Supplementary data
1. Design of specific PCR primers
Housekeeping and target genes were amplified using degenerate primers (Table S1) selected from conserved nucleotides of the same genes in several fish species phylogenetically close to Salariapavo (Table S1). For each PCR reaction, 1 µL of reverse transcribed product and Taq polymerase (Promega) were added in a final volume of 25 µL as follow: 94°C for 2 min, 94°C for 30 sec, annealing temperature (Table S1) for 30 sec and 72°C for 30 sec during 40 cycles, then 72°C for 10 min. Excision and purification steps were conducted from amplified products then, cloning was performed using pGEM-T plasmid vector (Promega). Sequencing was carried out by Beckman Coulter Genomics (Sanger sequencing). After sequence identifications, homology with corresponding genes identified from different fish species, was confirmed by BLAST analysis. Partial sequences obtained from peacock blennies were deposited in the EMBL databank (Accession Number in Table S1). For quantitative PCR, specific primers were identified using the Primer Express version 3® software (Table S2).
2. Real-time quantitative PCR (RT-QPCR)
Reactions of RT-QPCR were conducted using a Step One plus apparatus (Applied Biosystems). Activation of the polymerase was performed at 95 °C for 2 min followed by 45 cycles of amplifications at 95°C for 15 sec then, 60°C for 1 min. Each reaction was performed in a final volume of 15 µL containing 7 µL of diluted cDNA, 7.7 µL of SYBR Green Master Mix (Applied Biosystems) and 0.1 µM of each specific forward and reverse primer (Table S2). The dissociation curve was obtained after each amplification reaction by following the SYBR Green fluorescence level. The threshold cycles (Ct) was determined using the Step One plus version 2.1 software. The Ct slope method allowed the calculation of PCR reaction efficiency (E) for each gene that was expressed as: E= 10(-1/slope). Among 4 housekeeping genes (HKG) analysed in the current study, best ones were selected using the Bestkeeper Software (Pfaffl et al., 2004) which also allowed the normalization of target genes (TG). According to Livak and Schmittgen (2001), relative expression (RE) was determined by comparing the Ct method. Considering peacock blenny controls at time 0 (T0) as calibrator, relative expression (RE) was calculated as follow:RE= ((ETG) ΔCt TG)/(EHKG)ΔCt HKG) with ΔCt= CtT0- Ctsample(Pfaffl et al., 2004).
3. Checking of PCR efficiency and choice of housekeeping gene
When calculated efficiency is between 90 and 110%, it means that PCR reactions are excellent without any inhibition (Gašparičet al., 2008). Six target genes were normalized using 2 housekeeping genesselected as the most stable genes (EF1a and β-actin). They displayed a Ct standard deviation lower than 1 in addition to high pair-wise correlations (p<0.001). According to Derveaux et al. (2010), normalization of target gene transcriptions was carried out using the Bestkeeper index compiling EF1a and β-actin.
References
Derveaux S., Vandesompele J., Hellemans J. (2010), How to do successful gene expression analysis using real-time PCR, Methods, 50:227-230.
Gašparič M.B., Cankar K., Žel J., Gruden K. (2008), Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms, BMC Biotechnology, 8:26.
Pfaffl M.W., Tichopad A., Prgomet C., Neuvians T.P. (2004), Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol. Lett., 26:509-515.
Table S1. Primers used for cDNA fragment characterizations for the different genes; T (°C), annealing temperature; length (bp), amplified fragment length.
Gene / Forward primer (5’-3’) / Reverse primer (5’-3’) / T (°C) / Accession N° / Length (bp)EF1a / TTCATCAAGAACATGATCACTGG / TTCTTGATGTARGYGCTCACTTCC / 58 / LN864688 / 248
β -actin / TCATGAAGATCCTGACAGAGCG / ATTGCCRATRGTGATGACCTG / 58 / LN864686 / 191
α-tubulin / ACTCYGACTGYGCCTTCATGG / AAGTTGGTCTGGAACTCBGTCA / 58 / LN864687 / 186
18s / ATCCGGCGGCGTTATTC / TTCCTTTAAGTTTCAGCTTTGCAA / 58 / FN356969 / 101
mt1 / ATGGACCCTTGCGACTGCKCCAA / TCTTCCCTTTGCASACRCAGCC / 58 / LN864697 / 157
mt2 / AAACTGCTCCTGCACCASCTGCAA / ACAAGTCTTCCCTTTGCASACGC / 58 / LN864698 / 100
mnsod / ACCACAGCAAGCACCATGCCAC / AAGATRGTGTGGTTAATGTGGCC / 58 / LN864694 / 150
cuznsod / GAAGAACYATGGTGATYCAYGAG / CCACAGGCYAGRCGTCCACCAG / 58 / LN864693 / 100
cat / AACTACTTTGCRGAGGTGGAGC / GTTGGGATAGTARTTTGGAGC / 58 / LN864695 / 252
gpx / TGCAAYCAGTTYGGVCATCAGGAG / TGATCAGCTTGGGGTCGTTGATC / 58 / LN864696 / 220
Table S2. Specific primers used for qRT-PCR and length of amplified amplicons.
Gene / Forward primer (5’-3’) / Reverse primer (5’-3’) / Length (bp)EF 1a / GCTGACTGTGCTGGGCTGAT / TCACGGGTCTGACCGTTCTT / 80
β -actin / TGACAGAGCGTGGCTATTCCT / CCCATCTCCTGCTCGAAGTC / 106
α-tubulin / CCGCAGGAACCTGGACAT / CGAGACAATCTGGCCAATGA / 70
mt1 / TGCAACTGCGGATCATCCT / GCAGCTCTTCTTGCAGTTGGT / 63
mt2 / CAGCTGCAAGAAGAGCTGCTG / TTTGCACACGCAGCCAGAGGC / 73
mnsod / CACCATGCCACTTATGTCAACA / GCAGTCACATCTCCCTTTGC / 80
cuznsod / TGATTCACGAGAAGGCTGATGA / TTACCCGTTTTCAGGCTCTCTT / 64
cat / ACCGCCTGGGAGCAAAC / GCCACCTTGGTTGTCAAACA / 110
gpx / CCTTGAGAAGGTGGACGTGAA / TCCGTCAGGAACGCAAACA / 60