After washing, the hybridized microarrays were scanned using an Agilent DNA Microarray Scanner and data was extracted using Agilent Feature Extraction software. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.0 software package (Agilent Technologies). After quantile normalization of the raw data, mRNAs that had ‘Present’ or ‘Marginal’ flags in at least one out of two samples were selected for differentially expressed mRNA screening. To identify differentially expressed mRNAs, we performed fold change filtering between the two samples using a threshold fold change of ≥ 2.0. Further data analysis was performed using Agilent GeneSpring GX v12.0 software; this analysis was performed by KangChen Biotech, Shanghai, PR China.
Genespring gx v12
Manufactured by Agilent Technologies
Sourced in United States, China
GeneSpring GX v12.1 is a software application designed for the analysis and visualization of gene expression data. It provides a comprehensive suite of tools for data normalization, statistical analysis, and clustering, enabling researchers to gain insights into complex biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Feature extraction software, by Agilent Technologies (44 mentions)
Agilent feature extraction software, by Agilent Technologies (21 mentions)
Agilent one color microarray based gene expression analysis protocol, by Agilent Technologies (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (13 mentions)
Agilent scanner g2505c, by Agilent Technologies (11 mentions)
Dna microarray scanner, by Agilent Technologies (9 mentions)
Rneasy mini kit, by Qiagen (9 mentions)
Mrna only eukaryotic mrna isolation kit, by Illumina (8 mentions)
Agilent dna microarray scanner, by Agilent Technologies (8 mentions)
Flash rna labeling kit, by Arraystar (8 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (7 mentions)
Agilent array platform, by Agilent Technologies (7 mentions)
Quick amp labeling kit, by Agilent Technologies (5 mentions)
Agilent feature extraction software version 11.0.1.1, by Agilent Technologies (5 mentions)
Surehyb, by Agilent Technologies (4 mentions)
Hybridization oven, by Agilent Technologies (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Mirna isolation kit, by Thermo Fisher Scientific (3 mentions)
Human lncrna microarray v4, by Arraystar (3 mentions)
Scanner g2505c, by Agilent Technologies (3 mentions)
154 protocols using genespring gx v12
1
Microarray Data Analysis Protocol
2
Glucose-induced lncRNA profiling in HUVECs
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HUVECs were exposed to 30 or 5.5 mM D-glucose for 24 h. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.1 software package (Agilent Technologies, Inc.). A NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.) was used to measure the quantity and quality of the RNA. Arraystar Human lncRNA Microarray V3.0 was designed for the global profiling of human lncRNAs. Following the isolation of rRNA (using the mRNA-ONLY™ Eukaryotic mRNA Isolation kit, Epicentre), mRNA was purified from total RNA. The RNA was amplified and transcribed into fluorescent cRNA by utilizing a random priming method (Arraystar Flash RNA Labeling kit, Arraystar). Each labeled cRNA was fragmented by the addition of Blocking Agent and Fragmentation Buffer (Agilent Technologies, Inc.), and the mixture was then heated at 60°C for 30 min. The labeled cRNA was diluted by 2xGE Hybridization buffer (Agilent Technologies, Inc.). The hybridization solution was then dispensed into the gasket slide and assembled to the lncRNA expression microarray slide for 17 h at 65°C in Hybridization Oven (Agilent Technologies, Inc.). Finally, the hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (no. G2505C; Agilent Technologies, Inc.).
3
Transcriptomic Analysis of Ischemia-Reperfusion Injury
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Data were presented as mean±SEM. Statistical analysis was performed using a t test between two cohorts. Data processing and subsequent quantitative normalization were performed using GeneSpring GX v12.1 software (Agilent Technologies). Differentially expressed mRNAs and lncRNAs between control group and I/R group were identified through fold change filtering. P value <0.05 was considered statistically significant. Hierarchical clustering and combined analysis was performed with homemade scripts (Multi-Experiment Viewer clustering).
4
lncRNA Expression Profiling in TNBC
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For lncRNA expression profiling, we profiled 3 TNBC patient samples and 3 non-TNBC patient samples with Arraystar lncRNA microarrays as described previously [38 (link)]. Briefly, RNA was purified from 1 mg of total RNA after removal of rRNA (mRNA-ONLY Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent RNA along the entire length of the transcripts without bias utilizing a random priming method. The labeled RNAs were hybridized onto the Human LncRNA Array v3.0 (Agilent SureHyb). After washing, the arrays were scanned by the Agilent LncRNA Microarray Scanner, and Agilent Feature Extraction software (11.0.1.1) was used to subsequently collect the raw values of the microarray probe signal. Finally, Agilent GeneSpring GX v12.1 software was employed to normalize the values, and then, lncRNAs and mRNAs, which had at least 1 out of 2 groups have flags in Present or Marginal, were chosen for further data analysis. Additionally, hierarchical clustering and combined analyses were performed using homemade scripts.
5
Agilent Mouse Gene Expression Analysis
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Gene chip expression analysis was performed by Kangcheng Biotechnology Co., Ltd. using the Agilent array platform. First, the RNA quantity and quality were assessed using a NanoDrop ND‐1000 UV Spectrophotometer (Implen). RNA integrity of each sample was identified by standard denaturing agarose gel electrophoresis. Second, sample labeling and chip hybridization were performed according to the Agilent One‐Color Microarray‐Based Gene Expression Analysis protocol (Agilent). The samples were labeled by using the Agilent Quick Amp Labeling kit and hybridization experiments were performed by using the Agilent SureHyb. Specifically, the total RNA of each sample was linearly amplified and labeled with Cy3‐UTP; the labeled complementary RNAs (cRNAs) were purified by using the RNeasy Mini Kit (Qiagen), and the concentration and activity were detected with a NanoDrop ND‐1000 UV spectrophotometer. The purified Cy3‐UTP‐labeled cRNAs were hybridized with Agilent mouse 4 × 44 K gene expression profiling chip v2 (Agilent). The hybridization chip was washed, fixed, and scanned by using Agilent DNA Microarray Scanner (part number G2505C); Chip probe signal values were acquired by using Agilent Feature Extraction software (v11.0.1.1) to obtain raw data. Finally, the quantile normalization of raw data and subsequent data processing were performed by using GeneSpring GX v12.1 software (Agilent).
