miRNA profiling from freshly harvested duodenums

Correlation of miRNA expression patterns with clinically relevant information is greatly facilitated by the retrospective analysis of samples archived in tissue banks. Unfortunately, the quality of samples stored in tissue banks is variable due to heterogeneity in pre-analytical preparation of clinical specimens. Collectively, these variables will impact the reliability of the results of the analysis. To date no systematic studies have been performed to investigate the relationship between total RNA degradation and miRNA profiles determined from snap-frozen collections or freshly harvested tissues. To investigate this question, we compared miRNA expression profiles generated through delaying the extraction of RNA from liver and duodenum, to generate different RNA integrities as defined by RIN values. Duodenum samples were collected from mice, sliced into five identical pieces, transfered into eppendorf tubes and either processed immediately (T0) or maintained on ice and at latter time points [30 min (T30), 60 min (T60), 120 min (T120) and 240 min (T240)]. RNA was extracted by using Trizol and RNA integrity was assessed by Bioanalyzer electropherograms. We found that duodenal samples (which are rich in RNases) do show high susceptibility to degradation. These findings suggest that tissues such as duodenum or pancreas should either be processed immediately or snap-frozen and processed individually. miRNA expression profiling by microarray shows that miRNAs from freshly harvested duodenum are extensively degraded. Based on these data, we conclude that samples with low RIN values (less than 7) do not merit analysis on miRNA arrays.对组织库存档样本开展回顾性分析，可极大助力微小RNA（miRNA）表达谱与临床相关信息的关联研究。遗憾的是，由于临床标本分析前制备流程存在异质性，组织库储存样本的质量参差不齐。综合来看，上述变量会显著影响分析结果的可靠性。截至目前，尚未有系统性研究探讨速冻保存样本或新鲜采集组织的总RNA降解情况与miRNA表达谱之间的关联。为探究这一问题，本研究通过延迟肝脏与十二指肠组织的RNA提取时长，获得了不同RNA完整性数（RIN, RNA Integrity Number）的样本，并对其miRNA表达谱进行了比较。本研究从小鼠体内采集十二指肠组织样本，将其均分为5份，转移至Eppendorf离心管中，即刻进行处理（记为T0组），或置于冰上保存并分别于后续时间点[30 min（T30）、60 min（T60）、120 min（T120）及240 min（T240）]处理。采用TRIzol试剂提取总RNA，并通过生物分析仪电泳图谱评估RNA完整性。本研究发现，富含核糖核酸酶（RNase）的十二指肠组织样本确实极易发生降解。上述结果表明，十二指肠或胰腺等组织应即刻进行处理，或经速冻保存后单独开展后续处理。基因芯片（microarray）介导的miRNA表达谱分析结果显示，新鲜采集的十二指肠组织中的miRNA发生了广泛降解。基于上述数据，本研究得出结论：RNA完整性数低于7的样本不适合开展miRNA芯片分析。