Deep Sequencing of the Small RNA Transcriptome of Normal and Malignant Human B cells Identifies Hundreds of Novel MicroRNAs: microarray analysis. Homo sapiens

A role for microRNAs has been recognized in nearly every biological system examined thus far. A complete delineation of their role must be preceded by the identification of all microRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B cell samples that comprise the spectrum of B cell differentiation and common malignant phenotypes. We identified the expression of 333 known microRNAs, which is over twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel microRNAs in normal and malignant B cells. These microRNAs were validated at a high rate (92%) using quantitative PCR and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. Here, we analyzed the expression of 100 diffuse large B-cell lymphoma (DLBCL) samples using microarrays. Overall design: Total RNA was extracted from frozen patient samples. The RNA was labeled, fragmented and hybridized to Human Gene 1.0 ST arrays.

迄今为止，几乎所有已被研究的生物系统中均已证实微小RNA（microRNAs）发挥着关键调控作用。若要完整阐明这类分子的生物学功能，必先明确任一系统中存在的全部微小RNA。本研究通过对31份涵盖B细胞分化全谱系及常见恶性表型的人类正常与恶性B细胞样本开展深度测序，成功阐明了正常与恶性B细胞的完整小RNA转录组。本研究共鉴定出333种已知微小RNA的表达，该数量较此前任一组织中已报道的已知微小RNA总数高出两倍以上。此外，本研究还在正常与恶性B细胞中鉴定出286种候选新型微小RNA。本研究通过定量PCR以92%的高验证率完成了这些微小RNA的验证，并证明其可用于区分淋巴瘤的临床相关亚型。本研究采用微阵列技术分析了100份弥漫大B细胞淋巴瘤（diffuse large B-cell lymphoma, DLBCL）样本的表达水平。
实验整体设计：总RNA提取自冻存的患者样本。对提取的RNA进行标记、片段化处理后，与人类基因1.0 ST（Human Gene 1.0 ST）阵列进行杂交。