Total RNA was isolated with the RNAqueous-96 Automated Kit (Ambion) on the Janus automated liquid handling system (Perkin Elmer Inc.), quantified by NanoDrop 6000 spectrophotometer and quality checked by Agilent Bioanalyzer. 300ng of each of the samples with RIN value >7 were converted to biotinylated cRNA with the Illumina TotalPrep-96 RNA Amplification Kit (Ambion) using a standard T7-based amplification protocol and hybridized on the Human Genome U219 96-Array Plate (Affymetrix). Hybridization, washing, staining and scanning of the array plates were performed on the GeneTitan Instrument (Affymetrix) according to manufacturer’s protocols.
Genetitan instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneTitan instrument is a fully automated microarray platform designed for high-throughput gene expression analysis. It automates the entire workflow, from sample preparation to data analysis, to provide efficient and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Ambion wt expression kit, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Affymetrix genechip wt terminal labeling kit, by Thermo Fisher Scientific (2 mentions)
Mirna 4.1 array platform, by Thermo Fisher Scientific (2 mentions)
Genechip 3 ivt plus reagent kit, by Thermo Fisher Scientific (2 mentions)
Genechip ht mg 430 pm arrays, by Thermo Fisher Scientific (2 mentions)
Totalprep 96 rna amplification kit, by Thermo Fisher Scientific (1 mentions)
Janus automated liquid handling system, by PerkinElmer (1 mentions)
Human genome u219 96 array plate, by Thermo Fisher Scientific (1 mentions)
Affymetrix gene 1.1 st arrays, by Thermo Fisher Scientific (1 mentions)
Human gene 1.1 st array plate, by Thermo Fisher Scientific (1 mentions)
Encore biotin module, by Tecan (1 mentions)
Ovation pico wta system v2, by Tecan (1 mentions)
Affymetrix wt terminal labeling and hybridization user manual, by Thermo Fisher Scientific (1 mentions)
Genechip expression console software, by Thermo Fisher Scientific (1 mentions)
Genechip wt pico reagent kit, by Thermo Fisher Scientific (1 mentions)
Genechip command console, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
33 protocols using genetitan instrument
1
High-throughput Transcriptomic Profiling
2
Whole Transcriptome Profiling of Mouse Samples
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Total RNA (100 ng) was labelled by a Whole Transcript Sense Target Assay and hybridized to mouse whole-genome Affymetrix Gene 1.1 ST arrays targeting 21,115 unique genes (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning of all Affymetrix Genechips was performed according to standard Affymetrix protocols. Scans of the Affymetrix arrays were processed using the Affymetrix GeneTitan Instrument.
Quality control was performed on raw data by assessing the signal distribution by using scatter plot, MA-plot and a normal probability plot. Positive (landmark) and negative (blank) spots were used in the quality control and not used in further analyses. Normalized data were visualized by Principal Component Analysis (PCA) for additional quality assessment. The gene expression data have been deposited in NCBI's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/ ) and are accessible through GEO Series accession number GSE102016.
Quality control was performed on raw data by assessing the signal distribution by using scatter plot, MA-plot and a normal probability plot. Positive (landmark) and negative (blank) spots were used in the quality control and not used in further analyses. Normalized data were visualized by Principal Component Analysis (PCA) for additional quality assessment. The gene expression data have been deposited in NCBI's Gene Expression Omnibus (
3
Microarray Gene Expression Analysis Protocol
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The analyses were conducted in the analytic center using the vendors’ recommended methods. The Ovation Pico WTA System v2 (NuGEN Technologies, San Carlos CA, USA) was used with 50 ng of input total RNA, providing linear expansion of all transcripts without interference from ribosomal or globin RNAs. The resulting cDNA was labeled with biotin (Encore Biotin Module; NuGEN) and hybridized to Human Gene 1.1 ST Array Plates (Affymetrix, Santa Clara CA, USA) followed by scanning on an Affymetrix GeneTitan instrument and inspection of quality control metrics and probeset summarization with Expression Console software. Microarray data sets were analyzed using Partek Genomics Suite (Partek, St. Louis MO, USA) with normalization by the RMA algorithm (Irizarry et al., 2003 (link)).
