Development and utilization of a custom PCR array workflow: analysis of gene expression in mycoplasma genitalium and guinea pig (Cavia porcellus).
Development and utilization of a custom PCR array workflow: analysis of gene expression in mycoplasma genitalium and guinea pig (Cavia porcellus).
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DOI：
10.1007/s12033-014-9813-6
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发表时间：
2015-02
影响因子：
2.6
通讯作者：
Pyles, Richard B.
中科院分区：
文献类型：
--
作者：
Veselenak, Ronald L.;Miller, Aaron L.;Milligan, Gregg N.;Bourne, Nigel;Pyles, Richard B.
关键词：
转录组分析是用于评估对特定生物过程和疾病状态的成熟至关重要的分子途径的有力工具。最近，基于PCR的阵列补充了微阵列和RNA-seq方法，用于研究基因表达水平的变化。PCR阵列是一种更具成本效益的替代方案，然而，商业上可获得的组件通常仅限于少数更广泛研究的物种（例如，大鼠、人和小鼠）。因此，在创建此类测定之前，对新兴或研究不足的物种的调查受到阻碍。为了满足这一需求，我们提出的数据记录了一个开发的工作流程的成功，具有增强的潜力，以创建和验证新的RT-PCR阵列的代表性不足的物种与整个或部分基因组注释。利用这种增强的工作流程，我们已经实现了超过400个引物对的第一轮设计的成功率为80%。其中，约160个不同的靶标被序列确认。使用两种独特阵列的概念验证研究，一种靶向病原性细菌生殖支原体，另一种特异性针对豚鼠（豚鼠），使我们能够鉴定通过随后的qPCR验证的mRNA表达的显著（P < 0.05）变化。这种灵活且适应性强的平台为基因表达分析提供了一种有价值且具有成本效益的替代方案。本文的在线版本（doi：10.1007/s12033-014-9813-6）包含补充材料，可供授权用户使用。
Transcriptome analysis is a powerful tool for evaluating molecular pathways central to maturation of specific biological processes and disease states. Recently, PCR-based arrays have supplemented microarray and RNA-seq methodologies for studying changes in gene expression levels. PCR arrays are a more cost efficient alternative, however commercially available assemblies are generally limited to only a few more widely researched species (e.g., rat, human, and mouse). Consequently, the investigation of emerging or under-studied species is hindered until such assays are created. To address this need, we present data documenting the success of a developed workflow with enhanced potential to create and validate novel RT-PCR arrays for underrepresented species with whole or partial genome annotation. Utilizing this enhanced workflow, we have achieved a success rate of 80 % for first-round designs for over 400 primer pairs. Of these, ~160 distinct targets were sequence confirmed. Proof of concept studies using two unique arrays, one targeting the pathogenic bacterium Mycoplasma genitalium and the other specific for the guinea pig (Cavia porcellus), allowed us to identify significant (P < 0.05) changes in mRNA expression validated by subsequent qPCR. This flexible and adaptable platform provides a valuable and cost-effective alternative for gene expression analysis. The online version of this article (doi:10.1007/s12033-014-9813-6) contains supplementary material, which is available to authorized users.
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影响因子：
3.1
作者：
McGowin, Chris L.;Spagnuolo, Rae Ann;Pyles, Richard B.
通讯作者：
Pyles, Richard B.
影响因子：
2.4
作者：
Kwon, Y. K.;Lipatov, A. S.;Swayne, D. E.
通讯作者：
Swayne, D. E.
DOI：
10.1016/j.diagmicrobio.2009.01.032
发表时间：
2009-06-01
影响因子：
2.9
作者：
McGowin, Chris L.;Whitlock, Gregory C.;Pyles, Richard B.
通讯作者：
Pyles, Richard B.
影响因子：
1.2
作者：
Adachi, Satoshi;Hashimoto, Tomomi;Iwata, Hiroyuki
通讯作者：
Iwata, Hiroyuki
影响因子：
4
作者：
Finotello, Francesca;Di Camillo, Barbara
通讯作者：
Di Camillo, Barbara