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

Bacchetti De Gregoris T, Borra M, Biffali E, Bekel T, Burgess JG, Kirby RR, Clare AS - BMC Mol. Biol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Marine Science and Technology, Ridley Building, Newcastle University, Newcastle upon Tyne, England, UK. t.bacchetti-de-gregoris@ncl.ac.uk

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.

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Gene Ontology mapping for Balanus amphitrite proteins. Relative distribution of ESTs within the three main subclasses existing in the GO classification: A) Biological process; B) Cellular component; C) Molecular function.
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Figure 1: Gene Ontology mapping for Balanus amphitrite proteins. Relative distribution of ESTs within the three main subclasses existing in the GO classification: A) Biological process; B) Cellular component; C) Molecular function.

Mentions: Balanus amphitrite is one of the most extensively studied barnacles and has been suggested as a candidate genetic model for larval settlement and metamorphosis [19]. Recently, Thiyagarajan and Qian used a proteomics approach to investigate settlement regulation in this organism [20]. However, they argued that the lack of deposited gene sequences hindered a full appreciation of their results. The creation of an adult B. amphitrite cDNA library, the sequence of 960 clones (of which 55 were excluded from our analysis as they showed insert length shorter than 50 bp) and the detection of 530 singlets and 79 tentative contiguous (TC) sequences (a summary of the EST survey is given in table 1) is thus an important first-step towards understanding the molecular ecology of this barnacle. Among the 609 different genes we report, 107 appeared to be of mitochondrial origin and 75 showed similarities with previously published ribosomal sequences. Gene ontology entries [21] revealed that the main categories of the genes found in our library appear to be involved in electron transport, protein biosynthesis, catalytic activity, metal ion binding, metabolism and the biogenesis of structural elements such as muscle and cuticle (Figure 1). Approximately 38% of the unique sequences we obtained have been functionally annotated and a corresponding gene name proposed, and informative annotation has been given for an additional 18%. Of the remaining ESTs, 8% had a BLAST match with uncharacterized transcripts and 37% showed no appreciable similarity to previously published sequences. This distribution of ESTs among known/uncharacterized/unknown genes does not differ substantially from that found in recent EST surveys on other marine invertebrates [22,23]. We also determined several transcripts that were highly similar to sequences derived from the deposited complete mitochondrial genomes of the two barnacles Megabalanus volcano and Tetraclita japonica.


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

Bacchetti De Gregoris T, Borra M, Biffali E, Bekel T, Burgess JG, Kirby RR, Clare AS - BMC Mol. Biol. (2009)

Gene Ontology mapping for Balanus amphitrite proteins. Relative distribution of ESTs within the three main subclasses existing in the GO classification: A) Biological process; B) Cellular component; C) Molecular function.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713238&req=5

Figure 1: Gene Ontology mapping for Balanus amphitrite proteins. Relative distribution of ESTs within the three main subclasses existing in the GO classification: A) Biological process; B) Cellular component; C) Molecular function.
Mentions: Balanus amphitrite is one of the most extensively studied barnacles and has been suggested as a candidate genetic model for larval settlement and metamorphosis [19]. Recently, Thiyagarajan and Qian used a proteomics approach to investigate settlement regulation in this organism [20]. However, they argued that the lack of deposited gene sequences hindered a full appreciation of their results. The creation of an adult B. amphitrite cDNA library, the sequence of 960 clones (of which 55 were excluded from our analysis as they showed insert length shorter than 50 bp) and the detection of 530 singlets and 79 tentative contiguous (TC) sequences (a summary of the EST survey is given in table 1) is thus an important first-step towards understanding the molecular ecology of this barnacle. Among the 609 different genes we report, 107 appeared to be of mitochondrial origin and 75 showed similarities with previously published ribosomal sequences. Gene ontology entries [21] revealed that the main categories of the genes found in our library appear to be involved in electron transport, protein biosynthesis, catalytic activity, metal ion binding, metabolism and the biogenesis of structural elements such as muscle and cuticle (Figure 1). Approximately 38% of the unique sequences we obtained have been functionally annotated and a corresponding gene name proposed, and informative annotation has been given for an additional 18%. Of the remaining ESTs, 8% had a BLAST match with uncharacterized transcripts and 37% showed no appreciable similarity to previously published sequences. This distribution of ESTs among known/uncharacterized/unknown genes does not differ substantially from that found in recent EST surveys on other marine invertebrates [22,23]. We also determined several transcripts that were highly similar to sequences derived from the deposited complete mitochondrial genomes of the two barnacles Megabalanus volcano and Tetraclita japonica.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Marine Science and Technology, Ridley Building, Newcastle University, Newcastle upon Tyne, England, UK. t.bacchetti-de-gregoris@ncl.ac.uk

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.

Show MeSH
Related in: MedlinePlus