Genespring gx software version 11

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GeneSpring GX software version 11.5 is a data analysis solution for genomic research. It provides tools for visualizing, analyzing, and interpreting gene expression data from a variety of platforms.

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14 protocols using genespring gx software version 11

Laser Capture Microdissection and RNA Profiling of Mouse Tumor Models

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First, 12 μm frozen sections were prepared, dehydrated, and stained with hematoxylin. Subsequently, cancer tissues were dissected with the LMD6500 laser capture microdissection device (Leica Microsystems), from which RNA was immediately extracted with an RNeasy Micro Kit (Qiagen).
The quality of extracted total RNA was evaluated by RIN, which is calculated with Bioanalyzer (Agilent 2100). RNA sample scoring RIN > 6.0 were used for the following microarray analysis.
Gene expression profiling was compared among CDX2P‐G22Cre;Apc^flox/flox;Braf ^{LSL‐V600E/+} (n = 3), CDX2P‐G22Cre;Apc^flox/flox;Kras^LSL‐G12D/+ (n = 3), and CDX2P‐G22Cre;Apc^flox/flox (n = 3) mouse tumors with a Mouse Gene 1.0 ST Array (Affymetrix). The arrays were scanned using a GeneArray scanner (Affymetrix), and gene expression data were analyzed using GeneSpring GX software version 11 (Agilent Technology). The robust multichip analysis algorithm was used for normalization to remove artifactual differences between arrays, and cut‐off values were set at less than 20% to eliminate poorly reproducible entities between chips.

Kochi M., Hinoi T., Niitsu H., Miguchi M., Saito Y., Sada H., Sentani K., Sakamoto N., Oue N., Tashiro H., Sotomaru Y., Yasui W, & Ohdan H. (2020). Oncogenic mutation in RAS‐RAF axis leads to increased expression of GREB1, resulting in tumor proliferation in colorectal cancer. Cancer Science, 111(10), 3540-3549.

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Microarray Analysis of Gastrocnemius Muscle in 2-AA Mice

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As previously described (32 (link)), data of the scanned image files hybridized with probes from RNA extracted from the gastrocnemius muscle isolated at the specified times from the 2-AA-treated and untreated control mice (n=3) were converted to cell intensity files (CEL files) with the Microarray suite 5.0 (MAS; Affymetrix). The data were scaled to a target intensity of 500, and all possible pairwise array comparisons of the replicates to normal control mice were performed using a MAS 5.0 change call algorithm. Probe sets that had a signal value difference >100 and for which one of the two samples being compared was not termed 'absent', were scored as differentially modulated when i) the number of change calls in the same direction were at least 3, 4 and 6, when the number of comparisons were 4, 6 and 9, respectively; and ii) the other comparisons were unaltered. Based on the ratios of 100 genes determined to be invariant in most conditions tested (Affymetrix), an additional constraint of a minimum ratio of 1.65 was applied to control the known false positives at 5%. The microarray data are available at http://www.ncbi.nlm.nih.gov/geo/info/linking.html and the accession number is GSE43779. Gene Ontology (GO) analysis was performed using GeneSpring GX software (version 11) by Agilent Technologies (Santa Clara, CA, USA).

BANDYOPADHAYA A., CONSTANTINOU C., PSYCHOGIOS N., UEKI R., YASUHARA S., MARTYN J.A., WILHELMY J., MINDRINOS M., RAHME L.G, & TZIKA A.A. (2016). Bacterial-excreted small volatile molecule 2-aminoacetophenone induces oxidative stress and apoptosis in murine skeletal muscle. International Journal of Molecular Medicine, 37(4), 867-878.

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Transcriptome Response to Cholesterol Modulation

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Huh7 cells were cultured in DMEM containing 10% FBS and 120 μg/ml nLDL (to cholesterol enrich), 5 μM statin (to cholesterol deplete, Sigma #S6196) or vehicle control for 24 h. Following this, cells were harvested in Trizol (Invitrogen #15596026) and RNA was purified using the RNeasy Isolation Kit (Qiagen #74104). The purity and integrity of total RNA was verified using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) and hybridized to the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array according to the manufacturer’s instructions. Two biological replicates for each condition were used for microarray analysis. Raw data were normalized and analyzed by GeneSpring GX software version 11.5 (Agilent Technologies). mRNAs showing altered expression with nLDL/statin treatment compared to control were identified using unpaired two-sided Student’s t test (P ≤ 0.05, FC ≥ 1.1 or ≤–1.1). Functional annotation clustering of up- and downregulated genes was performed using DAVID (see below).

Goedeke L., Canfrán-Duque A., Rotllan N., Chaube B., Thompson B.M., Lee R.G., Cline G.W., McDonald J.G., Shulman G.I., Lasunción M.A., Suárez Y, & Fernández-Hernando C. (2021). MMAB promotes negative feedback control of cholesterol homeostasis. Nature Communications, 12, 6448.

