Bacchetti De Gregoris Tristano, Borra Marco, Biffali Elio, Bekel Thomas, Burgess J Grant, Kirby Richard R, Clare Anthony S
School of Marine Science and Technology, Ridley Building, Newcastle University, Newcastle upon Tyne, England, UK.
BMC Mol Biol. 2009 Jun 24;10:62. doi: 10.1186/1471-2199-10-62.
Balanus amphitrite is a barnacle commonly used in biofouling research. Although many aspects of its biology have been elucidated, the lack of genetic information is impeding a molecular understanding of its life cycle. As part of a wider multidisciplinary approach to reveal the biogenic cues influencing barnacle settlement and metamorphosis, we have sequenced and annotated the first cDNA library for B. amphitrite. We also present a systematic validation of potential reference genes for normalization of quantitative real-time PCR (qRT-PCR) data obtained from different developmental stages of this animal.
We generated a cDNA library containing expressed sequence tags (ESTs) from adult B. amphitrite. A total of 609 unique sequences (comprising 79 assembled clusters and 530 singlets) were derived from 905 reliable unidirectionally sequenced ESTs. Bioinformatics tools such as BLAST, HMMer and InterPro were employed to allow functional annotation of the ESTs. Based on these analyses, we selected 11 genes to study their ability to normalize qRT-PCR data. Total RNA extracted from 7 developmental stages was reverse transcribed and the expression stability of the selected genes was compared using geNorm, BestKeeper and NormFinder. These software programs produced highly comparable results, with the most stable gene being mt-cyb, while tuba, tubb and cp1 were clearly unsuitable for data normalization.
The collection of B. amphitrite ESTs and their annotation has been made publically available representing an important resource for both basic and applied research on this species. We developed a qRT-PCR assay to determine the most reliable reference genes. Transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, although other genes also performed well and could prove useful to normalize gene expression studies.
纹藤壶是生物污损研究中常用的一种藤壶。尽管其生物学的许多方面已得到阐明，但缺乏遗传信息阻碍了对其生命周期的分子理解。作为揭示影响藤壶附着和变态的生物成因线索的更广泛多学科方法的一部分，我们对纹藤壶的第一个cDNA文库进行了测序和注释。我们还对从该动物不同发育阶段获得的定量实时PCR（qRT-PCR）数据标准化的潜在参考基因进行了系统验证。
我们构建了一个包含成年纹藤壶表达序列标签（EST）的cDNA文库。从905个可靠的单向测序EST中获得了总共609个独特序列（包括79个组装簇和530个单序列）。使用BLAST、HMMer和InterPro等生物信息学工具对EST进行功能注释。基于这些分析，我们选择了11个基因来研究它们对qRT-PCR数据标准化的能力。从7个发育阶段提取的总RNA进行逆转录，并使用geNorm、BestKeeper和NormFinder比较所选基因的表达稳定性。这些软件程序产生了高度可比的结果，最稳定的基因是mt-cyb，而tuba、tubb和cp1显然不适合数据标准化。
纹藤壶EST的收集及其注释已公开提供，是该物种基础研究和应用研究的重要资源。我们开发了一种qRT-PCR检测方法来确定最可靠的参考基因。编码细胞色素b和NADH脱氢酶亚基1的转录本表达最稳定，尽管其他基因也表现良好，可能有助于基因表达研究的标准化。
