Limits...
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.

Show MeSH

Related in: MedlinePlus

Determination of the most stable reference genes using NormFinder. The NormFinder algorithm ranks the set of candidate normalization genes according to their expression stability in a given experimental design. Blue bars represent the stability values of our candidate genes, while purple bars indicate their standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2713238&req=5

Figure 7: Determination of the most stable reference genes using NormFinder. The NormFinder algorithm ranks the set of candidate normalization genes according to their expression stability in a given experimental design. Blue bars represent the stability values of our candidate genes, while purple bars indicate their standard error.

Mentions: NormFinder attempts to identify the optimal normalization gene among a set of candidates and provides a measure of the stability of genes' expression in different groups and at the same time estimates any bias in the expression of the genes between the groups based on two-way ANOVA [18]. When all data were analysed together, the most stable RG candidates in our essay using NormFinder were mt-cyb (stability value = 0.127), rpl15 (0.137) and mt-acp (0.165), as shown in figure 7. We then repeated the analysis grouping samples by developmental stage to assess intergroup variability. In this case, the best genes that allows comparison of different developmental stages and/or treatments in B. amphitrite, which was the goal of this study, were mt-cyb (0.159), mt-nd1 (0.167) and mt-acp (0.168), suggesting that these are the most suitable genes for data normalization. A last examination was performed adding the data to NormFinder as two subgroups (the two biological replicates for each developmental stage). As a result, the software produced the same gene ranking by their stability values, showing a very low variability between replicates, which was also confirmed by further statistical analysis; Pearson correlation coefficients of biological replicates for all RGs tested ranged between 0.711 and 0.979, with 9 genes out of 11 showing a significant correlation at the 0.01 level (2-tailed). Although the use of only 7 data points may affect the examination, our results suggested that the implemented protocol is effective in capturing meaningful differences in gene expression throughout B. amphitrite development.


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)

Determination of the most stable reference genes using NormFinder. The NormFinder algorithm ranks the set of candidate normalization genes according to their expression stability in a given experimental design. Blue bars represent the stability values of our candidate genes, while purple bars indicate their standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Determination of the most stable reference genes using NormFinder. The NormFinder algorithm ranks the set of candidate normalization genes according to their expression stability in a given experimental design. Blue bars represent the stability values of our candidate genes, while purple bars indicate their standard error.
Mentions: NormFinder attempts to identify the optimal normalization gene among a set of candidates and provides a measure of the stability of genes' expression in different groups and at the same time estimates any bias in the expression of the genes between the groups based on two-way ANOVA [18]. When all data were analysed together, the most stable RG candidates in our essay using NormFinder were mt-cyb (stability value = 0.127), rpl15 (0.137) and mt-acp (0.165), as shown in figure 7. We then repeated the analysis grouping samples by developmental stage to assess intergroup variability. In this case, the best genes that allows comparison of different developmental stages and/or treatments in B. amphitrite, which was the goal of this study, were mt-cyb (0.159), mt-nd1 (0.167) and mt-acp (0.168), suggesting that these are the most suitable genes for data normalization. A last examination was performed adding the data to NormFinder as two subgroups (the two biological replicates for each developmental stage). As a result, the software produced the same gene ranking by their stability values, showing a very low variability between replicates, which was also confirmed by further statistical analysis; Pearson correlation coefficients of biological replicates for all RGs tested ranged between 0.711 and 0.979, with 9 genes out of 11 showing a significant correlation at the 0.01 level (2-tailed). Although the use of only 7 data points may affect the examination, our results suggested that the implemented protocol is effective in capturing meaningful differences in gene expression throughout B. amphitrite development.

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