miRNA profiling from freshly harvested duodenums. Mus musculus

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. Overall design: 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.

对存档于组织库的样本开展回顾性分析，可极大助力微小RNA（microRNA, miRNA）表达谱与临床相关信息之间的关联研究。然而，由于临床样本前处理流程存在异质性，组织库储存样本的质量参差不齐。综上，上述变量会对分析结果的可靠性产生不利影响。截至目前，尚未有系统性研究探讨总RNA降解与经速冻保存样本或新鲜采集组织所得的miRNA表达谱之间的关联。

为解答这一科学问题，本研究通过延迟肝脏与十二指肠组织的RNA提取时长，获得了以RNA完整性数值（RNA Integrity Number, RIN）定义的不同RNA完整性水平的样本，并对其miRNA表达谱进行了比较。
实验整体设计：从小鼠体内采集十二指肠组织样本，将其均切为5份，转移至艾本德（Eppendorf）离心管后，要么立即进行处理（记为T0组），要么置于冰上静置，分别于后续时间点[30分钟（T30）、60分钟（T60）、120分钟（T120）及240分钟（T240）]进行处理。采用Trizol试剂提取RNA，并通过生物分析仪（Bioanalyzer）电泳图谱评估RNA完整性。

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，十二指肠或胰腺这类组织应立即进行处理，或经速冻保存后单独开展后续实验。通过微阵列进行的miRNA表达谱分析显示，新鲜采集的十二指肠组织中的miRNA已发生广泛降解。基于上述实验数据，本研究得出结论：RNA完整性数值低于7的低质量样本，不适用于miRNA微阵列分析。