Microarray analyses were performed using the Whole Mouse Genome Oligo Microarray (Mouse GE 4x44K v2 Microarray Kit; G4846A) (Agilent Technologies, Santa Clara, CA) [27 ]. A full list of cDNAs is available online (www.agilent.com ). Protocols for sample preparation and hybridization of the mononuclear cells were adaptations of those in the Agilent Technical Manual. Briefly, Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using a Low Input Quick-AMP labeling kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with a NanoDrop spectrophotometer. Thereafter, Cy3-labelled cRNA was fragmented at 60°C for 30 min. Fragmented cRNA samples were hybridized onto chips by means of 17 hr of incubation at 65°C with constant rotation, followed by a two-step microarray wash of 1 min in two washing buffers (Agilent Technologies). Hybridized microarrays were scanned in an Agilent Technologies Scanner (model G2505C), and the scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 026655_D_F_20100123) to obtain background subtracted and spatially de-trended Processed Signal intensities.
Rnaeasy column purification
Manufactured by Qiagen
Sourced in United States
The RNAeasy column purification is a lab equipment product designed for the extraction and purification of RNA from various biological samples. It utilizes a silica-based membrane technology to efficiently capture and purify RNA molecules, while removing contaminants and inhibitors.
Automatically generated - may contain errors
Lab products found in correlation
Low input quick amp labeling kit, by Agilent Technologies (5 mentions)
One color low rna input linear amplification plus kit, by Agilent Technologies (3 mentions)
Oligo dt directed reverse transcription, by Thermo Fisher Scientific (3 mentions)
Icycler, by Bio-Rad (3 mentions)
Feature extraction software 10, by Agilent Technologies (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Gene expression hybridization kit, by Agilent Technologies (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Agilent dna microarray scanner, by Agilent Technologies (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Scanner model g2505c, by Agilent Technologies (1 mentions)
Washing buffer, by Agilent Technologies (1 mentions)
Dna microarray scanner, by Agilent Technologies (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Feature extraction software 11, by Agilent Technologies (1 mentions)
Agilent 2200 tapestation, by Agilent Technologies (1 mentions)
One color quick amp labeling kit, by Agilent Technologies (1 mentions)
Genespring gx 12, by Agilent Technologies (1 mentions)
Agilent 4 44 k whole human genome oligo microarrays, by Agilent Technologies (1 mentions)
12 protocols using rnaeasy column purification
1
Whole Genome Oligo Microarray Analysis
2
Whole Rat Genome Microarray Hybridization
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Microarray hybridization was performed as described below. Cyanine-3 labeled complementary RNA (cRNA) was prepared from 1 μg RNA using the One-Color Low RNA Input Linear Amplification PLUS Kit (Agilent Technologies) according to the manufacturer’s instructions followed by RNAeasy column purification (Qiagen, Valencia, California, USA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). The labeled cRNA sample was hybridized on a Whole Rat Genome Microarray 4×44K v3 (Probe Name version, Design ID #028282, GPL14746 platform, Agilent Technologies) using a Gene Expression Hybridization Kit (Agilent Technologies) following the manufacturer’s instructions.
3
RNA-seq Analysis of Human Keratinocytes
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For RNA-seq, total RNA was isolated from triplicate sets of human keratinocytes at 48h after transfection using RNAeasy column purification (Qiagen, Germantown, MD). cDNA library was prepared via oligo-dT-directed reverse transcription (Ambion, Grand Island, NY), and subject to deep sequencing on Illumina HiSeq2000 (50bp single-read sequencing) and analyzed at Duke Genome Sequencing & Analysis Resource. The analysis pipeline includes the initial QC to remove sequencing adaptors and low quality bases to facilitate mapping using fastx toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html ). High quality reads were mapped to the human reference genome (hg19) using tophat (Trapnell et al., 2009 (link)). The gene differential expression analysis was performed between control and treatment using cuffdiff with upper quartile normalization, multi-hit correction and 0.5% FDR(Trapnell et al., 2013 (link)). Three biological replicates were used for statistical analysis of differential expression for each comparison. Pathway analyses were performed using WEB-based GEne SeT AnaLysis Toolkit (Wang et al., 2013 (link); Zhang et al., 2005a (link)). SYBR green-based real-time RT-PCR was performed in Bio-Rad iCycler with primers listed in (Table S4 ). Ribosomal 18S RNA or GAPDH was used as an internal control.
4
Transcriptomic Response to Ionizing Radiation
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Cells were treated with 10 Gy IR and collected at 15 min and 4 h post IR. Unirradiated cells were used as controls. Total RNA was isolated from cells using RNeasy Kit (QIAGEN, 74104) following manufacturer’s protocol. Integrity of total RNA was determined by Nanodrop ND-1000 analysis. Cyanine-3 (Cy3)-labeled cRNA was prepared from 0.5 μg RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent, 5188-5339) according to the manufacturer’s instructions, followed by RNAeasy column purification (QIAGEN, 79656). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. One microarray was performed for each sample, with no pooling. Samples were hybridized to ArrayStar GPL15692 genechip platform (Arraystar). Microarrays were scanned immediately after washing on a DNA Microarray Scanner (Agilent, G2505B) using one color scan setting for 1 × 44k array slides (scan area 61 × 21.6 mm, scan resolution 10 μm, Dye channel set to Green and Green PMT set to 100%).
