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SUPPLEMENTARY DATA
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1. Design of specific PCR primers for gene expression study
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In order to design specific primers for quantitative RT-PCR analysis, a preliminary amplification of both housekeeping and target genes was
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performed using degenerate primers based on the conserved nucleotides of the same genes in Cerastoderma edule (Table S1). Specific primers
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for CO1 were designed from the sequence available in GENBANK (HQ432846). PCR were conducted using one microliter of reverse
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transcribed product and Taq polymerase (Promega) in a final volume of 25µl as following: 94°C for 2 min, 94°C for 30 sec, annealing
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temperature (Table S1) for 30 sec and 72°C for 30 sec during 40 cycles, followed by 72°C for 10 min. Amplified products of the expected sizes
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were excised from agarose gel and purified using the QIAEX II gel extraction kit (Qiagen), then cloned into pGEM-T plasmid vector (Promega)
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and sequenced by Beckman Coulter Genomics (Sanger sequencing). BLAST analysis confirmed the close sequence homology of these
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amplicons with the corresponding genes in different bivalve species. All sequences obtained were deposited on the EMBL databank upon
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accession number indicated in Table S1. Thereafter specific primers usable for quantitative PCR were defined using the Primer Express version
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3® software based on the characterized partial sequences (Table S2).
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2. Detailed protocol for real-time quantitative PCR
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Real-time PCR reactions were performed using a Step One plus apparatus (Applied Biosystems) in a final volume of 10 µl. The amplification
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program consisted of an activation of the polymerase at 95°C for 20 sec, and then 40 cycles of amplification at 95°C for 3 sec, and 60°C for 20
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sec. Each 10µL reaction contained 4.86 µL of diluted cDNA, 5µL of Fast SYBR Green Master Mix (Applied Biosystems) and 0.1 µM of each
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specific forward and reverse primers (Table S2). The absence of non-specific PCR products and primer dimmers was confirmed by examination
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of the dissociation curve generated after the amplification cycles were completed and by amplicon sequencing. This curve was obtained by
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following the SYBR Green fluorescence level during a gradual heating of the PCR products from 60 to 95°C. The threshold cycle (Ct) was
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determined by the Step One plus version 2.1 software. The efficiency of the PCR reaction was calculated for each gene using the Ct slope
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method, which involves generating a dilution series of the target template and determining the Ct value for each dilution. A plot of Ct versus log
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(concentration) was constructed and efficiency (E) expressed as E= 10(-1/slope). Out of the four housekeeping gene (HKG) candidates, the most
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stable were selected using the Bestkeeper Software (Pfaffl et al., 2004). This software determines the optimal housekeeping genes employing
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pair-wise correlations and calculates the geometric mean of the best suited ones for accurate normalization of the target genes (TG) The
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calculation of the relative expression (RE) was based on the comparative Ct method (Livak and Schmittgen, 2001), the calibrator being control
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cockles at time 0 (T0): RE= ((ETG) ΔCt TG)/(EHKG) ΔCt HKG) with ΔCt = CtT0- Ctsample (Pfaffl et al., 2004).
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3. Checking of PCR efficiency and housekeeping gene choice
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Efficiencies calculated for all genes (90≤E≤110) indicated correct PCR reactions without inhibition (Gašparič et al., 2008). Three out of the 4
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housekeeping genes studied were used to normalize the 7 target genes. Indeed, the Elongation Factor 1α displayed a high Ct standard deviation
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(>1) and weak pair-wise correlations (p>0.05). On the contrary, the 3 other genes (β-Actin, α-Tubulin, β-Tubulin) displayed a Ct standard
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deviation lower than 1 and high pair-wise correlations (p<0.001). A Bestkeeper index compiling these 3 housekeeping genes was calculated and
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used to normalize accurately the target gene transcriptions as recommended by Derveaux et al. (2010).
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References
Derveaux S, Vandesompele J, Hellemans J (2010) How to do successful gene expression analysis using real-time PCR. Methods 50:227-230. doi:
10.1016/j.ymeth.2009.11.001
Gašparič MB, 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. doi: 10.1186/1472-6750-8-26
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408.
doi: 10.1006/meth.2001.1262
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (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.
