Total RNA extracted from CFPAC-1 cells with or without BACH1 knockdown were subject to analysis by the Agilent human lncRNA+mRNA array V.4.0 platform (Agilent Technologies). The raw and processed microarray data have been submitted to the Gene Expression Omnibus (accession number GSE111506). For Gene Ontology (GO) analysis, differentially expressed protein-coding genes were uploaded to online software DAVID (http://david.abcc.ncifcrf.gov/ ) and P<0.05 was considered significant for the biological process enrichment. For Gene Set Enrichment Analysis (GSEA), gene profiling data were uploaded to GSEA 3.0. Hallmark gene sets were obtained from MSigDBv6.1 (http://software.broadinstitute.org/gsea/msigdb/index.jsp ). False discovery rate (FDR) <0.25 was considered significant for enriched biological pathways.
Agilent human lncrna mrna array v4
Manufactured by Agilent Technologies
Sourced in China
The Agilent human lncRNA+mRNA Array V4.0 is a microarray platform designed for the comprehensive analysis of human long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs). The array contains probes that target over 14,000 lncRNAs and 34,000 protein-coding mRNAs, providing a powerful tool for the study of gene expression in human samples.
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6 protocols using agilent human lncrna mrna array v4
1
Transcriptome profiling of BACH1 knockdown
2
Differential lncRNA and mRNA Expression in Huh7 Cells
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TRIzol reagent was used to extract the total RNA of Huh7 cells treated with siHINT1 and siCTRL. The cDNA obtained by reverse transcription was hybridized to Agilent human lncRNA + mRNA Array V4.0(Boao, Beijing, China), which was designed with four identical arrays per slide (4×180K format) with each array containing probes interrogating about 41,000 human lncRNAs and about 34,000 human mRNAs. Microarray hybridization and computational analysis was performed as previously described [18 (link)].
3
Dexamethasone-induced Transcriptome Analysis
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The hBMSCs were cultured in complete DMEM containing 10% FBS and 100 units/mL of penicillin-streptomycin along with 10−6 mol/L Dex (Dex-induced group, Dex) or with the solvent of Dex (control group, Control). After treatment for 7 days, total RNA from was extracted from the hBMSCs in the two groups using a RNAiso plus kit (Takara Bio Inc., Kusatsu, Japan), following the manufacturer’s instructions. The purity and concentration of the RNAs were assessed at OD260/280 using a spectrophotometer (NanoDrop ND-1000). Total RNA was reverse-transcribed into cDNA, which was labeled using a fluorescent dye (Cy5 and Cy3-dCTP) and hybridized with the Agilent human lncRNA+mRNA Array V4.0 designed with four identical arrays per slide (4 × 180 K format). The microarrays were washed and then scanned using a G2565CA Microarray Scanner (Agilent). The lncRNA+mRNA array data were analyzed for data summarization, normalization, and quality control using GeneSpring software V13.0 (Agilent). A fold change of ≥ 2.0 or ≤ 2.0 and a P value (t test) of < 0.05 were used as threshold values to select differentially expressed lncRNAs and mRNAs. The experiment was performed and data were analyzed in triplicate.
4
Differential Expression Profiling of lncRNAs and mRNAs in Melanoma
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Total RNA was extracted from 3 pairs of primary melanoma and nevus tissues using Trizol reagent (Life Technologies, Carlsbad, CA) according to manufacturer’s protocol. Agilent human lncRNA+mRNA Array v4.0 (Agilent Technologies, Santa Clara, CA) was used for microarray hybridization performed by Capital Biotech (Beijing, China). The array data were analyzed for summarization, normalization and quality control by using the GeneSpring software V13.0 (Agilent Technologies, Santa Clara, CA). Genes having a fold change ≥ 2 or ≤ −2 and a P < 0.05 were considered as differentially expressed.
