Construction of an adult barnacle (Balanus amphitrite) cDNA library and selection of reference genes for quantitative RT-PCR studies
© Bacchetti De Gregoris et al; licensee BioMed Central Ltd. 2009
Received: 12 December 2008
Accepted: 24 June 2009
Published: 24 June 2009
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.
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.
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.
Many marine invertebrates have a pelagobenthic life cycle and biofouling by many of these species has a considerable economic impact in marine environments . Consequently, it is essential to understand the mechanisms regulating the transition between the free-living planktonic larvae and the benthic adult stage. The barnacle Balanus amphitrite  is a sessile gregarious species that is a model organism for both fundamental and applied larval settlement studies due to its invasive behaviour, its worldwide distribution, and the relative simplicity of manipulating its reproduction in the laboratory. The life cycle of B. amphitrite is characterized by the presence of six planktonic naupliar stages (naupliar instar I-VI) followed by a non-feeding larval stage, the cyprid, that is specialized to explore the substratum in order to locate a suitable place for permanent attachment. A number of behavioural studies have shown that B. amphitrite cyprids respond to biotic and abiotic factors as they explore the substratum [3–5]. To date however, the paucity of genomic information available for this organism has hindered in-depth mechanistic studies of the surface colonization process.
Expressed sequence tag (EST) surveys are fundamental for discovering new genes  and they represent an essential step for the molecular characterization of the species of interest. In addition, EST-derived information supports genomic sequence annotation by suggesting intron/exon boundaries and the existence of previously undescribed transcription units; consequently, mRNA sequences are invaluable in comparative genomics . We have therefore prepared an un-substracted cDNA library from adult B. amphtrite to identify the most expressed genes within the first few hundreds ESTs. We hope that the application of molecular probes developed from this EST library, in combination with standard methods for behavioural analysis, will allow us to better understand the timing and intensity of gene expression during different life history stages of B. amphitrite. Furthermore, few studies have investigated the regulation of the pelagobenthic life-cycle at a molecular level [8, 9], despite its broad distribution in marine invertebrates . Barnacles are good candidates to become a model system for this purpose, and the development of new molecular tool for these organisms could help to answer fundamental biological questions related to marine life.
Quantitative real-time PCR (qRT-PCR) is regarded as the most sensitive and reliable method to determine levels of mRNA transcription [11, 12]. The application of qRT-PCR has proved particularly useful for comparative studies, where the expression of genes of interest (GOIs) in different samples is measured against the expression of endogenous reference genes (RGs). This normalization procedure is fundamental to minimize inherent variability introduced during the RNA extraction or the reverse transcription steps [13, 14]. Ideally, RGs should both maintain a stable transcription level in all cells, tissues or individuals under investigation and should not be influenced by the experimental conditions. Unfortunately, many studies have shown that universal RGs for data normalization do not exist and for this reason, the selection of the best RGs should be validated for every new qRT-PCR assay .
Here, we describe the first characterization of the B. amphitrite transcriptome that is based on the creation of an EST library from adult individuals. The sequencing and annotation of 960 clones provides the background for further analysis of life-cycle regulation in this organism. We also established a qRT-PCR assay to monitor gene expression in different developmental stages and in individuals exposed to morphogenetic cues. The ability of 11 B. amphitrite transcripts to normalize qRT-PCR data was determined by comparing relative quantities obtained from cDNAs representing 14 different samples and 7 developmental stages. The software geNorm , BestKeeper and Normfinder  were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for qRT-PCR data normalization in B. amphitrite.
Results and discussion
Annotation of sequences from the ESTs library
Classification of Balanus amphitrite ESTs
N° of sequences
Blast2n vs nt
Blast2x vs SP
Blast2x vs KEGG
Fragment assembly generated a total of 79 TC comprising 375 ESTs. Sequences belonging to 8 different TCs were particularly frequent in our library, with the most common being an unassigned mitochondrial gene partly similar to 16S rRNA (with 52 entries), followed by cytochrome c oxidase subunit I (31), cysteine proteinase (17), cytochrome c oxidase subunit II (14), cytochrome b (12), a ribosomal RNA internal transcribed spacer (12), cytochrome c oxidase subunit III (11) and the elongation factor 1-α (11). The longest TC generated was 1703 nucleotides and translated for the18S rRNA gene. Considering the 609 unique sequences we obtained, a total of 280 had a match in the NCBI nucleotide database. A taxonomic subdivision of the first hit produced by these 280 transcripts showed that 109 of them matched sequences from barnacles. The remaining sequences were represented among insects, vertebrates, arachnids, plants, fungi and various other groups (79, 53, 6, 8, 5 and 19 sequences, respectively). To annotate B. amphitrite's genes, the proposed nomenclature for Drosophila melanogaster was used as a guide http://flybase.org and the corresponding gene symbol established in D. melanogaster was used when possible. However, in a slight departure, we decided to use the prefix mt- to identify mitochondrial genes.