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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)
Elongation factor 1a
GGTKTTGGACAAACTGAAG
TGGCACYGTTCCAATACCTCC
60
HF947020
616
β Actin
CCGAAGCGTGGTTACTCATTCA
CGGGAAGCTCGTAGCTCTTCT
60
HF947015
154
α Tubulin
TTCAAGGTCGGCATCAACTACC
TATGCACACACGGCTCTCTGG
63
HF947013
83
β Tubulin
TGACCGTGGCCTGTATGTTC
CATTCGACGAAGTAGCTGCTGTT
62
HF947014
106
MT
TCGAATCTCAACAACCATCCAG
CCACCGACAAGTACTGGGCTCG
62
HF947022
322
ABCB1
TGAACTTGGCATGGCTGAGAT
TCTGTTTCTGTCCCCCACTC
60
HF947016
237
HSP70
AAAATCAGTGAAGAAGACAAGAAAACC
ATTCAAATTCTTCCTTCTCTGCAGAT
59
HF947019
100
CAT
TATGAAYGGMTAYGGWAGYCACAC
CAAAKGGRTTCCAHCKGAA
61
HF947021
260
MnSOD
TAATCACTCTGTCTTTTGGACGG
TACTCTTAATCGACCACTCACTGG
60
HF947018
196
CuZnSOD
TGGTCCAGATGGAAAAGCTGAAA
CCACCCTCATGAACCACAACTGT
60
HF947017
102
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Table S2. Specific primers used for qRT-PCR and length of amplified amplicons.
Gene
Elongation factor 1a
β Actin
α Tubulin
β Tubulin
MT
ABCB1
HSP70
CAT
CO1
MnSOD
CuZnSOD
Forward primer (5’-3’)
GGATGGCACGGAGACAACAT
CGAAGCGTGGTTACTCATTCAC
TTCAAGGTCGGCATCAACTACC
TGACCGTGGCCTGTATGTTC
GAAATGTAGCTGTTCCGGATCAT
AACATCGCCTACGGAGACAACT
AAAATCAGTGAAGAAGACAAGAAAACC
TCCATCTGAACTTCTCTGCCTCTT
CTTGGCTATTTTTGCGCTACATC
CGGTTCTCAGTCCGAATGGA
CCAGATGGAAAAGCTGAAATTAACA
Reverse primer (5’-3’)
TGGTCTTTCCAGAAGCGTTTC
GCCATTTCCTGCTCGAAGTC
ATGCACACACGGCTCTCTGG
CATTCGACGAAGTAGCTGCTGTT
CCTCGACCATGCAAGGTTAAC
CTGGCAGACTGGCAATAAACTG
ATTCAAATTCTTCCTTCTCTGCAGAT
TGTACAACGCCATTGAGAAAGG
CCATCGTTGTCTCTGTCGCATA
GTAAGCTCCGCCTTCATTTGA
CAGAAGCGCTCTCCTGTCAGA
Length (bp)
100
106
83
100
115
100
100
80
101
100
66
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53
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Table S3. Correlation matrix between Cd bioaccumulation, mRNA expression values and CI of cockles exposed to 50 µg/L of Cd and controls.
[Cd]
CAT CuZnSOD
MT
HSP70 ABCB1
[Cd]
1
0,558
0,419
0,549
0,595
0,358
CAT
1
0,463
0,558
0,522
0,472
0,551
CuZnSOD 0,419
1
0,638
0,522
0,361
0,439
MT
1
0,330
0,549
0,472
0,361
0,571
HSP70
0,463
0,638
1
0,187
0,595
0,571
ABCB1
0,330
0,187
1
0,358
0,551
0,439
COI
0,078
0,285
0,292
0,259
0,530
0,604
MnSOD
0,123
0,135
0,142
-0,092
0,301
0,440
CI
-0,222
0,066
-0,091
-0,189
-0,289 -0,100
Pearson’s correlation coefficients. p-values<0.05 are indicated in bold.
COI MnSOD
CI
0,078
0,123 -0,222
0,285
0,066
0,440
0,292
0,135 -0,091
0,142 -0,189
0,530
0,259
-0,092 -0,289
0,301 -0,100
0,604
1
0,011 -0,040
0,011
1
0,240
-0,040
0,240
1
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Table S4. Correlation matrix between Cd bioaccumulation, mRNA expression values and CI of cockles exposed to 5 mg/L of Cd and controls.
[Cd]
CAT CuZnSOD
MT
HSP70 ABCB1
[Cd]
1
-0,053
0,687
0,678
0,710
0,617
CAT
1
-0,277
0,687
0,586
0,704
0,719
CuZnSOD 0,678
1
0,014
0,586
0,640
0,509
MT
1
-0,040
0,710
0,704
0,640
0,758
HSP70
1
-0,139
0,617
0,719
0,509
0,758
ABCB1
-0,053
-0,277
0,014
-0,040
-0,139
1
COI
-0,299
-0,145
0,097
0,100
0,050
0,628
MnSOD
0,038
-0,141
-0,041
-0,120
-0,128
0,188
CI
-0,286
-0,178
-0,307
-0,300
-0,232 -0,084
Pearson’s correlation coefficients. p-values<0.05 are indicated in bold.
COI MnSOD
CI
-0,299
0,038 -0,286
-0,145 -0,141 -0,178
0,097
-0,041 -0,307
0,100
-0,120 -0,300
0,050
-0,128 -0,232
0,188 -0,084
0,628
1
-0,194 -0,026
-0,194
1
0,205
-0,026
0,205
1