Hierarchical clustering analysis was conducted to elucidate the lncRNAs and mRNAs expression pattern. Differentially-expressed lncRNAs and mRNAs with statistical significance were displayed through volcano plot. The microarray data were deposited in Gene Expression Omnibus (GEO) database (accession No.: GSE183878) in the NCBI database.
Hierarchical clustering analysis was conducted to elucidate the lncRNAs and mRNAs expression pattern. Differentially-expressed lncRNAs and mRNAs with statistical significance were displayed through volcano plot. The microarray data were deposited in Gene Expression Omnibus (GEO) database (accession No.: GSE183878) in the NCBI database.
5
Profiling lncRNA and mRNA Expression
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Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) from lentivirus-mediated miR-155 overexpression NPCs and NC groups. The purity and concentration, as determined by the optical density (OD) 260/OD280 ratio, was ≥1.90 with total RNA ≥1 µg per sample, which was assessed with a NanoDrop 1000 device (NanoDrop Technologies; Thermo Fisher Scientific, Pittsburgh, PA, USA). Denaturing formaldehyde agarose gel electrophoresis (percentage of 1.2%) demonstrated clear RNA bands, with 28S to 18S exceeding 2 bands and good RNA integrity (data not shown). Then, lncRNA array detection was performed by Beijing Capitalbio Technology Co., Ltd. (Beijing, China).
The Agilent human lncRNA + mRNA Array v4.0 (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to detect lncRNAs in the present model system. Cy5 and Cy3 were used to label the molecule types on the same array at 37°C for 1.5 h and 70°C for 5 min, respectively. The Agilent array contained probes capable of detecting ~41,000 human lncRNAs and 34,000 human mRNAs, in a 4×180 K format. The array also contained 4,974 internal Agilent control probes. The experiments were performed according to the manufacturer's protocols and conducted twice. The fluorescent signals of Cy3 and Cy5 were obtained, and differentially expressed mRNAs and lncRNAs were screened, at a fold change cutoff of 2 and P<0.05.
The Agilent human lncRNA + mRNA Array v4.0 (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to detect lncRNAs in the present model system. Cy5 and Cy3 were used to label the molecule types on the same array at 37°C for 1.5 h and 70°C for 5 min, respectively. The Agilent array contained probes capable of detecting ~41,000 human lncRNAs and 34,000 human mRNAs, in a 4×180 K format. The array also contained 4,974 internal Agilent control probes. The experiments were performed according to the manufacturer's protocols and conducted twice. The fluorescent signals of Cy3 and Cy5 were obtained, and differentially expressed mRNAs and lncRNAs were screened, at a fold change cutoff of 2 and P<0.05.
6
Dexamethasone-Induced lncRNA and mRNA Expression in hBMSCs
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The hBMSCs were cultured in complete DMEM containing 10% FBS and 100 units/ml of penicillinstreptomycin along with 10 -6 mol/L Dex (Dex-induced group, Dex) or with the solvent of Dex (control group, Control). After treatment for 7 days, total RNA from was extracted from the hBMSCs in the two groups using a RNAiso plus kit (Takara Bio Inc., Kusatsu, Japan), following the manufacturer's instructions. The purity and concentration of the RNAs were assessed at OD260/280 using a spectrophotometer (NanoDrop ND-1000). Total RNA was reverse-transcribed into cDNA, which was labeled using a uorescent dye (Cy5 and Cy3-dCTP) and hybridized with the Agilent human lncRNA+mRNA Array V4.0 designed with four identical arrays per slide (4×180K format). The microarrays were washed and then scanned using a G2565CA Microarray Scanner (Agilent). The lncRNA+mRNA array data were analyzed for data summarization, normalization, and quality control using GeneSpring software V13.0 (Agilent). A fold change of ≥ 2.0 or ≤ 2.0 and a P value (t-test) of < 0.05 were used as threshold values to select differentially expressed lncRNAs and mRNAs. The experiment was performed and data were analyzed in triplicate.
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