Arraystar Mouse LncRNA Microarray version 3.0 was designed for the global profiling of mouse lncRNAs and protein-coding transcripts, with ~35,923 lncRNAs and 24,881 coding transcripts detected. Sample preparation and microarray hybridization were performed based on manufacturer standard protocols with minor modifications. An Arraystar RNA Flash Labeling Kit (Arraystar, Rockville, MD, USA) was used for sample labeling. Hybridization was performed in SureHyb Hybridization Chambers (Agilent Technology). After washing, the arrays were scanned using an Agilent DNA Microarray Scanner (Agilent Technology).
Surehyb hybridization chamber
Manufactured by Agilent Technologies
Sourced in United States
The SureHyb Hybridization Chambers are designed for uniform and reproducible hybridization of nucleic acid samples. They provide a controlled, temperature-regulated environment for incubating samples during hybridization experiments.
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Lab products found in correlation
Quick amp labeling kit, by Agilent Technologies (9 mentions)
Agilent dna microarray scanner, by Agilent Technologies (9 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Agilent quick amp labeling kit, by Agilent Technologies (4 mentions)
Feature extraction software, by Agilent Technologies (3 mentions)
Genespring gx v11, by Agilent Technologies (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Dna microarray scanner, by Agilent Technologies (3 mentions)
Whole genome oligo microarray, by Agilent Technologies (3 mentions)
Agilent dna microarray scanner g2505b, by Agilent Technologies (2 mentions)
Agilent array platform, by Agilent Technologies (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Feature extraction software version 10, by Agilent Technologies (2 mentions)
Agilent gene expression hybridization kit, by Agilent Technologies (2 mentions)
Flash rna labeling kit, by Arraystar (1 mentions)
Agilent quick amp kit, by Agilent Technologies (1 mentions)
Agilent whole rat genome oligo microarray, by Agilent Technologies (1 mentions)
Genespring gx, by Agilent Technologies (1 mentions)
Fe one color scenario, by Agilent Technologies (1 mentions)
25 protocols using surehyb hybridization chamber
1
Mouse lncRNA Microarray Profiling
2
Whole Genome Microarray Analysis
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Purified RNA samples (2 μg ea) were PCR amplified and labeled using an Agilent Quick Amp kit (Agilent Technologies, Santa Clara, CA, USA) and hybridized with Agilent Whole Rat Genome Oligo Microarray (4 × 44 K) in Agilent's SureHyb Hybridization Chambers. After hybridization and washing, processed slides were scanned by an Agilent DNA microarray scanner (part number G2505B) using manufacturer recommended settings.
3
Fluorescent Labeling of RNA Samples
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Sample labeling was performed using an RNA ligase method. Briefly, 1 microgram of each sample was 3′-end-labeled with Cy3 fluorescent label using Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA). The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C. After stopping the labeling procedure, the Cy3-labeled samples were hybridized to the Arraystar Human piRNA Array. Hybridization was performed at 65°C for 17 h in Agilent's SureHyb Hybridization Chambers. Slides were washed in an ozone-free environment and were fixed and scanned using the Agilent DNA Microarray Scanner (part number G2505C).
4
Agilent Microarray Transcriptome Analysis
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Agilent microarray hybridization and analysis was carried out by ArrayStar Inc., MD. Briefly, the RNA samples that passed quality control on the Nanodrop ND-1000 and Bioanalyser 2100 were amplified and labeled using the Agilent Quick Amp Labeling Kit and hybridized to Agilent whole genome oligo microarray in Agilent’s SureHyb hybridization chambers. After hybridization and washing, the processed slides were scanned using the Agilent DNA microarray scanner following Agilent Technologies’ guidelines. The resulting.txt files extracted from Agilent Feature Extraction Software (version 10.5.1.1) were imported into the Agilent GeneSpring GX software (version11.0) for further analysis. The microarray data sets were normalized in GeneSpring GX using the Agilent FE one-color scenario. After quantile normalization of the raw data, genes that were categorized as Detected (“All Targets Value”) and had flags in at least 20 out of 24 samples were chosen for further data analysis. Differentially expressed genes were identified through Fold-change screening. The p-value was calculated using t-test. The p-values were corrected for multiple comparisons using the Benjamini & Hochberg false discovery rate method [23 ]. The analysis of Gene Ontology (GO) and Pathway is based on Fisher’s exact test.
5
Microarray Analysis of Microglia Activation
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The RNA sample of primary cultured microglia with or without CatC stimulation was extracted by Trizol reagent (Life Technologies, USA). Then RNA quantity and quality were measured by NanoDrop ND-1000, and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. The Whole Mouse Genome Oligo Microarray was generated for test and control samples according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). These profiles were generated using customized 4 × 44 K oligonucleotide microarrays produced by Agilent Technologies (Palo Alto, CA, USA). The mouse whole genome microarrays covered more than 41,000 genes and transcripts. The RNA samples were amplified and microarray hybridization was performed in Agilent’s SureHyb Hybridization Chambers using the Agilent Quick Amp labeling kit. After hybridization and washing, the hybridized slides were scanned using an Agilent DNA microarray scanner. The microarray datasets were acquired in Agilent Feature Extraction Software (v11.0.0.1) and normalized in Agilent GeneSpring GX v12.1 Software which genes marked as present were chosen for further analysis. The procedure above was carried out by KangCheng Bio-Tech, Shanghai, China.
