For the analysis of the patients, CytoScan 750K Array Kit from Affymetrix was used, which was covering a total number of 750,436 markers with 200,436 SNP markers. Among these probes, 63 are covering PCDH19 gene. The purified DNA was then fragmented, labelled and hybridised overnight on to an array. The arrays were washed using an Affymetrix® GeneChip® Fluidics Station and scanned using an Affymetrix® GeneChip® scanner. gDNA was digested with Nsp1 and then ligated to a common oligonucleotide adaptor for amplification by PCR. After purification, the PCR products were fragmented and then labelled with a biotinylated deoxynucleotide analogue using the TdT enzyme followed by overnight hybridisation to the array. The arrays were washed using an Affymetrix® GeneChip® Fluidics Station (which included DNA selective staining with a streptadivin conjugated reporter molecule) and then scanned using an Affymetrix® GeneChip® scanner. The data files generated for each sample were analysed using Chromosome Analysis Suite (ChAS) Software (v1.0.1 or v1.2.2). All results were subjected to quality control with regard to SNPQC, MAPD, waviness, sex and signal intensity.
Genechip scanner
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneChip Scanner is a laboratory instrument used to detect and analyze gene expression data generated from DNA microarray experiments. It scans microarray slides and measures the intensity of fluorescent signals emitted by labeled samples, providing quantitative data on gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Fluidics station 450, by Thermo Fisher Scientific (9 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (9 mentions)
Genechip fluidics station, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Genechip wt plus reagent kit, by Thermo Fisher Scientific (5 mentions)
Ambion wt expression kit, by Thermo Fisher Scientific (5 mentions)
Fluidics station, by Thermo Fisher Scientific (4 mentions)
Genechip human genome u133 plus 2.0 array, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Genechip command console, by Thermo Fisher Scientific (3 mentions)
Human gene 1.0 st array, by Thermo Fisher Scientific (3 mentions)
Genespring 7, by Agilent Technologies (3 mentions)
Affymetrix rat genome 430 2.0 array, by Thermo Fisher Scientific (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Genechip hybridization oven 640, by Thermo Fisher Scientific (2 mentions)
Genechip mirna 4.0 array, by Thermo Fisher Scientific (2 mentions)
Wt expression kit, by Thermo Fisher Scientific (2 mentions)
Genechip mirna 2.0 array, by Thermo Fisher Scientific (2 mentions)
85 protocols using genechip scanner
1
Chromosomal Analysis Using CytoScan 750K
2
Microarray Analysis of Xenopus Embryo RNA
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RNA was isolated from control embryos and knockdown as described above at early gastrula stage 10.5. The quality of the RNA was verified using the Agilent 2011 Bioanalyzer (Agilent). Samples were processed in the University of York, Department of Biology Technology Facility. One microgram samples were processed in the University of York, Department of Biology Technology Facility. RNA was labelled using HSR FlashTag Biotin RNA labelling kit (Genisphere) according to manufacturer's instructions, which included the addition of spike‐in RNA controls to act as a method control. Samples were then hybridised to Genechips miRNA 2.0 (Affymetrix) overnight, and washed on a Fluidics Station 450 (Affymetrix), all carried out according to manufacturer's instructions. Scanning of the chips was carried out using an Affymetrix Genechip Scanner. CEL files were processed using Affymetrix QC tools software to provide background detection and quantile normalisation with a final median polish and log transformation. Xenopus feature data were extracted and statistical comparisons undertaken using a paired, two‐tail Student's t‐test. The complete triplicate summarization data set for the Xenopus features are shown in Supplementary Table S1. These data have been deposited in the ArrayExpress Archive (https://www.ebi.ac.uk/arrayexpress/ ) with accession number E‐MTAB‐3936.
3
Microarray analysis of INH-treated Hep3B cells
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Whole genome expression studies were performed by Microarray analysis. Total RNA from untreated and INH treated (20 mM) Hep3B cells was isolated manually by TriZol (Invitrogen). RNA quality and quantity were checked by formaldehyde gel electrophoresis and spectrophotometer respectively. RNA samples with approximately 2:1 ratio of 28 S: 18 S rRNA and 260/280 values ≥1.8 were used for gene expression analysis. First strand was synthesized from total RNA and then second strand was synthesized. Then cRNA was synthesized by in vitro transcription and purified. Further sense strand cDNA was synthesized from cRNA and purified. Now cDNA was fragmented, labeled and loaded on Human Gene 1.0 ST Array Chip and sealed with white tough tag. For hybridization, Chip was transferred to the Hybridization Chamber (Affymetrix Gene Chip Hybridization Oven 645) and incubated for 16 h at 45 °C with 60 rpm speed. After hybridization, Human Gene 1.0 ST Array Chip was washed in wash chamber (Affymetrix Gene Chip Fluidisc Station 450) to remove the unbounded fragmented cDNA. After washing, array was scanned (Affymetrix Gene Chip Scanner) and Microarray Data was analyzed by Gene Spring software and pathway analysis was performed by Ingunity Pathway Analysis (IPA) software from Qiagen. Eight differentially regulated genes were validated by quantitative real time PCR.