6
Microarray Analysis of lncRNA Expression
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The microarray hybridization was performed with service from KangChen Bio-tech (Shanghai, China), based on the manufacturer’s standard procedures. Briefly, mRNA was purified from 1 microgram of total RNA, and each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias utilizing random primers. The labeled cRNAs were then hybridized onto the mouse lncRNA microarray V2.0 (8 × 60K, Arraystar). The arrays were then scanned by the Agilent Scanner G2565BA, and the analysis of array images was performed by Agilent Feature Extraction Software. Data normalization and subsequent processing were performed with the GeneSpring GX v12.1 software package (Agilent Technologies, Santa Clara, CA, USA).
Differentially expressed lncRNAs and mRNAs were identified by performing a volcano plot filtering, with the threshold defined as fold-change >2.0 (Student’s t-test P < 0.05) [28 (link)]. Hierarchical clustering was carried out to show the distinguishable lncRNA expression profile between CS-exposed mice and control samples.
Differentially expressed lncRNAs and mRNAs were identified by performing a volcano plot filtering, with the threshold defined as fold-change >2.0 (Student’s t-test P < 0.05) [28 (link)]. Hierarchical clustering was carried out to show the distinguishable lncRNA expression profile between CS-exposed mice and control samples.
7
Extraction and Analysis of miRNA
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First, total RNA was extracted using the TRIzol technique from NP cells kept at a final concentration of 1 mg/mL. After purifying the miRNA component of the total RNA with the miRNA isolation kit (Ambion), the extracted miRNA samples were evaluated on the chip using the Agilent Company’s human miRNA chip (v.12.0). GeneSpring GX v12.1 software was used to process the data (Agilent Technologies).
8
Differential Expression of lncRNAs and mRNAs
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Raw data were extracted from the acquired array images using Agilent Feature Extraction software 11.0.1.1. Subsequently, GeneSpring GX v12.1 software (Agilent Technologies, Inc.) was used to normalize quantiles of raw data. lncRNAs and mRNAs with flags in ‘Present’ or ‘Marginal’ (‘All Targets Value’) in at least 5 out of 10 samples were chosen for further data analysis. Dysregulated lncRNAs and mRNAs between two groups with a fold change >1.5 were filtered based on their P-value/false discovery rate (<0.05).
Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analyses were applied to predict the roles of the dysregulated mRNAs by determining GO terms and biological pathways, respectively. In-house scripts of R3.5.0 were used to perform hierarchical clustering and to perform combined analysis. First,http://bioconductor.org/biocLite.R was sourced to install the clusterProfiler package and load org.Hs.eg.db. Subsequently, the function of enrichGO and enrichKEGG was used to obtain the results.
Coexpression analysis of the dysregulated lncRNAs subgroup was also performed to identify coregulated lncRNA-mRNA pairs. The subgroups of dysregulated RNA coregulation pairs that were dysregulated included large intervening noncoding RNAs (lincRNAs), antisense lncRNAs and their paired dysregulated mRNAs.
Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analyses were applied to predict the roles of the dysregulated mRNAs by determining GO terms and biological pathways, respectively. In-house scripts of R3.5.0 were used to perform hierarchical clustering and to perform combined analysis. First,
Coexpression analysis of the dysregulated lncRNAs subgroup was also performed to identify coregulated lncRNA-mRNA pairs. The subgroups of dysregulated RNA coregulation pairs that were dysregulated included large intervening noncoding RNAs (lincRNAs), antisense lncRNAs and their paired dysregulated mRNAs.
9
lncRNA Microarray Analysis Protocol
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Three pairs of samples were prepared for lncRNA microarray analysis using an Arraystar Human lncRNA Microarray V4.0 (Arraystar, Rockville, MD, USA). Approximately 40,173 lncRNAs and 20,730 coding transcripts can be detected by this third-generation lncRNA microarray. Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies, Santa Clara, CA, USA) with minor modifications. All microarray work was performed by Kangcheng Bio-Tec (Shanghai, China). Agilent Feature Extraction software (version 11.0.1.1) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.1 software package (Agilent Technologies, Santa Clara, CA, USA). Following quantile normalization of the raw data, lncRNAs and mRNAs for which at least 3 of 6 samples have flags in Present or Marginal (“All Targets Value”) were selected for further data analysis. Differentially expressed lncRNAs and mRNAs between the two groups were identified through P< 0.05 and a fold-change > 2.0. Hierarchical clustering and combined analysis were performed using in-house scripts.
10
Microarray Analysis of Gene Expression
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Total RNA from each sample was quantified using the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. For microarray analysis, Agilent Array platform was employed. The sample preparation and microarray hybridization were performed based on the manufacturer’s standard protocols. Briefly, total RNA from each sample was amplified and transcribed into fluorescent cRNA with using the manufacturer’s Agilent’s Quick Amp Labeling protocol (version 5.7, Agilent Technologies). The labeled cRNAs were hybridized onto the Whole Human Genome Oligo Microarray (4 × 44 K, Agilent Technologies). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.1 software (Agilent Technologies). Differentially expressed genes were identified through Fold Change filtering and Volcano filtering. Pathway analysis and GO Analysis were applied to determine the roles of these differentially expressed genes.
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