4
Microarray Hybridization Sample Preparation
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Sample preparation for microarray hybridization was carried out as described in the Ambion WT Expression Kit Protocol (Thermo Scientific) and the Affymetrix WT Terminal Labeling and Hybridization User Manual (Affymetrix, Inc., Santa Clara, CA, USA). In brief, 200–300 ng of extracted RNA were reverse transcribed, using an rRNA-depleted random primer mix, as provided in the Ambion WT Expression Kit, followed by an in vitro transcription reaction. 8–12 μg of in vitro transcribed antisense RNA were purified and reverse transcribed into dUTP-containing sense-strand- (ss-) cDNA. Purified ss-cDNA was fragmented using a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1), followed by terminal labeling with biotin. This process yielded between 1 and 3 μg fragmented and labeled ss-cDNA, which were hybridized to Affymetrix Human Gene 1.1 ST PEG plate arrays. Hybridization, washing, staining, and array scanning were performed with a GeneTitan® instrument (Affymetrix).
Summarized probe set signals were calculated by applying the RMA [21 (link)] algorithm as implemented in the Affymetrix GeneChip Expression Console Software. The result file was exported in .txt format, and fold change/significance calculations were done in Microsoft Excel.
Summarized probe set signals were calculated by applying the RMA [21 (link)] algorithm as implemented in the Affymetrix GeneChip Expression Console Software. The result file was exported in .txt format, and fold change/significance calculations were done in Microsoft Excel.
5
Microarray Analysis of Microglia-Depleted Mouse Cortex
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RNA (2 ng) isolated from laser-assisted microdissected tissue (somatosensory as well as motor cortex derived from four control and four d10 microglia-depleted mice) was transcribed, amplified and labeled using the GeneChip WT Pico Reagent Kit (Affymetrix) according to the manufacturer’s instructions and subsequently hybridized to a GeneChip® Mouse Gene 2.1 ST 16-Array Plate (Affymetrix) according to the manufacturer’s protocol. For microarray processing (washing, hybridization, signal detection), a GeneTitan® instrument (Affymetrix) was used. The raw data were processed by the Affymetrix Gene Chip Command Console (AGCC) and normalization was performed using the Affymetrix Expression Console (EC). Additionally, quality control was done with the EC by calculating several metrics (AUC, PM-mean, BG-mean, MAD, RLE, spike-ins). After quantile normalization, the data were exported to CHP files, which can be read by the Affymetrix Transcriptome Analysis Console (TAC).
6
Affymetrix Mouse Gene 1.1 ST Array Analysis
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Total RNA was harvested from the cells using an RNeasy Plus kit (Qiagen, Crawley, UK), according to the manufacturer's instructions. RNA was quantified and quality-controlled using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to determine RNA purity and integrity. Replicate 250-ng samples of total RNA, derived from two separate wells/time-point, were first processed using the Ambion WT Expression Kit (Life Technologies, Carlsbad, CA, USA) to generate amplified and biotinylated sense-strand DNA targets from the entire genome without bias. Sense-strand DNA samples were then labeled and hybridized to the Affymetrix Mouse Gene 1.1 ST Array Plate using the GeneChip WT terminal labeling and hybridization kit (Affymetrix, Santa Clara, CA, USA), according to the manufacturer's recommendation. Individual arrays interrogate >28,000 annotated transcripts using >770,000 distinct probes. Hybridization, washing, and scanning of the 64 arrays were performed in a single run using the Affymetrix GeneTitan instrument, according to the manufacturer's recommendations.
7
Lung Cancer Transcriptomic Profiles
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Preprocessed and normalized data for RNAseq gene expression, microRNA expression, copy number alterations, protein expression, somatic mutations, and clinical data for lung adenocarcinomas characterized by The Cancer Genome Atlas (TCGA) were obtained from the TCGA Web site. Processed gene expression data, somatic mutations, and clinical information for other publicly available data sets were downloaded from Gene Expression Ominbus (GEO) and ArrayExpress or from individual Web sites, as listed in Supplementary Table S1 (Supplementary Digital Content 1, http://links.lww.com/JTO/A585 ). In cases where data were presented as linear expression values, log2 transformed values were used. The status of LKB1 loss in NSCLC cell lines was taken from various previous studies given in Supplementary Data File 1 (Supplementary Digital Content 2, http://links.lww.com/JTO/A586 ).
