Construction of an adult barnacle (Balanus amphitrite) cDNA library and selection of reference genes for quantitative RT-PCR studies
| Autor: | Thomas Bekel, Elio Biffali, Richard R. Kirby, J. Grant Burgess, Anthony S. Clare, Tristano Bacchetti De Gregoris, Marco Borra |
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| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
0106 biological sciences
InterPro Aging lcsh:QH426-470 Molecular Sequence Data Gene Expression 01 natural sciences 660.6 03 medical and health sciences Reference genes Gene expression Animals Genomic library Selection Genetic lcsh:QH573-671 Gene Molecular Biology 030304 developmental biology Gene Library Genetics Expressed Sequence Tags 0303 health sciences Expressed sequence tag biology cDNA library Reverse Transcriptase Polymerase Chain Reaction lcsh:Cytology 010604 marine biology & hydrobiology Thoracica NADH dehydrogenase lcsh:Genetics biology.protein Research Article |
| Zdroj: | BMC Molecular Biology, Vol 10, Iss 1, p 62 (2009) BMC Molecular Biology |
| ISSN: | 1471-2199 |
| Popis: | Background 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. Results 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. Conclusion 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. |
| Databáze: | OpenAIRE |
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