5
Transcriptomic Analysis of Mouse Mammary Epithelial Cells
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Total RNA was extracted from MMECs with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Cyanine‐3‐labeled cRNA was prepared from 0.2 µg RNA using the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions, followed by RNAeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND‐1000 Spectrophotometer. The microarray analysis was carried out with Whole Mouse Genome microarray 4 × 44K version 2.0 (G2519F #26655; Agilent). The fluorescence intensity was measured with Agilent DNA Microarray Scanner (G2539A). Data were normalized and filtered with 3 filters using GeneSpring software 12.1 (Agilent). In brief, raw data were normalized with their 75 percentile values. These microarray data were deposited in the Gene Expression Omnibus database (GSE144328). The normalized microarray results were analyzed by the weighted average difference method.20 Enrichment analysis by EnrichR (version 2.1) was undertaken by R.21 , 22 Gene set enrichment analysis (A) was carried out on signal to noise metrics using gene sets obtained from H hallmark gene sets and C2 curated gene sets, which are freely available at http://www.broadinstitute.org/gsea/index.jsp .23
6
Marmoset Hippocampal RNA Profiling
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Total RNA was extracted from eleven marmoset hippocampus tissues using the TRIzol Reagent with the PureLink RNA Mini Kit (Thermo Fisher Scientific, USA). RNA quality was assessed using the Agilent 2200 TapeStation (Agilent, USA). RNA samples with a RIN value greater than 5.7 and 28S/18S ratio greater than 0.9 were selected for subsequent experiments (Supplementary Table S2 ). The microarray analysis was performed following the manufacturer's protocol. Briefly, 0.2 µg of total RNA was used to prepare Cyanine-3 (Cy3)-labeled cRNA using the Low Input Quick Amp Labeling Kit (Agilent, USA), followed by RNAeasy column purification (Qiagen, USA). Dye incorporation and cRNA yield were measured using the NanoDrop ND-1000 Spectrophotometer. The fragmented Cy3-labeled cRNA was then hybridized to the Marmoset microarray 8x60K (G4858A#84626, Agilent, USA). After hybridization, the microarrays were washed and scanned using the Agilent DNA Microarray Scanner. The scanned images were analyzed with Feature Extraction Software 11.0.1.1 (Agilent, USA) using default parameters, and the obtained data were normalized and filtered using three filters with GeneSpring software 14.9 (Agilent, USA).
The microarray data have been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE236481.
The microarray data have been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE236481.
7
RNA-seq Analysis of Human Keratinocytes
8
Cyanine-3 Labeled cRNA Microarray
9
Microarray Analysis of Cyanine-3 Labeled cRNA
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Cyanine-3 (Cy3)-labeled cRNA was prepared from 0.5 μg total RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent), followed by RNAeasy column purification (QIAGEN, Valencia, CA). A total of 1.65 μg of Cy3-labeled cRNA (specific activity >6.0 pmol) was fragmented and hybridized to Agilent 4 × 44K Whole Human Genome Oligo Microarrays (G2600D) using the Gene Expression Hybridization Kit (Agilent). After hybridization, the microarrays were washed with the Gene Expression Wash Buffer Kit (Agilent) and scanned with Agilent's Feature Extraction 9.1 software with default parameters. The microarray data have been deposited in NCBI's Gene Expression Omnibus with the series accession number GSE63596.
10
Microarray Analysis of ES Cell Transcripts
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Microarray analysis was performed using total RNA prepared from indicated ES cell clones using the TRIzol reagent (Invitrogen Carlsbad, CA) as follows: cyanine-3 (Cy3) labeled complementary RNA (cRNA) was prepared from 0.1 μg total RNA using the Low Input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by RNAeasy column purification (Qiagen, Valencia, CA). Cy3-labeled cRNA (0.6 μg) was fragmented and hybridized to SurePrint G3 Mouse GE 8×60K Microarray (Agilent Technologies) for 17 hr at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 1 min at room temperature with GE Wash Buffer 1 (Agilent Technologies), then for 1 min with GE Wash buffer 2 (Agilent Technologies) at 37°C, and dried immediately by brief centrifugation. Slides were scanned immediately on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8× 60k array slides (scan area 61 × 21.6 mm, scan resolution 3 µm, dye channel was set to Green and Green PMT was set to 100%). The scanned images were analyzed with the Feature Extraction Software 10.10.1.1 (Agilent Technologies) using default parameters to obtain background subtracted and spatially detrended processed signal intensities.
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