Total RNA from individual livers were isolated by TRIzol method (Invitrogen, Carlsbad, CA) couped with the GenEluteTM Mammalian Total kit from SIGMA. RNA integrity was examined by electrophoresis, and concentrations were determined by spectrophotometry. Absence of genomic DNA contaminations was checked by PCR amplification of RNA samples without prior cDNA synthesis.
Label
Cy3
Label protocol
Agilent low RNA input linear amplification kit PLUS, one-color –cyanine 3-CTP-was used for generating fluorescent complimentary RNA (cRNA) along with Agilent's One-Color RNA Spike-in Kit following the manufacturer's protocols. A total of 1 μg of pooled RNA (n=9) was used for the cDNA synthesis of each four technical replicates. Then, the samples were simultaneously amplified and labeled with cyanine 3-CTP. Qiagen´s RNeasy mini spin columns were used for the purifications of the labeled/amplified cRNA samples. Then, cRNAs were quantified using NanoDrop® ND-1000 spectrophotometer with the Nanodrop 3.0.1 software. The examined yield and specific activity of each reaction were always higher than the recommended values to proceed to the hybridization step.
Hybridization protocol
Hybridizations were carried out in Whole mouse genome oligonucleotide microarrays, 4x44K (4 arrays/slide), 60-mer (Agilent) using the Agilent gene expression hybridization kit with GEx hybridization buffer HI-RPM, according to the manufacturer´s protocol. The fragmentation step used 1.65 μg of cyanine 3-labeled, linearly amplified cRNA and the hybridization were for 17 h in a rotating hybridization oven. Washes were conducted as recommended by the manufacturer using Agilent gene expression wash buffer kit.
Scan protocol
Slides were dried with helium gas and immediately scanned with the GenePix® 4000B microarray scanner (Axon Instruments Inc. Foster City, CA) with GenePixTM Pro 4.1 software, Molecular Devices using the following parameters: 532 nm, 5 μm of resolution, 100 % laser power at various photomultiplier tube (PMT) gains (or voltage settings). The subsequent analysis was performed with that images that had the highest number of valid spots, that is, PMT of 450 V for SOL arrays and 500 V for PS , DR1. DR4 and DR6 arrays.
Description
Gene expression of pooled total RNA (n= 9) of male Mus spretus livers captured in a polluted site
Data processing
Spot intensities and other quality control features were extracted with Agilent's Feature Extraction Software version 9.5.3.1, using defaults for all parameters. To analyze the array data GeneSpring GX v. 9.0.2 (Agilent Technologies, Inc., Santa Clara, CA) was utilized. Briefly, a new experiment was generated after importing data. For preprocessing of input data defaults parameters were used, that is, threshold raw signals: 5 –minimum signal intensity-; normalization algorithms: median shift to percentile 75 of the measurements taken from each chip to reduce chip-wide variation in intensity (per chip normalization) and finally baseline transformation option: baseline to median of all samples –for each probe the median of the log summarized values from all the samples is calculated and subtracted from each of the samples- (per gene normalization). Only data from spots designated as “present” were used. Entities in which at least 75% of the values in any 2 out of 2 or 4 conditions (SOL and PS or SOL and DR1, DR4, DR6) had acceptable values were retained for further analysis. To define a set of statistically significant, differentially expressed genes, a unpaired t-test was applied with Bonferroni FWER (family-wise error rate, type of Error control that allow very few occurrences of false positives) as the multiple testing correction (p= 0.01) (asymptotic p-value computation). Fold changes were calculated as the ratio between the signal averages of polluted site and control SOL site. The normalized data on the logarithm base 2 scales were used in the proceeding analyses. A filter on the fold change (probes with fold differences ≥ 2 were considered significant) was next applied to the list of genes and a new list of genes was generated containing genes that exhibited ≥ 2-fold increase or decrease expression compared with control.
