Combining next‐generation sequencing with microarray for transcriptome analysis in rainbow trout gonads
Combining next‐generation sequencing with microarray for transcriptome analysis in rainbow trout gonads
复制标题
将新一代测序与微阵列相结合进行虹鳟性腺转录组分析
DOI：
10.1002/mrd.22127
复制
发表时间：
2012
影响因子：
2.5
通讯作者：
G. Yoshizaki
中科院分区：
文献类型：
--
作者：
M. Hayashi;Mana Sato;Y. Iwasaki;M. Terasawa;Masami Tashiro;S. Yokoyama;N. Katayama;Sakiko Sadaie;Misako Miwa;G. Yoshizaki
微阵列技术是研究全基因组基因表达的有力工具。然而，由于许多鱼类的基因组尚未确定，因此只能从有限的表达序列标签数据设计cDNA微阵列。在这项研究中，我们根据从虹鳟鱼（Oncorhynchus mykiss）胚胎生殖嵴，睾丸和卵巢中提取的RNA的下一代测序获得的测序数据（337，466读段）设计了一个微阵列。这些数据（307，264个读段）组装成28，668个重叠群; 3，298个读段不能组装，26，904个读段是不与其他读段聚类的独特序列。基于这些信息，为微阵列设计了55，928个微阵列探针，并通过与从A型精原细胞（A-SG）和睾丸体细胞提取的RNA的杂交实验进行了验证。经证实，A-SG中已知精原细胞标志物的表达高于睾丸体细胞，而支持细胞标志物在睾丸体细胞中的表达水平更高。该微阵列分析显示，8，068个转录物在A-SG中显示出比睾丸体细胞高至少四倍的信号。通过定量RT-PCR，17个随机选择的转录本中有14个在A-SG中的表达水平显著高于体细胞。此外，原位杂交分析的三个转录本在未成熟的鳟鱼睾丸中显示出A-SG-特异性信号，其中一个在A-SG中表现出异质性表达模式。因此，在这项研究中开发的虹鳟鱼性腺微阵列似乎是一个有用的工具，以了解虹鳟鱼配子发生。摩尔Reprod. Dev. 79：870-878，2012.© 2012 Wiley Periodicals，Inc.
Microarray technology is a powerful tool for studying genome‐wide gene expression. As the genome of many fish has not yet been determined, however, cDNA microarrays can only be designed from limited expressed sequence tag data. In this study, we designed a microarray based on the sequencing data (337,466 reads) obtained by next‐generation sequencing of RNA extracted from rainbow trout (Oncorhynchus mykiss) embryonic genital ridge, testis, and ovary. These data (307,264 reads) were assembled into 28,668 contigs; 3,298 reads could not be assembled and 26,904 reads were unique sequences that did not cluster with other reads. Based on this information, 55,928 microarray probes were designed for a microarray, which was validated by hybridization experiments with RNA extracted from type A spermatogonia (A‐SG) and testicular somatic cells. Expression of known spermatogonial markers was confirmed to be higher in A‐SG than in testicular somatic cells whereas supporting‐cell markers were expressed at higher levels in testicular somatic cells. This microarray analysis revealed that 8,068 transcripts showed at least fourfold higher signal in A‐SG than testicular somatic cells. Fourteen of 17 randomly selected transcripts were expressed at significantly higher‐levels in A‐SG than somatic cells, by quantitative RT‐PCR. In addition, three transcripts analyzed with in situ hybridization showed A‐SG‐specific signals in immature trout testis, with one of them exhibiting a heterogeneous expression pattern in A‐SG. The rainbow trout gonad microarray developed in this study therefore appears to be a useful tool to understand gametogenesis in rainbow trout. Mol. Reprod. Dev. 79: 870–878, 2012. © 2012 Wiley Periodicals, Inc.
影响因子：
2.7
作者：
Sawatari, Etsuko;Shikina, Shinya;Yoshizaki, Goro
通讯作者：
Yoshizaki, Goro
虹鳟鱼生殖细胞的性可塑性
DOI：
--
发表时间：
2010
期刊：
Anim.Reprod.
影响因子：
--
作者：
G.Yoshizaki;et al.
通讯作者：
et al.
影响因子：
4.6
作者：
Yoshizaki, Goro;Ichikawa, Masaki;Okutsu, Tomoyuki
通讯作者：
Okutsu, Tomoyuki