A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses
A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses
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作者：
Grundhoff, A;Sullivan, CS;Ganem, D
我们已经开发了一种基于生物信息学和基于阵列技术的方法来鉴定microRNAs (miRNAs)，而不使用cDNA克隆。该方法设计用于小尺寸（< 2mb）的基因组，在被KSHV和EBV两种淋巴性疱疹病毒感染的细胞上进行了测试。使用我们开发的算法（VMir）对病毒基因组进行前mirna的计算扫描。该分析提出的候选发夹随后在微阵列上合成为寡核苷酸，并与来自感染细胞的小rna杂交。在阵列上获得阳性的候选mirna然后进行验证性Northern blot分析。使用这种方法，鉴定了10种已知的KSHV pre-miRNA，以及一种先前未被检测到的新型pre-miRNA。该方法还鉴定出7个新的ebv编码的pre- mirna；通过使用额外的计算方法，我们共鉴定出18个新的EBV前miRNA，它们产生22个成熟的miRNA分子，从而使迄今已知的EBV miRNA总数增加了四倍多。讨论了该方法的优点和局限性。
We have developed an approach to identify microRNAs ( miRNAs) that is based on bioinformatics and array-based technologies, without the use of cDNA cloning. The approach, designed for use on genomes of small size (< 2 Mb), was tested on cells infected by either of two lymphotropic herpesviruses, KSHV and EBV. The viral genomes were scanned computationally for pre-miRNAs using an algorithm (VMir) we have developed. Candidate hairpins suggested by this analysis were then synthesized as oligonucleotides on microarrays, and the arrays were hybridized with small RNAs from infected cells. Candidate miRNAs that scored positive on the arrays were then subjected to confirmatory Northern blot analysis. Using this approach, 10 of the known KSHV pre-miRNAs were identified, as well as a novel pre-miRNA that had earlier escaped detection. This method also led to the identification of seven new EBV-encoded pre-miRNAs; by using additional computational approaches, we identified a total of 18 new EBV pre-miRNAs that produce 22 mature miRNA molecules, thereby more than quadrupling the total number of hitherto known EBV miRNAs. The advantages and limitations of the approach are discussed.