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 optimal number of reference genes for data normalization. Bar values indicate the magnitude of the change in the normalization factor after the inclusion of an additional reference gene. The authors of geNorm suggest that V > 0.15 should be considered as the threshold to include an extra RG into the assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Determination of the optimal number of reference genes for data normalization. Bar values indicate the magnitude of the change in the normalization factor after the inclusion of an additional reference gene. The authors of geNorm suggest that V > 0.15 should be considered as the threshold to include an extra RG into the assay.

Mentions: The software geNorm provides a ranking of the tested genes based on their stability measure (M), determining the two most stable RGs or a combination of multiple stable genes for normalization. The value M represents the mean pair-wise variation between a gene and all other tested candidates. The gene with the highest M is then excluded from the analysis and the calculation is repeated in a stepwise fashion that allows genes ranking until the best two genes are found. According to geNorm, the two most stable genes in our assay were mt-nd1 and mt-cyb (Figure 4), with an M value of 0.41. The threshold value M for considering a gene to be unsuitable for data normalization is suggested to be ≥ 1.5 [16]. Low values of the pair-wise variation V between two sequential normalization factors containing an increasing number of genes showed it was unnecessary to include another RG in our protocol (Figure 5). However, as mt-nd1 and mt-cyb are both contained in the mitochondrial genome, it may be advisable to include a nuclear gene in the normalization strategy. In this case, geNorm suggested that either act or ef1a should be used.


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 optimal number of reference genes for data normalization. Bar values indicate the magnitude of the change in the normalization factor after the inclusion of an additional reference gene. The authors of geNorm suggest that V > 0.15 should be considered as the threshold to include an extra RG into the assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Determination of the optimal number of reference genes for data normalization. Bar values indicate the magnitude of the change in the normalization factor after the inclusion of an additional reference gene. The authors of geNorm suggest that V > 0.15 should be considered as the threshold to include an extra RG into the assay.
Mentions: The software geNorm provides a ranking of the tested genes based on their stability measure (M), determining the two most stable RGs or a combination of multiple stable genes for normalization. The value M represents the mean pair-wise variation between a gene and all other tested candidates. The gene with the highest M is then excluded from the analysis and the calculation is repeated in a stepwise fashion that allows genes ranking until the best two genes are found. According to geNorm, the two most stable genes in our assay were mt-nd1 and mt-cyb (Figure 4), with an M value of 0.41. The threshold value M for considering a gene to be unsuitable for data normalization is suggested to be ≥ 1.5 [16]. Low values of the pair-wise variation V between two sequential normalization factors containing an increasing number of genes showed it was unnecessary to include another RG in our protocol (Figure 5). However, as mt-nd1 and mt-cyb are both contained in the mitochondrial genome, it may be advisable to include a nuclear gene in the normalization strategy. In this case, geNorm suggested that either act or ef1a should be used.

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