Transcriptional profiling using a novel cDNA array identifies differential gene expression during porcine embryo elongation
Transcriptional profiling using a novel cDNA array identifies differential gene expression during porcine embryo elongation
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DOI：
10.1002/mrd.20291
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发表时间：
2005-06-01
影响因子：
2.5
通讯作者：
Tuggle, CK
中科院分区：
文献类型：
--
作者：
Lee, SH;Zhao, SH;Tuggle, CK
一种新的猪cDNA阵列，含有1,015个PCR产物选择胚胎表达，用于转录分析的概念，在围着床期发育的四个阶段。总孕体RNA从小的球形，大的球形，管状，和丝状阶段进行扩增，转化为cDNA，并杂交到膜。首先，比较使用来自总RNA或来自扩增的RNA的cDNA获得的归一化信号强度。与对每个基因进行的测试相关的P值的均匀分布表明没有证据表明扩增引入了偏倚。通过使用扩增的靶点和新阵列获得的数据分析确定了不同阶段差异表达的基因。这些基因通过在基因特异性混合模型中检验显著阶段效应来鉴定。共有9个基因被宣布差异表达。九个基因中有六个的P值小于0.001，大约17%的错误发现率与该显著性阈值相关。当使用Bonferroni方法来控制一个或多个假阳性的概率时，六个基因中的两个是显著的。其他三个基因的P值在0.001和0.01之间，并且在阶段之间表现出大于两倍的差异。通过使用定量真实的时间RT-PCR，证实了选择用于确认的所有四个基因（类固醇生成急性调节蛋白、白细胞介素I β、转化生长因子β 3和胸腺素β 10）的差异表达。我们的研究表明，RNA扩增是有用的限制猪胚胎RNA的转录谱，这种新的靶向阵列可以检测滋养层细胞伸长过程中的差异基因表达。最后，我们的研究结果有助于增加对控制孕体发育的已知基因表达的时间模式的理解，以及识别在植入过程中也受到差异调节的新基因。(c)2005 Wiley-Liss，Inc.
A novel porcine cDNA array, containing 1,015 PCR products selected for embryonic expression, was used for transcriptional profiling of conceptuses at four stages of peri-implantation development. Total conceptus RNA from small spherical, large spherical, tubular, and filamentous stages was amplified, converted to cDNA, and hybridized to membranes. Initially, normalized signal intensities obtained using cDNA from total RNA or from amplified RNA were compared. Uniform distribution of P-values associated with Mests conducted for each gene indicated no evidence that amplification introduced bias. Analysis of data obtained by using amplified targets and the novel array identified genes differentially expressed across stages. Such genes were identified by testing for significant stage effects in gene-specific mixed models. A total of nine genes were declared differentially expressed. Six of the nine genes had P-values less than 0.001, and a false discovery rate of approximately 17% was associated with this significance threshold. Two out of six genes were significant when using the Bonferroni method to control the probability of one or more false positives. The other three genes had P-values between 0.001 and 0.01 and exhibited differences greater than twofold between stages. All four genes selected for confirmation (steroidogenic acute regulatory protein, interleukin I beta, transforming growth factor beta 3, and thymosin beta 10) were shown to be differentially expressed by using quantitative real time RT-PCR. Our study shows that RNA amplification is useful for transcriptional profiling with limiting porcine embryonic RNA, and that this novel targeted array can detect differential gene expression during trophoblastic elongation. Finally, our results contribute to an increased understanding of the temporal patterns of expression of known genes controlling conceptus development, as well as identify novel genes also differentially regulated during implantation. (c) 2005 Wiley-Liss, Inc.