4
Microarray Analysis of Glioma and Normal Cells
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NHAs were grown as adherent cultures and glioma TS cells were grown as spheroids for 4 days prior to harvesting for RNA using the RNeasy Mini Kit (Qiagen). RNA purity and quality was quantitated using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano Kit (Agilent). RNA was only used for microarrays if it had 260/280 ratios of 1.7–2.1, 28S/18 S ratios around 2, and an RNA integrity number of at least 7. Gene expression profiling was performed on PrimeView Microarrays using the GeneChip 3′ IVT Express Labeling Kit (Affymetrix). Microarrays were stained and washed on a GeneChip Fluidics Station 450 and scanned on a GeneChip Scanner (Affymetrix). See SI Text for microarray processing and data analysis and for quantitative PCR. Gene expression profiling data can be found in the GEO repository at http://www.ncbi.nlm.nih.gov/geo , Accession Number GSE79097.
5
RNA Isolation and Microarray Analysis of GC Tissue
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RNA from fresh-frozen GC tissue (n = 99) collected at the time of surgery was isolated with Trizol (Invitrogen) and column chromatography (RNeasy, Qiagen). Microarrays were hybridized using U133+ 2 chips (Affymetrix) and scanned with the Genechip Scanner (Affymetrix). Data were previously submitted to Gene Expression Omnibus (GEO; Series GSE5110510 (link)).
6
Microarray Analysis of Mesenchymal Stem Cells
7
Microarray Analysis of Mouse Intestine
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The GeneChip mouse 430 2.0 array; one-cycle target labeling and control reagents kit; and hybridization, wash, and stain kit were purchased from Affymetrix (Santa Clara, CA). Total RNA (5 μg) from the upper and lower intestine samples from five randomly selected mice was processed to prepare biotin-labeled complementary RNA using one-cycle target labeling and control reagents kit according to the manufacturer’s instructions. Biotin-labeled cRNA was fragmented and used as a probe for hybridization to the GeneChip mouse 430 2.0 array. Fluorescence data were scanned by a GeneChip Scanner controlled by the GeneChip operating software (GCOS) 1.2 (Affymetrix).
8
Affymetrix miRNA Expression Analysis
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Biotinylated RNA strands were hybridized at 48 °C for 18 h on an Affymetrix GeneChip miRNA 4.0 Array (Affymetrix, Santa Clara, CA, US). The arrays were analyzed using an Affymetrix GeneChip scanner with associated software. The miRNA expression levels were calculated using Transcriptome Analysis Console. Relative signal intensities were generated for each miRNA using the Robust Multi Array Average algorithm. The microarray data were analyzed using a row z-score.
9
Gene Expression Profiling by Microarray
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Gene expression profiles were determined using Whole-transcript Expression Analysis on GeneChip® Human Gene 2.0 ST Arrays (Affymetrix). Total RNA was extracted 48hours after lentiviral infection using the RNeasy Mini Kit (Qiagen). cDNA was generated using the WT Expression Kit (Ambion) and labeled using the GeneChip WT Terminal Labeling Kit (Affymetrix). The hybridisation protocols were performed on GeneChip Fluidics Station 450, and scanned using the Affymetrix GeneChip Scanner. Expression data was pre-processed and normalised using ‘affy’ and the RMA algorithm [32 (link)] and differential expression determined using ‘limma’ [33 ]. Unless otherwise stated, significant difference of expression is taken as q < 0.05 (false discovery rate adjusted) and fold-change greater than +/- 1.5.
For qRT-PCR, RNA was reverse transcribed using Protoscript M-MuLV First Strand cDNA Synthesis Kit (New England BIolabs) and analysed by quantitative real-time PCR (qPCR) with SYBR green fluorescence. Data were normalized to Actin and GAPDH expression as indicated. See supplementary experimental procedures for primer sequences.
Microarray data has been deposited in GEO as GSE53501 and ChIP-seq. data in EMBL-EBI as SRP035339.
For qRT-PCR, RNA was reverse transcribed using Protoscript M-MuLV First Strand cDNA Synthesis Kit (New England BIolabs) and analysed by quantitative real-time PCR (qPCR) with SYBR green fluorescence. Data were normalized to Actin and GAPDH expression as indicated. See supplementary experimental procedures for primer sequences.
Microarray data has been deposited in GEO as GSE53501 and ChIP-seq. data in EMBL-EBI as SRP035339.
10
Profiling FFPE Tumor miRNA Expression
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Total RNA was extracted from 20-μm-thick FFPE tumor sections using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE samples (Ambion, USA). Samples were then hybridized to Affymetrix GeneChip miRNA 2.0 Array (Affymetrix, USA), following the manufacturer instructions. Briefly, RNA samples were subjected to two cycles of cDNA conversion, amplified, and labeled with biotinylated ribonucleotide analogues, generating cRNA single strands. Synthesized strands were then purified, heat-induced fragmented, and finally hybridized to the microarray chip. Microarrays were scanned in a GeneChip Scanner (Affymetrix, USA), and data acquisition was performed by GeneChip Command Console (Affymetrix, USA).
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