Methodological research of RNA extraction and quantitative analysis of long non-coding RNA from formalin-fixed paraffin-embedded human brain specimens / 上海交通大学学报（医学版）

ABSTRACT

Objective: To compare the quality of RNA extracted from fresh and formalin-fixed paraffin-embedded (FFPE) brain tissues and to explore the long non-coding RNA (lncRNA) expression level. Methods: FFPE samples stored under various conditions and paired frozen brain tissues were collected and total RNA qualities were then detected. Amplification efficiency (AE) and expression stability of each RNA marker were calculated and analyzed based on real-time quantitative PCR. After selecting reference biomarkers, normalized △ Ct values of candidate makers within different amplicon size were measured to assess the possibility of lncRNA quantification in FFPE tissues. Results: The purity of RNA extracted from FFPE was relatively high, but the RNA integrity was lower than fresh samples. All biomarkers were successfully amplified and amplification efficiencies of long-chain RNA markers were correlated with amplicon sizes, sample treatment and preservation conditions, namely temperature and storage time. 5S, miR-9 and miR-125b achieved optimal AE and showed quite stable expression in all specimens, therefore they were chosen as control markers. Compared with fresh samples, the △ Ct values of only 2 lncRNA (HAR1F and MALAT1-L, whose amplicon size were both higher than 200 bp, respectively) increased in the FFPE samples kept in 4 ℃, while in FFPE tissues kept in room temperature, increments of the △ Ct values were significant for most target genes except for short amplicon markers (<60 bp), which showed consistently stable expression in all brain specimens. Conclusion: RNA integrity is affected by sample treatment and preservation conditions, but lncRNA expression levels in FFPE tissues can be accurately quantificated by using optimal amplicon sizes and considerable reference markers.