The hybridized slides were scanned by an Axon GenePix Laser Fluorescence Scanner and analyzed by GenePix Pro 5.1 (Axon Instruments) and GeneSpring 7.0 (Silicongenetics, Redwood City, CA, USA). Gene expression was considered as significant for genes showing more than a twofold difference in expression level between normal and diabetic rats. Genes showing similar patterns were distinguished by a Pearson's correlation analysis. The images thus obtained show that genes that are up-regulated are shown in red, whereas genes that are down-regulated are shown in green. First of all, we investigated the differences in angiogenesis gene expression in keratocytes between diabetic and normal rats. In addition, the angiogenesis-related genes that show different expressions between normal and diabetic keratocytes after treatment with IL-1α and TNF-α were also studied.
Genepix pro 5
Manufactured by Molecular Devices
Sourced in United States
GenePix Pro 5.0 is a software application designed for the analysis of microarray data. It provides tools for image acquisition, gridding, feature extraction, and data analysis. The software supports a variety of microarray platforms and enables users to efficiently process and interpret their experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Agilent low input linear amplification kit, by Agilent Technologies (2 mentions)
Agilent whole mouse genome arrays g4122f, by Agilent Technologies (2 mentions)
Genepix 4000b scanner, by Molecular Devices (2 mentions)
Oncyte avid, by Grace Bio-Labs (2 mentions)
Genepix 4000a, by Molecular Devices (2 mentions)
Agilent c scanner, by Agilent Technologies (1 mentions)
Mircury lna microrna array kit, by Qiagen (1 mentions)
Genepix 4200a microarray scanner, by Molecular Devices (1 mentions)
Genepix 4000a microarray scanner, by Molecular Devices (1 mentions)
Oligonucleotide microarray, by Agilent Technologies (1 mentions)
Genepix 4000b, by Molecular Devices (1 mentions)
Genepix microarray scanner, by Molecular Devices (1 mentions)
Genepix 4100a scanner, by Molecular Devices (1 mentions)
G2565ba, by Agilent Technologies (1 mentions)
Superscript 2 kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna 2 kit, by Macherey-Nagel (1 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
19 protocols using genepix pro 5
1
Angiogenesis Gene Expression in Diabetic Keratocytes
2
miRNA Expression Profiling via LNA Microarray
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The MiRNA expression profiling was performed using the miRCURY LNA microRNA array kit (Exiqon, Vedbaek, Denmark) according to the manufacturer's protocol. This array contains 3100 capture probes covering human miRNAs in miRBase v. 19.0. Slides were scanned using an Agilent C scanner (Agilent Technologies). Next, scanned images were imported into GenePix Pro 5.1 software (Axon Instruments, Foster City, CA) for grid alignment and data extraction. Expressed data were normalized using quantile normalization. After normalization, miRNAs that were significantly differentially expressed between the 2 groups were identified through fold changes (>1.5) and P-values (<.05). The sample number was limited; thus, we did not perform a multiple hypotheses testing, such as false discovery rate.
3
Microarray Analysis of Mouse Transcriptome
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This was performed as in our previous publications (Pendergrass et al., 2012 (link)). cDNA samples were amplified and labeled using the Agilent Low Input Linear Amplification kit (Agilent Technologies, Santa Clara, CA) and were hybridized against Universal Mouse Reference (UMR) (Strategene, La Jolla, CA) to Agilent Whole Mouse Genome arrays (G4122F) (Agilent Technologies, Santa Clara, CA) in a common reference based design. Microarrays were hybridized and washed in accordance with manufacturer’s protocols and scanned using a dual laser GenePix 4000B scanner (Axon Instruments, Foster City, CA). The pixel intensities of the acquired images were then quantified using GenePix Pro 5.1 software (Axon Instruments, Foster City, CA).
4
Microarray Analysis of Mouse Transcriptome
5
Microarray Analysis of Polysomal Fractions
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The human cDNA microarrays contained a set of ~10 000 human cDNA clones, manufactured in MRC Toxicology Unit Leicester. Fluorescently labelled DNA probes were generated from equal proportions of RNAs (~7 μg) of pooled polysomal fractions (fractions 1–5) and pooled non-polysomal fraction (fractions 6–11) or with fixed amount of universal commercial RNA control (UniRNA Supplier, Stratagene, San Diego, CA, USA). Microarray slides were scanned using a GenePix 4200A microarray scanner and GenePix Pro 5.1 software (Axon Instruments, Union City, CA, USA).
6
Colony Size Quantification Protocol
7
Total RNA Extraction and cDNA Labeling
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Total RNA was purified using phenol extraction [74] (link). Fluorescently labelled cDNA was prepared from total RNA using the SuperScript Plus Direct cDNA Labelling System (Life Technologies) as described by the manufacturer, except for the following modifications: 8 µg of total RNA was labelled in a reaction volume of 15 µl. 0.5 µl of 10× nucleotide mix with labelled nucleotide were used (1/3 of the recommended amount) and 1 µl of a home-made dNTP mix (0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP) was added to the reaction. All other components were used at the recommended concentrations. Note that these changes are essential to prevent dye-specific biases. Labelled cDNAs were hybridised to oligonucleotide microarrays manufactured by Agilent as described [63] (link). Microarrays were scanned with a GenePix 4000A microarray scanner and analysed with GenePix Pro 5.0 (Molecular Devices).
8
RPPA Analysis of Cell Lysates
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RPPA experiments were performed as previously described [47 , 74 (link)]. Briefly, cell lysates from three biological replicates for each condition were spotted in nitrocellulose-coated glass slides (Oncyte Avid, Grace-Biolabs, Bend, OR, USA) in technical triplicates. All the primary antibodies used were previously validated through Western blots to test their specificity. Signal intensities of spots were quantified using GenePixPro 5.0 (Molecular Devices, Sunnyvale, CA, USA). Intensity values were log2 transformed and plotted using morpheus software (https://software.broadinstitute.org/morpheus/ ). List of antibodies used is provided in Table S8
9
Reverse Phase Protein Array (RPPA) Experiment Protocol
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The RPPA experiments were performed as previously described [38 (link),39 (link)]. Briefly, cell lysates from three biological replicates for every condition were spotted on nitrocellulose-coated glass slides (Oncyte Avid, Grace-Biolabs, Bend, OR, USA) in technical triplicates each. All antibodies used were previously tested with Western blotting to validate their specificity. The signal intensities of the spots were quantified using GenePixPro 5.0 (Molecular Devices, Sunnyvale, CA, USA). Preprocessing and scaling of the RPPA data, background correction, and the merging of technical triplicates was performed in R using the software packages RPPanalyzer [40 (link)] and BlotIt [41 (link)].
10
Microarray Data Preprocessing and Analysis
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Microarrays were scanned using either AxonScanner 4200 or 4000B (Molecular Devices, Sunnyvale, CA). PMT levels were auto-adjusted to achieve 0.1–0.25% pixel saturation. Each element was located and analyzed using GenePix Pro 5.0 (Molecular Devices). Data were filtered to exclude elements that did not have a regression correlation of ≥0.6 between Cy5 and Cy3 signal over the pixels compromising the array element of and intensity/background ratio of ≥2.5 in at least one channel, for 60% of the arrays. For cluster and SAM analysis of Ago+/− miRNA IPs vs mock IPs, measurements corresponding to oligonucleotides that map to the same EntrezID were treated separately and the data were globally normalized per array, such that the median log2 ratio was 0 after normalization.
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