高通量测序分析miRNAs在胃癌新鲜组织与石蜡包埋组织中的表达

High throughput sequencing analysis of miRNAs expression in fresh gastric cancer tissues and paraffin embedded tissues

目的利用二代测序技术对FFPE标本进行高通量测序及相关的生物信息学分析,探讨miRNAs在来源于石蜡包埋胃癌组织(FFPE)中的标本检测的可靠性,探讨其临床意义。方法选取2015年和2016年2例胃癌患者手术切除同一组织的FFPE和新鲜冰冻标本,从标本中提取RNA并分离构建small RNA文库,采用Illumina测序平台测序的方法对其进行高通量测序,统计各已知miRNAs家族序列的表达量,构建已知miRNAs表达谱,对标本miRNAs进行Spearman相关性分析,评估miRNAs表达量的差异变化情况。结果新鲜冰冻标本中提取的总RNA质量较高,RNA保存度完整,显示28S是18S的1.3~1.4倍,从石蜡组中提取的RNA,结果显示有不同程度的降解,28S和18S的波峰不明显,miRNAs表达谱在冰冻和FFPE标本中,Spearman相关系数分别为0.90和0.86,两者miRNAs表达量较一致,具有相关性。结论 FFPE中的miRNAs仍然保持一定的完整性,且可通过二代测序进行肿瘤标本检测。

Objective To analyze formalin-fixed and paraffin-embedded（FFPE）sample by next generation sequencing（NGS）technology for high throughput DNA sequencing and related bioinformatics analysis,and to explore the reliability and clinic significance of miRNAs in the detection of sample from FFPE gastric cancer tissue.Methods FFPE and fresh frozen samples of the same tissue were excised from 2 cases of gastric cancer in 2015 and 2016.RNA was extracted from the samples and the small RNA library was isolated and constructed,and high throughput DNA sequencing was conducted with Illumina sequencing platform.The DNA sequencing results were analyzed by quantifying the expression of known miRNAs families sequence,and the expression profiling of known miRNAs was constructed.Spearman correlation analysis was performed on the miRNAs of the samples to assess the difference in the expression of miRNAs.Results The total RNA extracted from the fresh frozen sample had higher quality and complete RNA coverage,and the results showed that the 28 Swas 1.3-1.4 times of 18 S.The RNA extracted from the FFPE sample showed different degrees of degradation,and the peaks of 28 Sand 18 Swere not significant.The spearman correlation of miRNAs expression profiling in frozen and FFPE samples was 0.90 and 0.86 respectively,and the expression levels of miRNAs in two samples were consistent and had significant correlation.Conclusion miRNAs in FFPE sample can retain certain level of integrity and can be used in tumor test by NGS.