液相杂交检测B细胞淋巴瘤细胞系miR-28的表达_医学论文.docVIP

液相杂交检测B细胞淋巴瘤细胞系miR-28的表达_医学论文 液相杂交检测B细胞淋巴瘤细胞系miR-28的表达_医学论文 【摘要】 microRNA (miRNA)是一组在进化上高度保守、非编码蛋白、参与转录后调节的小分子RNA（19-25核苷酸）。为了进一步明确miRNA的加工机制及功能和定量研究miRNA的表达水平，应用液相杂交和Northern blot两种方法检测miR-28在B细胞淋巴瘤细胞系中的表达并进行了比较。结果表明：液相杂交和Northern blot检测miR-28均显出阳性结果，但液相杂交所用的细胞总RNA量（5 μg）明显少于Northern blot（30 μg），液相杂交的条带信号也较Northern blot的强。结论：液相杂交是一种较Northern blot更为快速、简便和敏感的检测miR-28的方法。 【关键词】 淋巴瘤 　　Expression of miR-28 in B Cell Lymphoma Cell Lines Detected by Solution Hybridization 　　Abstract microRNA (miRNA) is evolutionarily conserved，non-coding small RNA (19-25 nt) involved in post-transcriptional gene regulation.To further understand the biogenesis and functions of miRNA，and to quantify miRNA expression levels，the expression of miR-28 in B lymphoma cell lines was detected by solution hybridization and Northern blot，and their detected results were compared.The results indicated that detection of miR-28 by solution hybridization and Northern blot showed positive，but total RNA amount used for solution hybridization detection was significantly lower than that for Northern blot detection (5 μg vs 30 μg)，signal of solution hybridization was stronger than that of Northern blot.It is concluded that the solution hybridization for detection of miR-28 in B cell lymphoma cell lines is a faster and more sensitive method as compared with standard Northern blot technique. 　　Key words solution hybridization Northern blot miRNA lymphoma miR-28 　　microRNA(miRNA)是一种大小约19-25个碱基的单链小分子RNA，是由具有发夹结构的约70-90个碱基大小的单链RNA前体经过切酶（dicer）加工后生成［1］。miRNA在线虫、果蝇、小鼠和人等多个物种中被广泛发现，而且在进化上高度保守，在人类已经发现有222个 miRNA。目前对miRNA的研究不断深入，并认为这些普遍存在的小分子在真核基因表达调控中有着广泛的作用。 由于miRNA是一类很小的分子，它的表达水平可能很低，因而需要极为灵敏而定量的分析工具。由于其分子很小，用RT-PCR的方法来定量研究非常困难，目前研究人员多采用Northern blot方法来检测miRNA的存在［2］。传统的Northern blot方法是用探针检测固相支持物（膜）上的目标分子，由于用探针检测液相中的目标分子远比检测固相中的目标更为灵敏，本研究采用液相杂交（solution hybridization）的方法检测4种B细胞淋巴瘤细胞系中miR-28的表达。miR-28基因是22个碱基的单链小分子RNA，是由具有发夹结构的86个碱基的单链RNA前体经过切酶加工后生成，位于染色体3q27-28的含LIM的脂肪瘤多见融合伙伴（LIM-containing lipoma preferred partner