Research article

Construction of an adult barnacle (Balanus amphitrite) cDNA library and selection of reference genes for quantitative RT-PCR studies

Tristano Bacchetti De Gregoris^1,2 , Marco Borra³ , Elio Biffali³ , Thomas Bekel⁴ , J Grant Burgess² , Richard R Kirby⁵ and Anthony S Clare¹

¹School of Marine Science and Technology, Ridley Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, England, UK

²Dove Marine Laboratory, Newcastle University, Cullercoats, Tyne and Wear, NE30 4PZ, England, UK

³Stazione Zoologica Anton Dohrn, Napoli, Villa Comunale, 80121, Italy

⁴Center for Biotechnology (CeBiTec), Bielefeld University, D-33594 Bielefeld, Germany

⁵School of Biological Sciences, University of Plymouth, Plymouth, PL4 8AA, England, UK

author email corresponding author email

BMC Molecular Biology 2009, 10:62doi:10.1186/1471-2199-10-62

Published:	24 June 2009

Abstract

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.