Genome-wide expression analysis was performed using Agilent Mouse Gene Expression 4 × 44 K microarrays at the Genome Technology Access Center (GTAC), Department of Genetics in Washington University in St. Louis. Quantitative real-time RT-PCR was conducted on an ABI 7900 using TaqMan Gene Expression Assay system (Applied Biosystems) in accordance with the manufacturer's recommendations. Expression was normalized to β-actin and relative expression was calculated in comparison to a calibrator (RNA-probe from a mixture of lymphoid organs from B6wt mice). At least a 2-fold threshold in relative gene expression was used for evaluation. For 47S RT-PCR, total RNA were calculated using a standard curve generated from serial dilutions of a known quantity of cDNA.24 (link)
Agilent mouse gene expression 4 44 k microarrays
Manufactured by Agilent Technologies
The Agilent Mouse Gene Expression 4 × 44 K microarrays are a high-density, whole-genome microarray platform designed for comprehensive transcriptional profiling of mouse samples. The arrays contain approximately 44,000 probe sets that target well-characterized mouse genes and transcripts, enabling researchers to analyze the expression of thousands of genes simultaneously.
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2 protocols using agilent mouse gene expression 4 44 k microarrays
1
Genome-wide expression analysis of B cells
2
Bladder Macrophage Transcriptome Analysis
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Sorted bladder macrophages were transferred to buffer RLT with β‐mercaptoethanol (Sigma, M6250), with at least 6 × 103 cells being per sorted bladder cell population. RNA was then isolated from the cells and frozen, per the manufacturer's protocols (RNeasy Mini kit, Qiagen), and assessed for quality using the Bioanalyzer 2100 (Agilent). With the Sigma WTA2 RNA amplification kit, 1.0 ng of total RNA were amplified and then 3 µg of cDNA were chemically labeled with Kreatech ULS Fluorescent Labeling Kit for Agilent arrays (Kreatech Diagnostics). Finally, 1.7 µg of labeled cDNAs were subsequently hybridized onto Agilent Mouse Gene Expression 4 × 44 K Microarrays (cat. G4122F‐014868). Slides were scanned on an Agilent C‐class Microarray scanner to detect Cy5 fluorescence, according to manufacturer's specifications. Gridding and analysis of images was performed using Feature Extraction (v11.5.1.1, Agilent Technologies). The data was then analyzed, and normalized heat maps were generated, using the R software package, with a cutoff of P ≤ 0.05 and fold change of >2.
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