Validation of best reference genes for qRT-PCR
Our main interests focus on elucidating those genes involved in barnacle settlement. In this respect, qRT-PCR is particularly suitable to monitor how external cues, such as environmental variables, the presence of conspecific individuals or the occurrence of biofilm and/or of certain microorganisms, influence gene expression prior to and during settlement and metamorphosis. Since most of the annotated ESTs we found represent highly expressed housekeeping genes, this suggests that information from a few hundred clones derived from a cDNA library is sufficient to validate RGs for subsequent qRT-PCR studies.
List of primers and reference genes under investigation
Fructose bisphosphate aldolase
NADH dehydrogenase subunit 1
NADH dehydrogenase subunit 4L
Cysteine protease 1
Myosin 1-light chain
Presence of primer dimers
60s ribosomal protein L15
Elongation Factor 1 alpha
As a general consideration, although geNorm, BestKeeper and NormFinder have the same aim, they employ different strategies to calculate the most stable genes and it is unlikely that they will give the same results. For example, looking at the absolute ranking of best genes, mt-acp scored 5th, 9th and 3rd. However, its stability values as determined by the tree software do not change substantially from that of the genes ranked closely (e.g. the value obtained by BestKeeper for mt-acp (9th) was 1.98 and that of act (4th) was 1.78). Finally, it was noted that Ct values for the best RGs tended to increase during the life cycle. This was particularly evident with the cDNA derived from adult barnacles, which required ~3 to 4 more cycles to reach the PCR exponential phase in comparison to the cDNA from larvae that had just hatched (Figure 3). While we cannot exclude the possibility that the genes analysed are down-regulated in the adult stage, this trend could also be explained by the presence of reverse transcription inhibitors that concentrate or are synthesized in later stages of B. amphitrite development, as RT-inhibitors are known to be one of the main sources of variability in qRT-PCR experiments . Although the CT value shifts remained in an acceptable range, it may be advisable to include a reference assay to rule out the presence of inhibitors. This is commonly achieved by adding an aliquot of the RNA under investigation to a well characterised exogenous RNA and measuring the effect on the amplification of the cDNA derived from the latter [24, 25].
Balanus amphitrite is already established as a model organism to study the pelagobenthic life cycle. Here, we have presented the first cDNA library sequenced from adult B. amphitrite. We are currently generating three further normalized EST libraries for the developmental stages of nauplius I, cyprid and adult, and we estimate that another 15,000 sequences will be available soon. The addition of this genetic information will serve as an invaluable tool to investigate gene expression in barnacles. The three programs implemented to analyse qRT-PCR results indicated that tuba, tubb and cp1 are unsuitable genes for data normalization. They also showed that mt-cyb itself, and the pair mt-cyb – mt-nd1, were the genes expressed most stably throughout life cycle of B. amphitrite, and so we recommend their use as reliable reference genes in future qRT-PCR experiments. Other genes that performed well in our analyses were mt-acp, rpl15, mt-nd4L, ef1a, ubc and act.
