Sureprint g3 human ge 8 60k microarray

Samples were processed for microarray analysis at the Core Instrumentation Facility of Keio University School of Medicine. Total RNA was extracted from cells with the use of the Trizol reagent (Invitrogen) and the RNeasy Mini Kit (Qiagen). Cy3-labeled cRNA probes were synthesized from the total RNA and subjected to hybridization with a SurePrint G3 Human GE 8 × 60 K microarray (Agilent). Raw intensity data for each experiment were analyzed with the use of GeneSpring GX software (Tomy Digital Biology). Microarray data are available in the GEO database under the accession number GSE113860.

Total RNA was extracted from cells using an RNeasy kit (Qiagen, Valencia, CA, USA) and subjected to a cDNA microarray analysis using SurePrint G3 Human GE 8 × 60 K Microarray (Agilent Technologies, Santa Clara, CA, USA). The mRNA expression of genes encoding enzymes involved in glutathione metabolism was also evaluated by RT-PCR. The genes analyzed in this study were glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), glutathione synthase (GSS), glutathione reductase (GSR) and glutathione S-transferase pi 1 (GSTP1). Primer sequences are listed in Table 1. RT-PCR reactions were performed using an RNA LA PCR kit (Takara). The PCR conditions were as follows: 94 °C for 5 min, then increasing cycle numbers of 94 °C for 30 s, 51 °C for 30 s and 72 °C for 1 min, and a final extension step at 72 °C for 7 min.Table 1

Sequences of primers used in RT-PCR analysis

Gene	Primer orientation	Sequence
GCLC	forward	5'-GTTCTCAAGTGGGGCGATGA-3'
	reverse	5'-CCGGCTTAGAAGCCCTTGAA-3'
GCLM	forward	5'-AAGTGCAGTTGACATGGCCT-3'
	reverse	5'-TGACCGAATACCGCAGTAGC-3'
GSS	forward	5'-GACCAGCGTGCCATAGAGAA-3'
	reverse	5'-ACCCTCTCTCTGGGGCTTTA-3'
GSR	forward	5'-GTGGCACTTGCGTGAATGTT-3'
	reverse	5'-AGTTTTCGGCCAGCAGCTAT-3'
GSTP1	forward	5'-CAAGTTCCAGGACGGAGACC-3'
	reverse	5'-GCCATTGATGGGGAGGTTCA-3'
GAPDH	forward	5'-ATCACCATCTTCCAGGAGCGA-3'
	reverse	5'-GCTTCACCACCTTCTTGATGT-3'

RNA microarray analysis of PDX tissue was performed as previously described [19 (link)]. Data are available at GEO accession number GSE41193. Total RNA samples were prepared and processed using Agilent's One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labelling v6.0. An input of 100 ng of total RNA was used to generate cyanine-3-labeled cRNA. Samples were hybridized on Agilent SurePrint G3 Human GE 8×60K Microarray (Design ID, 028004). Arrays were scanned with the Agilent DNA Microarray Scanner at 3-μm scan resolution and data were processed with Agilent Feature Extraction 11.0.1.1. The processed signal was quantile normalized with Agilent GeneSpring 12.0.

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA. The RNA integrity of each sample was evaluated by an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). A MaestroNano spectrophotometer (Maestrogen, Las Vegas, NV, USA) was used to check if A260/A230 and A260/A280 ratios of RNA ranged 1.8~2.1. Fresh samples of ampullary cancer and surrounding normal duodenal tissues were analyzed by a complementary (c)DNA microarray. During the in vitro transcription process, RNA from normal samples was labeled with Cy3, and that from ampullary adenocarcinomas was tagged with Cy5 dye (PerkinElmer, Waltham, MA, USA). An Agilent SurePrint G3 Human GE 8×60K microarray was hybridized with Cy-labeled cDNA. The scanning wavelength of the microarray was set to 535 nm for Cy3 and 625 nm for Cy5. Lowes' method with a rank-consistency filter was used to normalize scanned images. Data were analyzed with Agilent Genespring software as we previously described 19 (link)-22 (link).

Five pairs of ampullary adenocarcinoma and normal duodenal tissues isolated from the same patients were sent for cDNA microarray analysis (Additional file 2: Table S2). The RNA from normal duodenal tissue was labeled with Cy3 (CyDye, PerkinElmer, Waltham, MA USA), and the RNA from ampullary adenocarcinoma was labeled with Cy5 during the in vitro transcription process. A total of 0.3 μg of Cy-labeled cRNA was fragmented to an average size of 50–100 nucleotides and hybridized to an Agilent SurePrint G3 Human GE 8 × 60 K microarray (Agilent, Santa Clara, CA, USA) at 65 °C for 17 h. The microarrays were scanned at 535 nm for Cy3 and 625 nm for Cy5. Scanned images were analyzed and quantified. The results were substantially normalized using the rank-consistency-filtering LOWESS method, and the data was analyzed using GeneSpring software (Agilent). The Ingenuity Pathway Analysis (IPA6.0; Ingenuity Systems, Redwood City, CA, USA; www.ingenuity.com) was used to identify networks of interacting genes.

Gene expression profiling of cells were performed in biological triplicates with an Affymetrix (for LNCaP androgen deprivation) or Agilent (for STM-reprogrammed cells) human gene array platforms. Control parental LNCaP were grown in media supplemented with 10% charcoal-stripped serum (CSS) and 10 pM R1881, while androgen deprived LNCaP cells were cultured for 15 days in media supplemented with CSS alone before RNA extraction. RNAs were labeled using the WT labeling and control kit (Affymetrix) and were hybridized to GeneChip^® Human Gene 1.0 ST Arrays. Arrays were scanned using Affymetrix Microarray Scanner System. To compare gene expression between parental PCa cells and STM-reprogrammed cells (LNCaP, VCaP, 22rv1 and LAPC4), RNAs from biological triplicate samples of parental cells (cultured in phenol red-free media with FBS) and corresponding STM-reprogrammed cells (> 14 days in STM) were extracted. Samples for gene expression analysis were prepared following Agilent's One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling v6.0. An input of 100ng of total RNA was used to generate Cyanine-3 labeled cRNA. Samples were hybridized on Agilent SurePrint G3 Human GE 8×60K Microarray (AMDID 028004). Arrays were scanned with the Agilent DNA Microarray Scanner at a 3 μm scan resolution and data was processed with Agilent Feature Extraction 11.0.1.1.

To further investigate the specific genes affected by miR-145-5p and miR-145-3p, we performed a combination of in silico and genome-wide gene expression analyses. We attempted to identify target genes using a BC cell line transfected with these miRNAs. A Sure Print G3 Human GE 8 × 60K Microarray (Agilent Technologies) was used for expression profiling of miR-145-5p and miR-145-3p transfectants. The microarray data were deposited into GEO (http://www.ncbi.nlm.nih.gov/geo/) and were assigned GEO accession number GSE66498. Next, we selected putative miRNA target genes using the microRNA.org database (August, 2010 release, http://www.microrna.org). Finally, to identify upregulated genes in BC, we analyzed publicly available gene expression data sets in GEO (accession numbers: GSE11783, GSE31684). The data were normalized and analyzed with Gene Spring software (Agilent Technologies) as described previously [22 (link), 23 (link), 40 (link)–42 (link)]. The strategy for investigation of the target genes is shown in Figure 3.