Supplementary material
Citrate gold nanoparticle exposure in the marine bivalve Ruditapes philippinarum: Uptake,
elimination and oxidative stress response
Environmental Science and Pollution Research
Moritz Volland1*, Miriam Hampel2,3, Juan A. Martos-Sitcha1, Chiara Trombini1, Gonzalo Martínez-Rodríguez1, Julián
Blasco1
1
Instituto de Ciencias Marinas de Andalucia (CSIC), 11510 Puerto Real (Cadiz), Spain
2
Department for Physical Chemistry, Faculty of Marine and Environmental Sciences, University of Cadiz, Polígono Rio
San Pedro s/n, 11510 Puerto Real, Cadiz, Spain
3
Andalusian Center of Marine Science and Technology (CACYTMAR), Campus Universitario de Puerto Real, 11510
Puerto Real, Cadiz, Spain
Corresponding author: Moritz Volland*
E-mail: moritz.volland@icman.csic.es
Telephone: 0034 956832612 Ext. 257
Telefax: 0034 956 834701
TABLE OF CONTENTS
Supplementary material and methods
AuNP suspensions preparation and characterization
RNA extraction, cDNA synthesis and q-PCR protocol
Supplementary references
Supplementary material and methods
AuNP suspensions preparation and characterization
A 300 mg L-1 (as Au) AuNPs suspension was prepared based on the synthesis method by Frens
(1972), as adapted from Turkevich et al. (1951). In detail, 0.031 g of H(AuCl4) 3H2O (Aldrich,
99,9%) was dissolved in 50 mL Milli-Q and heated to boiling under reflux conditions. Following
the addition of 0.052g sodium citrate (Sigma Aldrich, 99%) dissolved in 1 mL Milli-Q water, the
solution was allowed to stand for 10 min during which the reduction to Au0 took place. From this
starting solution, dilutions were made immediately after preparation. From this starting solution
stock dilutions were prepared immediately, without allowing the colloid to age.
AuNP suspensions were characterized by UV-VIS spectroscopy (Perkin Elmer Lambda 12,
PerkinElmer, Waltham, USA), as well as transmission electron microscopy (TEM). Primary particle
size and morphology were characterized using a TEM microscope (Philips CM200) operating at 200
kV. For sample preparation, 5-10 µL of a particular AuNP suspension was pipetted onto a carbon
coated copper TEM grid and left to dry in air. The average diameter of 21.5 ± 2.9 nm was
established using the Gatan Digital Micrograph program (Gatan, Pleasanton, United States)
following a Lognormal fitting of the size distribution histogram, based on a minimum of four
independent measurements of a minimum of 50 particles.
RNA extraction, cDNA synthesis and q-PCR protocol
Total RNA was extracted from R. philippinarum digestive gland tissue (n=1, 50-100 mg) in 1 mL
Tri Reagent® (Sigma Aldrich), using an Ultra-Turrax® T10 (IKA®-Werke, Staufen, Germany)
applying the Sigma® standard protocol. Following RNA concentration determination in a Qubit®
2.0 Fluorometer using a Qubit® RNA Assay Kit (Invitrogen), RNA clean-up was performed using
the RNeasy® Mini Kit (Qiagen) according to manufacturer’s instructions. In short, a volume of 100
µL of RNA containing less than 1 µg µL-1 was added to 450 µL RLT buffer and 175 µL ethanol
(99%), loaded on a spin column, washed two times with 500 µL RPE buffer and finally eluted in 50
µL RNAse free water. RNA quantity was measured as described above and its quality was
determined in a Bioanalyzer 2100 with the RNA 6000 Nano kit (Agilent Technologies). Total RNA
(750 ng) was reverse-transcribed in 20 µL reaction volume using the QuantiTect® Reverse
Transcription Kit (Qiagen) following the standard protocol. Subsequently cDNA was diluted 15-fold
to obtain the final concentration of 2.5 ng µL-1 of input RNA, using TE buffer (20 mM Tris and 1
mM EDTA, pH 8.0 in sterile H2O), aliquoted and stored at -80° C for quantification analysis.
For the relative quantification of gene expression using real-time PCR (q-PCR) of selected genes
(Table 1) specific primer pairs were designed using Primer 3 version 4.0.0 software (Untergasser et
al. 2012) (available at http://bioinfo.ut.ee), unless stated otherwise. Subsequently their
oligonucleotide sequences were synthesized by Biomers®. For metallothionein (MT: AJ249686)
primers were used as described by Binias et al. (2014), whereas for Glutathione S-transferase (GST:
FJ516741.2) and β-Actin (EF520696.1) primers were used as described by Xu et al. (2010) and for
Allograft Inflammatory Factor-1 (AIF1: GQ384410.1) as described by Zhang et al (2011). In order
to optimize the q-PCR conditions, two different primer concentrations (200 and 400 nM), as well as
a primer annealing temperature gradient (48 – 65 °C) were applied. Further, to assess assay linearity
(r2) and amplification efficiency (E) different cDNA concentrations, as estimated from a six 1:5 step
dilution series of input RNA (1 ng µL-1, 0.2 ng µL-1, 40 pg µL-1, 8 pg µL-1, 1.6 pg µL-1, 0.32 pg µL1
), were measured in triplicates, with r2 ? 0.99 and E: 0.93 - 1.12 (see Table 1) for a minimum range
of 1 ng µL-1 to 8 pg µL-1. To perform q-PCR tests, 4 µl of each putative cDNA concentration, 0.5 µl
specific forward and reverse primers (previously diluted till both test concentrations used), and 5 µl
of Maxima SYBR® Green q-PCR Master MixTM (Thermo Scientific) were used. q-PCR reactions
(15 min at 95 °C; 40 cycles of denaturing for 15 sec at 95 °C, annealing for 30 sec at 48 – 65 °C and
extension for 30 sec at 72 °C; and a final melting curve from 60 °C to 95 °C for 20 min) was
performed on a Mastercycler®epgradient S Realplex2 with Realplex software (Eppendorf, version
2.2). The melting curve was used to ensure that a single product was amplified by each primer pair.
Moreover, two negative controls, using RNA (10 ng per reaction) and sterile water, were performed
to detect possible gDNA contamination or primer-dimer artefacts. Despite gene expression was
linear for all primer pairs used from 1 ng µL-1 to ?8 pg µL-1 cDNA per reaction, 1 ng µL-1 cDNA
was used for all reactions. q-PCR was carried out with a Fluorescent Quantitative Detection system
(Eppendorf Mastercycler ep realplex 2S). Each reaction mixture (10 µL) was treated as described
above for primer optimization, except for the annealing, which was established at 60° C for all
reactions.
R. philippinarum (Xu et al. 2010, Zhang et al. 2011, Tao et al. 2013, Binias et al. 2014) and also
owing its low variability (less than 0.3 CT) under our experimental conditions, β-actin
(EF520696.1) was used to normalize the results. In addition two-way analysis of variance identified
no significant differences between treatment and control (p = 0.595), between days of exposure (p =
0.392), nor an interaction between treatments and days of exposure (p = 0.377). Relative gene
quantification was performed using the 2-ΔΔCT method (Livak and Schmittgen 2001). Relative fold
changes (FC) in gene expression of AuNP treated digestive gland tissue samples (n=3) compared to
their respective control were calculated as follows: FC = Treatment 2-ΔΔCT/Control 2-ΔΔCT.
Supplementary references
Frens, G. (1972) Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold
Suspensions. Nature 24: 20-2
Turkevich J, Stevenson PC, Hillier J (1951) A study of the nucleation and growth processes in the
synthesis of colloidal gold. Discuss Faraday Soc 11: 55-75