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Transcriptome Profiling Using GeneChip Human Exon 1.0 ST Array

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The starting material for generating total RNA/Poly-A RNA controls was 1 μg/μl of total RNA, which was mixed with the GeneChip® Eukaryotic Poly-A Control Kit (Affymetrix, Inc., CA, USA). To enhance sensitivity, the majority of rRNA was eliminated from the total RNA samples prior to target labeling using the RiboMinusTM Human Transcriptome Isolation Kit (Invitrogen, CA, USA). Subsequently, cDNA synthesis was performed according to the manufacturer's instructions using the GeneChip® WT Sense Target Labeling and Control Reagents Kit (Affymetrix, Inc., CA, USA). The resulting sense cDNA was fragmented by UDG (uracil DNA glycosylase) and APE 1 (apurinic/apyrimidinic endonuclease 1), followed by biotin labeling with TdT (terminal deoxynucleotidyl transferase) using a GeneChip® WT Terminal Labeling Kit (Affymetrix, Inc., CA, USA). Once the biotin-labeled sense target DNA was prepared, it underwent hybridization to a gene chip known as The GeneChip® Human Exon 1.0 ST array. Hybridization involved incubating 5 μg of biotinylated target with a GeneChip® Hybridization Wash and Stain Kit and a GeneChip® Fluidics Station 450 (Affymetrix, Inc., CA, USA). Finally, the arrays were scanned using a GeneChip® Scanner 3000 7G(Affymetrix，Inc., CA，USA), and raw data were extracted from scanned images for analysis utilizing Agilent Technologies'GeneSpring GX software version11.5.

Guo J., Liang J., Wang Y., Guo T., Liao Y., Zhong B., Guo S., Cao Q., Li J., Flores-Morales A., Niu Y, & Jiang N. (2023). TNIK drives castration-resistant prostate cancer via phosphorylating EGFR. iScience, 27(1), 108713.

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Microarray Analysis of miR-183 Overexpression

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For the microarray analyses, groups were divided into the ESC with miR-183 overexpression and the control ones. Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. Gene expression profiling was conducted using PrimeView Human Gene Expression Array (Affymetrix). The array contains 530,000 probes covering more than 36,000 transcripts and variants, which represent more than 20,000 genes mapped through RefSeq or via UniGene annotation. All subsequent technical procedures and quality controls were performed by Genechem Co., Ltd., Shanghai, China. The arrays were scanned using a GeneChip Scanner 3000 (Affymetrix, Inc., CA, USA). Raw data were extracted from the scanned images and analyzed using GeneSpring GX software version 11.5 (Agilent Technologies, CA, USA).
The data were normalized using the PLIER default protocol. Significant differentially expressed genes were analyzed using an unpaired t-test. Hierarchical cluster analysis was used to assess correlations among samples for each identified gene set with Euclidean distance and average linkage statistical methods.

Chen J., Gu L., Ni J., Hu P., Hu K, & Shi Y.L. (2015). MiR-183 Regulates ITGB1P Expression and Promotes Invasion of Endometrial Stromal Cells. BioMed Research International, 2015, 340218.

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miRNA Expression Profiling in nHHDPCs

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In order to analyze the miRNA expression profile, nHHDPCs (2×10⁶) were seeded into 60-mm culture dishes and treated with 400 μM PPD. Following 24 h of treatment, total RNA was purified using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Total RNA was dephosphorylated and labeled with pCp-Cy3 using an Agilent miRNA Labeling kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Labeled RNAs were hybridized using a SurePrint G3 Human v16 miRNA 8×60K microarray (Agilent Technologies, Inc.) at 65°C for 20 h. The miRNA expression profile was digitized using Feature Extraction version 10.7 software (Agilent Technologies). Fold changes in miRNA expression levels were determined using GeneSpring GX software, version 11.5 (Agilent Technologies).

LEE O.K., CHA H.J., LEE M.J., LIM K.M., JUNG J.W., AHN K.J., AN I.S., AN S, & BAE S. (2015). Implication of microRNA regulation in para-phenylenediamine-induced cell death and senescence in normal human hair dermal papilla cells. Molecular Medicine Reports, 12(1), 921-936.

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RNA-seq Analysis of Cardiac Ventricles

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Total RNA was isolated from the ventricles using Trizol (Invitrogen) according to the manufacturer’s protocol and purifed with RNeasy Mini Kit (Qiagen). Microarray analysis was performed employing Affymetrix GeneChip assays using three independent experiments by the Affymetrix Gene Expression Service Lab (http://ipmb.sinica.edu.tw/affy/) supported by Academia Sinica, Taiwan using the Mouse Genome 430 2.0 Array. Differential expressions of genes were analyzed by GeneSpring GX software version 11.5 (Agilent Technologies) with Log2FC cutoff = 1, and a P value cutoff < 0.05. All data were submitted to GEO. Accession no. GSE157746.

