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

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.

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.

Figure 2

Comparative expression of the analysed genes. Box-and-whisker plot representing the expression level (threshold cycles) of candidate reference genes in B. amphitrite (n = 14). The box plot, obtained using the software Minitab, shows the smallest observation, lower quartile, median, upper quartile, largest observation and indicates Ct value that might be considered outliers.

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.

Figure 4

Gene expression stability M of candidate reference genes calculated by geNorm. The geNorm program proceeds to the stepwise exclusion of the genes whose relative expression levels are more variable among samples. Data points represent the average expression stability values of remaining reference genes.

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.

Figure 6

Results from BestKeeper correlation analysis. BestKeeper calculates the stability measure for each candidate gene and then ranks them from the most to the least stable (SD [± x-fold]). The coefficient of correlation (R) and the p-value measure the correlation between each gene and the BestKeeper index. For each variable presented in the figure (SD [± x-fold], R and p-value), genes that ranked comparatively better are highlighted with a more intense cell colour.

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.