Balanus amphitrite, culturing and RNA extraction
Wild B. amphitrite adults were collected from Beaufort, North Carolina, USA (courtesy of Prof. D. Rittschof). Brood stocks were maintained in semi-static culture in UV-irradiated, 10 μm filtered natural seawater. The adults were fed on newly-hatched Artemia sp. nauplii (Artemia International LCC, U.S.A.). To obtain barnacle nauplii, the adults were placed in a tank of fresh seawater and released larvae were attracted to a point light source and collected by pipette over a 2 h interval. Nauplii were cultured at the density of ~1 larva ml-1 in an incubator at 28°C on a 12:12 light:dark cycle. The larvae were fed each day with 1 l of a Skeletonema costatum culture (~2 × 105 cells ml-1) until they reached the cyprid stage (approx. for 4–5 days). Cyprids were collected by filtering through a tier of filters (pore sizes of 350 and 250 μm) in order to discard undeveloped cyprids and microalgae, and stored at 6°C until use. The different developmental stages we studied were:
N-1) naupliar instar I – just hatched;
N-6) naupliar instar VI – three-eyed stage;
C-0) young cyprids – recently metamorphosed;
C-3) mature cyprids – these are standard larvae for settlement assays and they are maintained for 72 h at 6°C after the C-0 stage;
C-I) mature cyprids (same as C-3) that have been exposed to sea water containing 10-5 M of 3-isobutyl-1-methylxanthine (IBMX) at 28°C for two hours ;
J) juveniles collected ~24 hours after settlement onto glass slides;
All larvae were isolated under a dissecting microscope and placed in a 1.5 ml tube kept on ice. The tubes were centrifuged briefly and after the residual seawater was removed the larvae were resuspended in TRIzol (Invitrogen) prior to storage at -20°C. Settled juveniles were collected by scraping them off the glass slides using a sterile scalpel. For the adult stage, the pooled soft tissues of ten individuals were dissected and ground under liquid nitrogen prior to RNA extraction.
After the larval tissues were homogenized and crushed by pipetting and vigorous shaking, the total RNA was extracted from each biological replicate using 1 ml TRIzol. The extracted RNA was then stored in 1 ml of isopropanol at -20°C. Prior to cDNA synthesis the stored RNA was precipitated by centrifugation at 12,000 g for 5 min at 4°, washed twice with 1 ml of 70% ethanol and then resuspended in milliQ water. The RNA purity and quality were evaluated using a NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies) and the quality was confirmed by gel electrophoresis (RNA picture provided in additional file 4).
EST library creation and sequencing
Whole soft tissues of B. amphitrite were ground under liquid nitrogen and the total RNA was extracted using TRIzol as above. An EST library was then prepared by standard methods. Briefly, total RNA was first treated with DNase-1 to remove contaminating DNA, followed by a LiCl precipitation step. Messenger RNA was then purified from the total RNA pool prior to reverse transcription. The cDNA was prepared using the first strand synthesis primer 5'-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAG T17-3' (complementary to the poly-A mRNA tail), which contains an Xho-1 restriction site (in bold) to facilitate directional cloning of the 3' end of the ds-cDNA insert into the vector. The first strand synthesis used me5-dCTP rather than ordinary dCTP. Non-methylated dCTP was then used in the second strand reaction to make the complementary cDNA strand. This method prevents internal cleavage of the cDNA when the linker is digested subsequently with Xho-1. Prior to cloning, a double stranded linker containing a 5-Eco-R1 overhang (5'-OH-AATT CGGCACGAGG-3', overhang given in bold) was blunt-end ligated onto the ds-cDNA. The lack of phosphate on the 5' overhang for the Eco-RI linker prevented concatemerization during linker ligation (this was phosphorylated in a subsequent step). The linker-ligated cDNA was then digested with Xho-I and cloned directionally into the multiple cloning site of the plasmid vector pBluescript II SK+, previously linearised by digestion with the restriction enzymes Eco-RI and Xho-I. The library was cloned into DH5α cells and a total of 960 positive clones were randomly chosen to be sequenced. Plasmid DNA was prepared using a standard alkaline lysis plasmid prep . Plasmids were sequenced using Sanger method (ABI BigDye Chemistry) and the sequencing reactions were run commercially on either an ABI-3700 capillary an ABI-3730 capillary or an ABI-377xl slab gel instruments using plasmid specific primers (Amplicon Express, USA). Both the quality-clipping and the subsequent base calling steps on the sequences were performed using the Phred13 software . The average read lengths were 836 nucleotides for raw reads and 533 for high quality data.