Gene expression analysis of A549 and H2122 cell lines transduced with pBABE, LKB1, or LKB1-K78I was performed using HT Human Gene 1.1 ST PM16 array plate using a GeneTitan instrument (Affymetrix; Santa Clara, CA). They were then scanned on the Affymetrix Gene Titan AGCC v. 3.2.3 and then analyzed on Affymetrix Expression Console v. 1.1 using an robust multi-array average (RMA) normalization algorithm producing log2 results. These data are available from GEO data repository (GSE51266).
Gene expression analysis of A549 and H2122 cell lines transduced with pBABE, LKB1, or LKB1-K78I was performed using HT Human Gene 1.1 ST PM16 array plate using a GeneTitan instrument (Affymetrix; Santa Clara, CA). They were then scanned on the Affymetrix Gene Titan AGCC v. 3.2.3 and then analyzed on Affymetrix Expression Console v. 1.1 using an robust multi-array average (RMA) normalization algorithm producing log2 results. These data are available from GEO data repository (GSE51266).
8
Transcriptomic Analysis of Laser-Treated Skin
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Microarray profiling was performed on 140 untreated or laser-treated samples using the HG-U219 array (Affymetrix) as similarly described for the aged dermis samples [8 (link)]. Flash-frozen, bisected 4 mm full-thickness skin punch biopsies were homogenized in TRIzol reagent (ThermoFisher, Waltham, MA) and total RNA was extracted according to the manufacturer’s protocol. RNA was further purified using RNEasy spin columns (QIAGEN, Hilden, Germany). Biotinylated cRNA libraries were synthesized from 500 ng of total RNA using the Affymetrix HT 3’ IVT Express kit (Affymetrix, Santa Clara, CA) and the Beckman Biomek FXp Laboratory Automation Workstation (Beckman Coulter, Brea, CA). Biotinylated cRNA was subsequently fragmented by limited alkaline hydrolysis and then hybridized overnight to the Affymetrix Human Genome U219-96 GeneTitan array. Plates were scanned using the Affymetrix GeneTitan Instrument utilizing AGCC GeneTitan Instrument Control software version 4.2.0.1566. Image data was summarized using Affymetrix PLIER algorithm with quantile normalization. Microarray data from this study as well as the aged dermis study have been deposited in the Gene Expression Omnibus (GEO) under GSE168760 and GSE139300, respectively.
9
Transcriptomic Profiling using Affymetrix Arrays
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RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 100 ng of total RNA spiked with bacterial RNA transcript positive controls (Affymetrix) was amplified and labeled using the GeneChip 3′ IVT express kit (Affymetrix). All steps were carried out according to the manufacturers protocol (Affymetrix). A mixture of purified and fragmented biotinylated amplified RNA (aRNA) and hybridisation controls (Affymetrix) was hybridized on Affymetrix Human Genome U219 Array Plate followed by staining and washing in the GeneTitan® Instrument (Affymetrix) according to the manufacturer’s procedures. To assess the raw probe signal intensities, chips were scanned using the GeneTitan® HT Array Plate Scanner (Affymetrix).
10
Gene Expression Profiling Protocol
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A minimum of two arrays was used for each test condition. Raw probe intensities were generated using Gene Chip Operating Software (GCOS) and the Gene Titan Instrument from Affymetrix. Normalized expression values for each probe set were obtained from R 2.7.0 downloaded from the Bioconductor project (http://www.bioconductor.org ) using robust multiarray averaging with correction for oligosequence (gcRMA). Standard correlation coefficients were calculated using GeneSpring GX 10.0.2. One-way ANOVA analysis with Tukey’s Post Hoc test was performed to extract differentially expressed genes. P values were calculated using Welch’s t-test after multiple test correction by the Benjamini–Hochberg method. A post-hoc test using Tukey’s Honestly Significant Difference test was conducted to determine significant differences between samples.
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