Chen C.Y., Lee D.S., Choong O.K., Chang S.K., Hsu T., Nicholson M.W., Liu L.W., Lin P.J., Ruan S.C., Lin S.W., Hu C.Y, & Hsieh P.C. (2021). Cardiac-specific microRNA-125b deficiency induces perinatal death and cardiac hypertrophy. Scientific Reports, 11, 2377.

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Macrophage mRNA Profiling in Ac-LDL Exposure

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Mouse thioglycollate-elicited peritoneal macrophages we incubated in presence of Ac-LDL (120 μg ml⁻¹) for 24 h. Total RNA was extracted using TRIzol (Invitrogen), and mRNA was purified using and mRNA isolation Kit (Qiagen). The purity and integrity of total RNA sample was verified using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). mRNA was hybridized to Illumina expression profiling microarray (Mouse WG-6 v2.0 Expression beads Chip) according to the manufacturer's directions. The data discussed in this publication have been deposited in NCBÍs Gene Expression Omnibus and are accessible through GEO Series accession number GSE83090 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3090). Raw data were normalized and analysed by GeneSpring GX software version 11.5 (Agilent Technologies).

Aryal B., Rotllan N., Araldi E., Ramírez C.M., He S., Chousterman B.G., Fenn A.M., Wanschel A., Madrigal-Matute J., Warrier N., Martín-Ventura J.L., Swirski F.K., Suárez Y, & Fernández-Hernando C. (2016). ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression. Nature Communications, 7, 12313.

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Differential miRNA Expression in Recurrent Miscarriage

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To compare miRNA expression between decidual tissues of RM (n = 14) and IA samples (n = 13), gene expression profiling was conducted using the PrimeView Human Gene Expression Array (Affymetrix), which contains 530,000 probes covering more than 36,000 transcripts and variants. Total RNA was hybridized according to the manufacturer's instructions. Six repeats were performed from each sample to ensure consistency of hybridization. All subsequent technical procedures and quality controls were performed by Genechem Co., Ltd., Shanghai, China. The arrays were scanned by a GeneChip Scanner 3000 (Affymetrix, inc., Santa Clara, CA, USA). GeneSpring GX software version 11.5 (Agilent Technologies, Palo Alto, CA, USA) was used to analyse the raw data obtained from each probe. Next, the data were normalized using the PLIER default protocol. Additionally, an unpaired t‐test was applied to analyse the significantly differentially expressed genes. Hierarchical clustering analysis was used to assess the relationship between significantly altered miRNAs in samples for each identified gene set with Euclidean distance and average linkage statistical methods.

Zhao W., Shen W., Cao X., Ding W., Yan L., Gao L., Li X, & Zhong T. (2017). Novel mechanism of miRNA‐365‐regulated trophoblast apoptosis in recurrent miscarriage. Journal of Cellular and Molecular Medicine, 21(10), 2412-2425.

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Affymetrix Human Exon 1.0 ST Array Protocol

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One µg/µl of total RNA was used as a starting material for making total RNA/Poly-A RNA controls, and was mixed with a GeneChip®Eukaryotic Poly-A Control Kit (Affymetrix, Inc., CA, USA). The majority of the rRNA was removed from the total RNA samples prior to target labeling, so as to increase sensitivity by RiboMinusTM Human Transcriptome Isolation Kit (Invitrogen, CA, USA); cDNA was synthesized using the GeneChip® WT Sense Target Labeling and Control Reagents Kit, as per the manufacturer's instructions (Affymetrix, Inc., CA, USA). The sense cDNA was then fragmented by UDG (uracil DNA glycosylase) and APE 1 (apurinic/apyrimidinic endonuclease 1), and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using a GeneChip® WT Terminal Labeling Kit. (Affymetrix, Inc., CA, USA). After the biotinlabeled sense target DNA was prepared, the sample was ready to hybridize to gene chip (The GeneChip® Human Exon 1.0 ST array). Hybridization was performed using 5 µg of biotinylated target, which was incubated with a GeneChip® Hybridization, Wash and Stain Kit and a GeneChip® Fluidics Station 450 (Affymetrix, Inc., CA, USA). The arrays were scanned using a GeneChip® Scanner 3000 7G (Affymetrix, Inc., CA, USA). Raw data were extracted from the scanned images and analyzed with GeneSpring GX software version 11.5 (Agilent Technologies, CA, USA).

Jiang N., Guo T., Wang M., Liao Y., Ma F., Gao J., Wang Y., Wang K., Wu Y., Morales A.F., & Wen S. (2020). TNIK swaths AR to WNT pathway and drives Castration-Resistant Prostate Cancer.

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