Beadarray reader

RNA was extracted from 28 tumors using MagAttract RNA Universal Tissue M48 Kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol using BioRobot M48. RNA concentrations were determined by spectrophotometry (NanoDrop, Thermo Scientific, Wilmingron, DE, USA) and quality was analysed using the 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA, USA).
Two hundred ng of total RNA from each sample was used for cRNA production by the Illumina TotalPrep RNA amplification kit (Ambion Inc., St. Austin, TX, USA) according to the provided protocol. The quality of purified cRNA was evaluated using the RNA 6000 p kit (Agilent Technologies) in the Agilent 2100 Bioanalyzer (Agilent Technologies). A total of 750 ng biotinylated cRNA was hybridized to the human HT12 Illumina Beadchip gene expression array (Illumina, San Diego, CA, USA) according to manufacturer’s protocol and scanned using the Illumina Bead Array Reader (Illumina). Expression array data was analysed using the Illumina BeadStudio V2011.1 software and samples were normalized using the cubic spline algorithm. Gene expression levels of the MX2 (ILMN_2231928), SMAD6 (ILMN_1767068) and SOCS3 (ILMN_1781001) genes were extracted from the arrays.

Total RNA was isolated from TRPC1 silencing vector (siTRPC1) and empty vector-transfected (control) cells following 48-h incubation using the instructions provided in the High Pure RNA Isolation Kit (Roche Applied Science). The incubation time was chosen based on our previous report [16 (link)]. 500 ng total RNA was amplified and biotin labelled using the Illumina Total Prep RNA Amplification Kit (Ambion). Biotinylated cRNA (750 ng) was hybridised at 58 °C for 16 h to HumanHT-12 v3 expression BeadChip (Direct Hybridization Assay Kit, Illumina). The BeadChip was washed, blocked, and scanned using (Illumina BeadArray Reader), and Cy3 signal intensity was measured.
Data quality was assessed using GenomeStudio, all system control values were within the expected ranges. Background fluorescence representing signals from non-specific dye binding and/or cross-hybridization were subtracted from all other probe intensities using GenomeStudio. R and BioConductor packages were used for analysis. Following quantile normalisation using lumi, Rank Product analysis [19 (link)] was performed using the RankProd package to determine differentially expressed genes. Pathway analysis was performed using DAVID Bioinformatics Resources 6.7 (functional annotation clustering) [20 (link)]. Raw and processed microarray data have been submitted to GEO database (GSE77386).

Rats which had been operated successfully were randomly divided into the following groups: Sham, MCAO, MCAO + EA group (n = 6 for each group). After reperfusion, the rats received EA treatment. At 28 d post-MCAO, RNA was isolated from each cerebral ischemic penumbra using the mirVana miRNA Isolation Kit (Ambion, USA). The miRNA microarray was implemented according to MicroRNA Expression Profiling Assay Guide (Illumina Inc., USA). After adding a stretch of poly A tail to the 3′-end of each sequence in the 1000 ng intact total RNA sample. Then the biotinylated cDNAs were linked with miRNA specific oligos in an assay specific extension plate. The oligos were hybridized to the cDNA, mis-hybridized and excess oligos were washed away. In this process, the BeadChips were hybridized using the hybridization chamber. The BeadChips were hybridized overnight in the Illumina hybridization oven, with a temperature ramp from 60 °C to 45 °C. The Illumina BeadArray Reader (Illumina Inc.) used a laser to excite the fluor of the single-base extension product on the beads of the BeadChip sections.

Gene expression profiles of frozen tissue specimens from 12 IIP patients, derived from the central part of the surgical lung biopsy, were analyzed by GP Biosciences Ltd. (Kanagawa, Japan). RNA quality was verified using the RNA6000 Nano Assay on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Illumina Human WG-6 v3 BeadArrays (Illumina Inc., San Diego, CA, USA) with about 48,000 transcripts were used according to the manufacturer’s instructions. An Illumina TotalPrep RNA amplification kit (Ambion, Inc., Austin, TX, USA) was used to obtain biotin-labeled cRNA from 500 ng total RNA. As a control probe, normal human lung poly(A) RNA (BD Biosciences Clontech) was amplified under the same conditions. cRNA was synthesized overnight (18 h), labeled, and hybridized to the chip at 58 °C overnight. Hybridized arrays were labeled with streptavidin-Cy3 (PA43001; Amersham, Buckinghamshire, UK) and scanned with an Illumina BeadArray reader (Illumina Inc.). Scanned images were imported into BeadStudio v3 software (Illumina Inc.) for extraction, quality adjustment, and quantile normalization. Satisfactory quality was observed for all arrays and samples.

Gene transcription profiling for the BN, GK, and chromosome 1 congenic strains was performed using Sentrix® BeadChip RatRef-12 v1 Whole-Genome Gene Expression Arrays (Illumina Inc., San Diego, California, USA).
Double-stranded cDNA and purified biotin-labeled cRNA were synthesized from 300 ng high quality total RNA using the Illumina® TotalPrep RNA Amplification Kit (Ambion Inc., Austin, Texas, USA). cRNA concentrations were determined using a NanoDrop spectrophotometer whilst cRNA quality and integrity were assessed using an Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany). Hybridizations onto Sentrix® BeadChip RatRef-12 v1 Arrays were carried out using 750 ng of each biotinylated cRNA in a 58°C hybridization oven for 18 hours. Following washing and staining with Streptavidin-Cy3, the BeadChip Arrays were scanned on the Illumina® BeadArray Reader (Illumina Inc., San Diego, USA). Resulting data were then preliminarily analysed using the Illumina® BeadStudio Application software before undergoing comprehensive statistical analysis.
Microarray experiments were compliant with MIAME (Minimum Information About a Microarray Experiment) and both protocol details and raw data have been deposited in ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) under the accession number E-MTAB-1048.