNA extracted from FF and FFPE brain specimens and to explore the possibility of lncRNA quantification in FFPE tissues, 10 paired of frozen and FFPE human brain tissues were collected from autopsies by our collaborating teams at the Institute of Criminal Scientific Technology in Shanghai (). We selected 2 endogenous control mRNA (ACTB and GAPDH) and 6 target lncRNA (MALAT1, H19, PTENP1, BC1, HAR1 and DD3) from literatures, and designed 2 short (<100 bp, non‐overlapping) and 1 long (about 200 bp) primers for each biomarker. After total RNA was extracted from all specimens, the expression level of each biomarker was analyzed by real‐time quantitative PCR (RT‐qPCR) and amplification efficiency (AE) was subsequently calculated. The degradation profiles of total RNA were evaluated by RNA integrity number (RIN), and RIN values of FFPE samples were significantly lower than FF samples (P<0.05). Following RT‐qPCR, short amplicon of all 6 RNA markers achieved optimal AE between 90% and 110% in FF samples, while AE of long amplicons differed remarkably with RNA type, and only 2 lncRNA (MALAT1 and BC1) and endogenous control mRNA reached the optimal efficiency. As for FFPE tissues, most short amplicons (except for HAR1) achieved the optimum efficiencies, however, AE of most long amplicons was overblown in FFPE samples, including ACTB and GAPDH. In present study, high concordance of RNA expression was also observed in 2 non‐overlapping short amplicons of both FF and FFPE samples. In conclusion, short amplicons of target lncRNA were amplified more efficiently than long amplicons and this study may provide an experimental basis for accurate lncRNA quantitative analysis with short amplicons in FFPE brain tissues.Support or Funding InformationAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical permission was granted through the Science and Ethics Committee of Shanghai University of Medicine & Health Sciences. This study was funded by the Seed Fund of Shanghai University of Medicine & Health Sciences and Open Foundation of Shanghai Key Laboratory of Forensic Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.