An antibody-based microarray assay for small RNA detection
An antibody-based microarray assay for small RNA detection
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DOI：
10.1093/nar/gkl142
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发表时间：
2006-01-01
影响因子：
14.9
通讯作者：
Leppla, Stephen H.
中科院分区：
文献类型：
--
作者：
Hu, Zonglin;Zhang, Aixia;Leppla, Stephen H.
在微阵列上检测RNA正迅速成为分子生物学家的标准方法。然而，目前的方法经常歧视结构化和/或小RNA种类。在这里，我们提出了一种绕过这些问题的方法。将未修饰的RNA直接与DNA微阵列杂交，并使用高亲和力、核苷酸序列非依赖性、DNA/RNA杂交特异性小鼠单克隆抗体S9.6进行检测。随后与荧光标记的抗小鼠IgG抗体或生物素标记的抗小鼠IgG以及荧光标记的链霉亲和素的反应产生可以在标准微阵列扫描仪中测量的信号。基于抗体的方法能够比常用的基于cDNA的方法更有效地检测大肠杆菌的低丰度小RNA。检测到0.25 μ mol量的特异性小RNA（即在25 μ l反应中浓度为10 μ M）。该方法是一种高效、稳健且廉价的技术，允许对基因表达进行定量分析，并且不歧视短的或结构化的RNA。
Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 mu mol (i.e. concentration of 10 mu M in a 25 mu l reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.