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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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Related in: MedlinePlus

Correlation between biological replicates for the five best reference genes. The Ct values (adjusted to primer efficiencies) obtained for the seven developmental stages we analysed were plotted for the five best reference genes. The size of the shape indicates the developmental stage: the smallest shapes represent values from just-released nauplii, whereas the largest represent values obtained from adult barnacles.
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Figure 3: Correlation between biological replicates for the five best reference genes. The Ct values (adjusted to primer efficiencies) obtained for the seven developmental stages we analysed were plotted for the five best reference genes. The size of the shape indicates the developmental stage: the smallest shapes represent values from just-released nauplii, whereas the largest represent values obtained from adult barnacles.

Mentions: The expression levels of RGs were obtained from qRT-PCR reactions in the form of threshold cycle (Ct) values (Figure 2). The 14 Ct values collected for each primer pair were derived from the two biological replicates of the seven developmental stages under investigation (raw Ct data are provided in additional file 2). These samples were initially considered independent and the data they generated were analyzed with geNorm [16], BestKeeper [17] and NormFinder [18] to determine the most steadily expressed genes. It can be argued that two biological replicates are not enough for statistical analysis, and this is the case for many biological systems (e.g. comparing different tissues, single individuals) where at least five replicates should be performed. In our investigation, however, the RNA was extracted from ten (the adult stage) to hundreds of pooled individuals, so that the RNA could be considered an average sample of the developmental stage analysed. In our opinion, the high correlation found between the two biological replicates, at least for the most stably expressed genes (figure 3), confirmed our expectations.


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)

Correlation between biological replicates for the five best reference genes. The Ct values (adjusted to primer efficiencies) obtained for the seven developmental stages we analysed were plotted for the five best reference genes. The size of the shape indicates the developmental stage: the smallest shapes represent values from just-released nauplii, whereas the largest represent values obtained from adult barnacles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Correlation between biological replicates for the five best reference genes. The Ct values (adjusted to primer efficiencies) obtained for the seven developmental stages we analysed were plotted for the five best reference genes. The size of the shape indicates the developmental stage: the smallest shapes represent values from just-released nauplii, whereas the largest represent values obtained from adult barnacles.
Mentions: The expression levels of RGs were obtained from qRT-PCR reactions in the form of threshold cycle (Ct) values (Figure 2). The 14 Ct values collected for each primer pair were derived from the two biological replicates of the seven developmental stages under investigation (raw Ct data are provided in additional file 2). These samples were initially considered independent and the data they generated were analyzed with geNorm [16], BestKeeper [17] and NormFinder [18] to determine the most steadily expressed genes. It can be argued that two biological replicates are not enough for statistical analysis, and this is the case for many biological systems (e.g. comparing different tissues, single individuals) where at least five replicates should be performed. In our investigation, however, the RNA was extracted from ten (the adult stage) to hundreds of pooled individuals, so that the RNA could be considered an average sample of the developmental stage analysed. In our opinion, the high correlation found between the two biological replicates, at least for the most stably expressed genes (figure 3), confirmed our expectations.

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