6
Profiling Human piRNA Expression Using ArrayStar HG19 Array
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The ArrayStar HG19 piRNA array was used to profile piRNA expression in humans (ArrayStar; Agilent Technologies, Santa Clara, CA, USA). This array contains probes for 23,677 piRNAs obtained from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/nuccore/?term=piRNA ) and landmark publications (15 (link),39 (link)). Human piRNAs were mapped to the HG19 genome sequence using UCSC Blat (https://genome.ucsc.edu/cgi-bin/hgBlat?command=start ). Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. Sample labeling was performed using an RNA ligase method (40 (link)). The labeled samples were hybridized onto Arraystar Human piRNA Array in Agilent's SureHyb Hybridization Chambers according to manufacturer's standard protocols (Agilent Technologies). After slides were scanned, data were extracted using Agilent Feature Extraction software. Raw signal intensities were normalized in quantiles by GeneSpring GX v11.5.1 (Agilent Technologies), and low intensity piRNAs were filtered. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze the acquired array images (40 (link)). Normalized intensity values were then log2-transformed.
7
Liver Transcriptome Analysis via Microarray
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Total RNA was harvested from the liver using TRIzol (Invitrogen) and the RNeasy kit (Qiagen); this procedure included a DNase digestion step. RNA was quantified using a Nanodrop ND-1000 apparatus (NanoDrop Technologies, Wilmington, DE, USA). RNA quality was verified with denaturing gel electrophoresis. Samples were amplified and labeled using the Agilent Quick Amp labeling kit and hybridized using an Agilent 4×44K whole-genome oligo microarray in Agilent SureHyb Hybridization Chambers. After hybridization and washing, microarray slides were scanned using the Agilent DNA microarray scanner (G2505B). Text files of results were extracted using Agilent Feature Extraction Software (version 10.5.1.1) and imported into Agilent GeneSpring GX software (version 10.0). Genes differentially expressed between the 2 groups were defined as >2.0 fold-change and P<0.05 between the 2 groups at each time point. Identified genes were analyzed using the KEGG PATHWAY Database (http://www.genome.jp/kegg/ ). Two-sided Fisher's exact test was used to classify enriched pathways. Enrichment was defined by (a/n)/(A/N), where a represents the number of target genes; n, the total number of genes in the particular pathway; A, the total number of differentially expressed genes in all the pathways; and N, the total number of genes in all the pathways.
8
Agilent Microarray Protocol for Transcriptome Analysis
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Total RNAs were harvested using TRIzol (Invitrogen) and the RNeasy kit (Qiagen) according to manufacturer's instructions, including a DNase digestion step. After having passed RNA measurement on the Nanodrop ND-1000 and denaturing gel electrophoresis, the samples were amplified and labeled using the Agilent Quick Amp labeling kit and hybridized with Agilent whole genome oligo microarray in Agilent's SureHyb Hybridization Chambers. After hybridization and washing, the processed slides were scanned with the Agilent DNA microarray scanner (G2505B) using settings recommended by Agilent Technologies. The resulting text files extracted from Agilent Feature Extraction Software (version 10.5.1.1) were imported into the Agilent GeneSpring GX software (version 11.0) for further analysis.
9
Transcriptomic Analysis of Rat Lung Tissue
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For transcriptomic analysis, lung tissue RNA was purified from six rats from each of the three experimental groups using a Qiagen RNeasy Micro Kit (Qiagen, Venlo, Netherlands). RNA integrity and quantity were verified using the Bioanalyzer 2100 (Agilent, Palo Alto, CA).
Total RNA was PCR amplified using First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) to conduct real-time PCR experiments, and labeled using Quick Amp kit (Agilent Technologies, Santa Clara, CA, U.S.A.) and hybridized with Agilent Whole Rat Genome Oligo Microarray (4 × 44 K) in Agilent’s SureHyb Hybridization Chambers. After hybridization, processed slides were scanned by an Agilent DNA microarray scanner (part number: G2505B) using manufacturer recommended settings. Samples from randomly chosen rats were analyzed using at least biological triplicates. All downstream microarray analyses were performed using Agilent GeneSpring GX software version 11.0. Microarray datasets were background subtracted and normalized by applying GeneSpring GX using the Agilent FE one-color scenario (mainly median normalization) Processed data were subsequently filtered through fold change (|log2 ratio|>1) and Student’s t test screening (P-value <0.05).
Total RNA was PCR amplified using First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) to conduct real-time PCR experiments, and labeled using Quick Amp kit (Agilent Technologies, Santa Clara, CA, U.S.A.) and hybridized with Agilent Whole Rat Genome Oligo Microarray (4 × 44 K) in Agilent’s SureHyb Hybridization Chambers. After hybridization, processed slides were scanned by an Agilent DNA microarray scanner (part number: G2505B) using manufacturer recommended settings. Samples from randomly chosen rats were analyzed using at least biological triplicates. All downstream microarray analyses were performed using Agilent GeneSpring GX software version 11.0. Microarray datasets were background subtracted and normalized by applying GeneSpring GX using the Agilent FE one-color scenario (mainly median normalization) Processed data were subsequently filtered through fold change (|log2 ratio|>1) and Student’s t test screening (P-value <0.05).
10
Transcriptome Analysis of Aspergillus fumigatus
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The Agilent Whole A. fumigatus Genome Expression 44K v.1 (AWAFUGE) microarray was used to analyze the transcriptome profiles from each sample [17 ]. From each time point studied, starting at a maximum of 100 ng per sample, RNA was labeled using the “Low Input Quick Amp WT Labeling kit, One-Color” kit (Agilent Technologies, Santa Clara, CA, USA). Then, cDNA was transcribed using T7 RNA polymerase in the presence of Cy3-CTP, and hybridized using the SureHyb hybridization chambers (Agilent Technologies) following the ozone barrier slide covers Agilent protocol. Finally, microarray slides were scanned using a GenePix 4100A scanner (Axon Instruments), and images were analyzed using the associated GenePix Pro 6.0 software (Molecular Devices).
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