For microarray hybridizations, 500 ng of total RNA from each sample was labeled with fluorescent dye (Cy3; Amersham Biosciences Corp, Piscataway, NJ) using the Low RNA Input Linear Amplification Labeling kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s protocol. The amount and quality of the fluorescently labeled cRNA was assessed using a NanoDrop ND-1000 spectrophotometer and an Agilent Bioanalyzer. According to manufacturer’s specifications, 1.6 mg of Cy3-labeled cRNA was hybridized to the Agilent Human Whole Genome Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA) for 17 h, prior to washing and scanning. Data was extracted from scanned images using Feature Extraction Software (Agilent Technologies, Inc., Palo Alto, CA).
Low rna input linear amplification labeling kit
Manufactured by Agilent Technologies
The Low RNA Input Linear Amplification Labeling kit is a laboratory product designed for the amplification and labeling of small amounts of RNA samples. The kit provides the necessary reagents and protocols to linearly amplify and label RNA, enabling the analysis of samples with limited starting material.
Automatically generated - may contain errors
Lab products found in correlation
Feature extraction software, by Agilent Technologies (2 mentions)
Agilent whole human genome oligo microarray, by Agilent Technologies (1 mentions)
Cyanine3 (cy3), by Cytiva (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Agilent Technologies (1 mentions)
Nanodrop, by Agilent Technologies (1 mentions)
Bioanalyser, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
G2565ba scanner, by Agilent Technologies (1 mentions)
3 protocols using low rna input linear amplification labeling kit
1
Microarray Hybridization and Analysis
2
Microarray Analysis of Gene Expression
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RNA was extracted with TRIzol and purified on RNeasy Micro Kit spin columns (Qiagen GmbH). The RNA amount, purity and integrity were evaluated by NanoDrop and Bioanalyser (Agilent Technologies, Inc.). For array hybridization, RNA samples (100 ng) were amplified and stained with fluorophores using the Low RNA Input Linear Amplification Labeling kit (Agilent Technologies, Inc.). Cy3-stained cRNA were hybridized on Agilent Human Whole Genome Oligo Microarray format 8×60K, design 028004 (Agilent Technologies, Inc.), then washed and scanned. Data were extracted by Feature Extraction software (v10.5.1.1; Agilent Technologies, Inc.) and analysis was performed by the limma package (30 (link)) of the Bioconductor Project. An intra-array normalization was performed, followed by a quantile inter-array normalization. The median of all probes for a given transcript was then recovered. Differential expression level analysis was performed using the following criteria: Absolute fold change >2 and corrected P-value [false discovery rate (FDR)] <0.05. The microarray data and protocols are available at the European Molecular Biology Laboratory European Bioinformatics Institute database (https://www.ebi.ac.uk/arrayexpress/ ) under accession no. E-MTAB-8777.
3
C57BL/6J Mice Hepatotoxicity Transcriptomics
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Details regarding animal treatments, RNA isolation, and microarray hybridizations can be found in the original publication by the Toxicogenomics Research Consortium.7 (link) All animal studies for this project were approved by each Institution’s respective Animal Care and Use Committee. Briefly, randomly assigned C57BL/6 J mice were dosed with 10 mL/kg body weight of vehicle (methylcellulose, 0.5% wt/vol), AMAP (300 mg/kg), or APAP (300 mg/kg). Mice were euthanized at 6, 12, or 24 hours after treatment. Total RNA was isolated from liver samples (left lateral lobe) using Qiagen RNeasy mini kits according to the manufacturer’s protocol. Total RNA from individual mouse liver samples was amplified and labeled with a fluorescent dye (Cy3), whereas a common reference of pooled C57BL/6 J liver mRNA was amplified and labeled with Cy5 using Agilent Technologies Low RNA Input Linear Amplification Labeling Kit following the manufacturer’s protocol. Equal amounts of Cy3 and Cy5-labeled cRNA were hybridized to an Agilent Mouse Oligonucleotide Microarray (~21,000 features, catalog # G4121) and scanned using an Agilent G2565BA scanner. Raw microarray data were processed and analyzed with tools in the Bioconductor27 (link) software package. This dataset (raw and normalized data files) is publicly available from cebs.niehs.nih.gov (accession number 009–00001–0010–000–1).
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