Clustering, assembly and functional annotation of the EST library
The bioinformatics analysis of the cDNA library BA23840 was performed using the sequence analysis and management system SAMS-2.0 . We first applied a clustering step based on pair-wise comparison on the DNA level using the TIGR default parameters  to avoid redundancies in the dataset. Individual ESTs fall in the same cluster if they show a similarity of at least 95% over a region of not less than 40 bp in a pair-wise alignment and unmatched flanking regions must not exceed a length of 20 bp. Each cluster was then assembled using CAP3 , to produce 79 TCs and 530 singlets that were nearly free of redundancies and allowed the following functional analysis to be constructed within SAMS. After applying a modified GenDB  annotation pipeline consisting of a collection of standard bioinformatics tools including BLAST , HMMer  and InterPro  on each sequence, we applied Metanor , the GenDB automatic function prediction program. Regarding BLAST, while BlastX has been used for protein databases (NR, SP, Kegg and KOG), the BlastN algorithm was used for scanning the nucleotide database NT. By interpreting all the tool results we obtained, we created consistent functional annotations and assigned gene products, EC numbers, GO terms and KOG functional categories . Finally, TCs and singlets were manually checked and gene names were given whenever possible. High quality ESTs were deposited in the EMBL database, accession numbers form FM882258 to FM883162. Assembly sets were also deposited to the EMBL under accession numbers FM994549 to FM994627. Access to sequences annotation via the SAMS interface will be provided upon request to the authors.
Tentative contiguous sequences (TCs) for RGs were analysed by Primer3 release 1.1.0 http://fokker.wi.mit.edu/primer3/input.htm using the following parameters: a) product size range: 80–180; b) primer size: min 16, opt 19, max 22; c) primer Tm: minimum 55°, optimum 60°, maximum 65°; d) primer GC%: minimum 40, optimum 50, maximum 60; e) all other parameters were left as the default. Oligonucleotides (synthesized by Invitrogen) were resuspended as stock solutions containing 0.7 pmol/μl of both the forward and reverse primers. The cDNA obtained from adult RNA was initially used to visualize the melting curve of PCR products and to determine the possible formation of PCR artefacts. PCR products were also sequenced to confirm specificity.
cDNA synthesis, primer efficiencies and cycle parameter for qRT-PCR
We reverse transcribed 1 μg of total RNA from each sample for 20 min at 42°C with the QuantiTect kit (Qiagen). After the genomic wipe-out step and prior to the reverse transcription we collected 1 μl from each reaction to be later used as a negative-RT control to check for genomic contamination. Serial dilutions of 1:5, 1:10, 1:25, 1:50, 1:250, 1:500, 1:5000 and 1:50000 were then made from the initial 20 μl of adult cDNA. Each qRT-PCR experiments comprised 12.5 μl of Faststart SYBR green (Roche Diagnostics Ltd), 10.5 μl of stock primers (final concentration 0.3 μM each) and 2 μl of cDNA. Reactions were performed in sealed 96-well plates using a Chromo4 Research thermocyler and analyzed with the Opticon Monitor 3 software (BioRad).
The qRT-PCR thermal profile consisted of an initial step at 95°C for 5 min, followed by 40 cycles of 15 s, at 95°C and 1 min, at 60°C. A final elongation step at 72°C was included before the melting curve was determined by monitoring SYBR green fluorescence during the temperature ramp 60 to 95°C with an increase of 0.5°C and a hold of 1 s. We determined primer efficiencies using five cDNA dilution points for each primer pair that were chosen according to the expected expression level of the corresponding gene. Triplicates were tested for each dilution point and primer pair, together with a duplicate negative control that contained sterile water instead of cDNA. The resulting efficiency graphs are given in the additional file 1 accompanying this paper. To determine the best RGs, 2 μl of the cDNA diluted 1:50 were used for all 14 samples and primers tested.
qRT-PCR data analysis
When required, raw Ct values were transformed to relative quantities by a comparative method based on the formula: 1/E(Ct value-lowest Ct); where E is the primer efficiency and the lowest Ct refers to the smallest value obtained with each specific primer pair. The most stable RGs were then determined using software geNorm 3.5 , BestKeeper  and NormFinder .
The authors would like to thank Dr T. Taybi and Dr J. D. Barnes from the Institute for Environment and Sustainability, Newcastle University, for providing help and access to Real-Time PCR equipments. We also thank Dr V. Mittard Runte, K. Henckel and Dr A. Goesmann from the CeBiTec, Bielefeld University, for their support and contribution throughout the annotation process. This study was funded by Marine Genomics Europe, EU Network of Excellence award to ASC (ref: 505403).
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