上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2018, Vol. 38 ›› Issue (4): 400-.doi: 10.3969/j.issn.1674-8115.2018.04.008

• 论著·基础研究 • 上一篇    下一篇

人体脑组织福尔马林固定石蜡包埋样本中长链非编码RNA的提取及定量分析方法

吕叶辉 1, 2, 3，李志宏 1，刘丽 1，黎世莹 2，李堃 1，杨智昉 1，陈忆九 2, 3   

  1. 1. 上海健康医学院基础医学院，上海201318；2. 司法鉴定科学研究院上海市法医学重点实验室，上海200063；3. 四川大学华西基础医学与法医学院，成都610065
  • 出版日期:2018-04-28 发布日期:2018-05-03
  • 通讯作者: 杨智 ，电子信箱：yangzf@sumhs.edu.cn；陈忆九，电子信箱：chenyj@ssfjd.cn。为共同通信作者。
  • 作者简介:吕叶辉（1989—），男，讲师，博士生；电子信箱：yeyeliushu@163.com。
  • 基金资助:
    上海健康医学院种子基金项目

Methodological research of RNA extraction and quantitative analysis of long non-coding RNA formalin-fixed paraffin-embedded human brain specimens

Lü Ye-hui1, 2, 3, LI Zhi-hong1, LIU Li1, LI Shi-ying1, LI Kun1, YANG Zhi-fang1, CHEN Yi-jiu2, 3   

  1. 1. School of Basic Medical Sciences, Shanghai University of Medicine & Health Science, Shanghai 201318, China; 2. Shanghai Key Laboratory of Forensic Medicine, Academy of Forensic Science, Shanghai 200063, China; 3. West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610065, China
  • Online:2018-04-28 Published:2018-05-03
  • Supported by:
    Seed Fund Project of Shanghai University of Medicine & Health Science

PDF (PC)

1093

摘要： 目的·比较脑组织新鲜样本和福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded，FFPE）样本中RNA的质量，并分析长链非编码RNA（long non-coding RNA，lncRNA）的表达情况。方法·收集不同保存条件下的FFPE及成对新鲜脑组织，提取并检测RNA质量，运用实时定量PCR（RT-qPCR）分析各RNA指标的扩增效率和表达稳定度，筛选内参指标并对比不同扩增长度候选指标的△Ct值改变情况，以明确FFPE样本中lncRNA真实表达水平。结果· FFPE样本中RNA纯度相对较高，但完整性明显低于新鲜样本。所有指标均成功扩增，长链RNA的扩增效率与产物长度、样本处理方式、保存温度及保存时间有关，而5S、miR-9及miR125b扩增效率均接近理想状态并在所有样本中稳定表达，适合作为内参指标。与新鲜样本相比，在4 ℃保存的FFPE样本中，只有HAR1F和MALAT1-L（>200 bp）的△Ct值升高；在室温保存的FFPE样本中，多数指标的△Ct值显著增加，但是短扩增（<60 bp）指标在所有样本中表达一致。结论·尽管处理方式和保存条件会影响组织RNA的完整性，但通过选择合适的扩增长度及内参指标，能够准确定量FFPE组织中lncRNA的表达水平。

关键词: 脑组织, 福尔马林固定石蜡包埋, RNA, 质量, 扩增效率, 内参指标, 长链非编码RNA

Abstract:

Objective · To compare the quality of RNA extracted fresh and formalin-fixed paraffin-embedded (FFPE) brain tissues and to explore the long non-coding RNA (lncRNA) 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 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 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-125bachieved optimal AE and showed quite s 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 s in all brain specimens. Conclusion · RNA integrity is affectedsample treatment and preservation conditions, but lncRNA levels in FFPE tissues can be accurately quantificatedusing optimal amplicon sizes and considerable reference markers.

Key words: brain tissue, formalin-fixed paraffin-embedded, RNA, quality, amplification efficiency, internal reference marker, long non-coding RNA

中图分类号: