Total RNA of Z. mobilis ZM4 cells was extracted using a Trizol reagent kit from Invitrogen (Carlsbad, CA, USA). Purity and concentrations of the RNA samples were measured by a ratio of OD260/280 readings using a NanoDrop ND-1000 spectrophotometer. DNA microarray was performed by CapitalBio Co., Beijing, China. cDNA fragments were hybridized with the probes and labeled with Cy3-dCTP fluorescent dye [26 (link)]. Data were analyzed using GeneSpring V12 Agilent Technologies (Santa Clara, CA, USA) with proper normalization and quality control procedures. Raw data were transformed into algorism phase based on Log2 and statistical analysis was carried out using software CLUSTER 3.0 [27 (link)]. Significance of differential expression levels compared with the control was examined using False Discovery Rate (FDR) test (p < 0.05) [28 ] and analysis of variance (ANOVA). A threshold of fluorescence intensity with more or less than twofold changes was regarded as significantly differentially expressed genes. Finally, a hierarchical clustering analysis was performed using Java Treeview (Stanford University, Stanford, CA, USA).
Genespring v12
Manufactured by Agilent Technologies
Sourced in United States
GeneSpring v12.0 is a software application developed by Agilent Technologies for the analysis and visualization of genomic data. It provides a comprehensive platform for processing, analyzing, and interpreting data from various genomic experiments, including gene expression, microarray, and next-generation sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Humanht 12 v4 expression beadchip, by Illumina (2 mentions)
Totalprep 96 rna amplification kit, by Thermo Fisher Scientific (2 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (2 mentions)
Trizol reagent kit, by Thermo Fisher Scientific (1 mentions)
Sureprint g3 mouse ge 8x60k microarray, by Agilent Technologies (1 mentions)
Ovation rna amplification system v2 kit, by Tecan (1 mentions)
Isogen, by Nippon Gene (1 mentions)
Humanht 12 v4 beadchip, by Illumina (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Mouse genome 430 2.0 array, by Thermo Fisher Scientific (1 mentions)
Ht mg 430 pm array, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Human lncrna array v3, by Agilent Technologies (1 mentions)
Human gene 1.0 st array, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Feature extraction v10, by Agilent Technologies (1 mentions)
Spss v17, by IBM (1 mentions)
19 protocols using genespring v12
1
Transcriptome Analysis of Z. mobilis ZM4
2
Microarray Data Analysis Protocol
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Dye-swapped duplicate data were mean averaged to control for color biases, and this value was used for subsequent analyses (GeneSpring v12, Agilent Technologies, Santa Clara, CA). The absolute fold change was converted by a (log2) transformation. p-value was then calculated for these fold changes, reflecting the probability of obtaining a given fold change for a probe by chance, and incorporated the control measurements from the spike-in controls and dye-swap variation. In Fig. 1 , p-values were normalized by a (− log10) conversion. mRNA signals required a mean fold change of >|2 |, and a p-value of < 0.05 in order to be considered experimentally significant.
3
Stomatal Kinetics and Transcriptomics
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The data were subjected to analyses of variance with no blocking (GenStat 6.2). The rates of stomatal closure or opening in response to long-term changes in CO 2 concentration and to DCMU feeding were compared by fitting the empirical functions of best fit among a series of standard curves to the g s kinetics over time. Regressions with groups (analysis of parallelism, GenStat 6.2) were analysed to determine whether a common regression should be fitted for the two genotypes (implying that there was no difference between them), or whether regressions with some or all their parameters separated should be selected (indicating that the kinetics differed in elevation or slope).
To fit the functions, all five or six replicate leaves from each genotype were included separately. Stomatal opening and closing rates were estimated as first derivatives of the selected functions with respect to time. The microarray data were processed using GeneSpring (v.12) software (Agilent). A statistical filter was applied to the data, which were subjected to an analysis of variance (P value<0.01) with Benjamini and Hochberg correction [49] for false positives (FDR).
To fit the functions, all five or six replicate leaves from each genotype were included separately. Stomatal opening and closing rates were estimated as first derivatives of the selected functions with respect to time. The microarray data were processed using GeneSpring (v.12) software (Agilent). A statistical filter was applied to the data, which were subjected to an analysis of variance (P value<0.01) with Benjamini and Hochberg correction [49] for false positives (FDR).
4
Microarray Data Analysis Pipeline
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A computational analysis of microarray data was performed using GeneSpring v12.0 (Agilent Technologies, Inc., Santa Clara, CA, USA). Based on a Student's t-test analysis, differentially expressed genes were filtered through statistical estimation of fold-changes from replicated samples (fold change ≥2.0) using a P-value threshold (P<0.05). Distinguishable gene expression of those samples was demonstrated via hierarchical clustering, followed by heatmap generation. Additionally, GO and pathway analyses of differentially expressed genes were performed to determine the potential signaling pathways underlying their biological functions. Public data from bioinformatics resources (http://www.geneontology.org/ ) were utilized for GO enrichment analysis. Ingenuity Pathway Analysis was utilized to identify genes whose expression was changed by at least 2-fold.
5
Microarray Data Analysis Pipeline
6
Microarray Data Analysis Workflow
7
Microarray Data Computational Analysis
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A computational analysis of microarray data was performed using GeneSpring v12.0 (Agilent Technologies, Inc., Santa Clara, CA, USA). Data are expressed as the mean ± standard error of the mean. Differences in the mean values were evaluated by Student's t-test (two means comparison). Differentially expressed miRNA were ltered through statistical estimation of fold-changes from replicated samples (fold change ≥ 2.0) using a P-value threshold (P < 0.05).
8
Transcriptomic Analysis of IVF Blastocysts
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Five Egfp-PE, Kdm4b-PE and IVF blastocysts (120 h) were lysed using ISOGEN (Nippongene) and RNA was extracted by phenol–chloroform and isopropanol precipitation. cDNA was synthesized using the Ovation RNA Amplification System V2 kit (NuGEN, West Cumbria, UK) and hybridized with SurePrint G3 Mouse GE 8x60K Microarray (Agilent Technologies). Analysis was conducted using GeneSpringV12.5 (Agilent Technologies). Transcripts were considered to be expressed if raw values were >100 and a flag was present in at least one of the groups.
9
Transcriptome Analysis with GeneSpring
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Data evaluation was done with Genespring V12.5 (Agilent Technologies). Values were log2 transformed and quantile normalized, before relative expression values were calculated by subtracting the median expression of each probe from the other values of this specific probe (baseline transformation). After removing outliers and transcripts without significant expression at any time point, ANOVA (P ≤ 0.005, FC ≥ 3) and FDR correction (Benjamini-Hochberg) was performed. These stringent parameters were chosen in order to identify important transcripts without (unnecessarily) expanding the data set.
10
Transcriptome Analysis with GeneSpring
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Data evaluation was done with Genespring V12.5 (Agilent Technologies). Values were log2 transformed and quantile normalized before relative expression values were calculated by subtracting the median expression of each probe from other values of this specific probe (baseline transformation). After removing outliers and transcripts without significant expression at any time point, ANOVA [P≤ 0.005, fold change (FC) ≥3] and false discovery rate (FDR) correction (Benjamini–Hochberg) was performed. To identify transcripts with similar expression profiles, K-means clustering (Pearson correlation, 30 clusters) was performed. In order to reduce the number of total clusters, the median values of each cluster were subjected to hierarchical clustering (Pearson correlation), and 10 new clusters were derived.
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