Microarray CGH analysis was performed as described previously, using a 180 k oligonucleotide array (SurePrint G3 Human CGH Microarray Kit 4 × 180 K, Agilent Technologies, Palo Alto CA, USA)16 (link). Images were acquired using a Microarray scanner G2505B (Agilent Technologies, Palo Alto CA, USA). Analysis and data extraction were quantified using feature extraction software (version 9.1, Agilent Technologies, Palo Alto CA, USA). Gains and losses were defined as deviations of 0.2 or more from log2 ratio = 0.0. High-level amplification was considered when at least 2 neighboring clones reached a log2 ratio of >2.0 and homozygous deletions (HDs) were defined as two or more consecutive oligonucleotides with a log2 ratio of <−3. The possibility of copy number variations (rather than copy number alterations) was excluded by using normal DNA of the same patient as reference. The microarray CGH data are accessible through GEO Series accession number GSE57201.
Microarray scanner g2505b
Manufactured by Agilent Technologies
Sourced in United States
The Microarray Scanner G2505B is a high-performance, fully automated microarray scanner designed for life science and genomics research applications. It utilizes dual-laser excitation and high-resolution optics to capture detailed, high-quality images of microarray samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Feature extraction software version 9.1, by Agilent Technologies (1 mentions)
Rna 6000 nano labchipvr reagent set, by Agilent Technologies (1 mentions)
Mirneasy mini kit for mirna, by Qiagen (1 mentions)
Gene expression hybridisation kit, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Gene expression hybridization kit, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Bioprime array cgh genomic labeling kit, by Thermo Fisher Scientific (1 mentions)
Agilent genomic workbench, by Agilent Technologies (1 mentions)
Cy5 dctp, by GE Healthcare (1 mentions)
Cy3 dctp, by GE Healthcare (1 mentions)
Sureprint g3 human cgh microarray kit, by Agilent Technologies (1 mentions)
4 protocols using microarray scanner g2505b
1
Microarray CGH Analysis of Copy Number Variations
2
RNA Isolation and Microarray Analysis
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RNA isolation was performed according to the manufacturer's instructions using the RNeasy Mini-Kit (Qiagen, Hilden, Germany) for mRNA and miRNeasy Mini-Kit for miRNA (Qiagen). The prepared RNA was preserved at −80 °C. Only samples with RNA integrity number >8.8 for miRNA and >8.7 for mRNA were accepted for further processing.
Array analyses were performed using RNA 6000 Nano LabChipVR reagent set (Agilent Technologies, Boeblingen, Germany), Low RNA Input Linear Amplification Kit PLUS, One-Color (Agilent Technologies), Gene Expression Hybridisation Kit (Agilent) and miRNA Labelling Reagent and Hybridisation Kit (Agilent Technologies).
The Microarray Scanner G2505B (Agilent Technologies) was used for washing and scanning of miRNA and mRNA arrays.
Array analyses were performed using RNA 6000 Nano LabChipVR reagent set (Agilent Technologies, Boeblingen, Germany), Low RNA Input Linear Amplification Kit PLUS, One-Color (Agilent Technologies), Gene Expression Hybridisation Kit (Agilent) and miRNA Labelling Reagent and Hybridisation Kit (Agilent Technologies).
The Microarray Scanner G2505B (Agilent Technologies) was used for washing and scanning of miRNA and mRNA arrays.
3
RNA Quantity, Quality, and Differential Expression
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RNA was quality and quantity was assessed with a Nanodrop 1000 (Thermo Scientific). 260/280 nm ratios were between 1.9 and 2.1. An Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to measure the RNA integrity number (RIN) and only RNA samples with values >8.7 were submitted to microarray analysis. For cy3-labeling the “Low RNA Input Linear Amplification Kit, PLUS, One-Color” (Agilent Technologies) was used, and for hybridization (17.5 h) the “Gene Expression Hybridization Kit” (Agilent Technologies, USA) was applied. Washing and scanning was done in the Micro Array Scanner G2505B (Agilent Technologies, USA), imaged files were processed with the NimbleScan Software 2.6 with using default setting of the Robust Multi-Array Analysis (RMA). Averages were made from the normalized microarray data from one experimental group, and ratios between two experimental groups were calculated with Excel 2013. In case the ratio is >1 it equals the fold change, if the ration is <1 the fold change is the negative reciprocal of the ratio. Fold changes were considered to represent a significantly differential expression if the t-test p-value was <0.05.
4
Genome-wide DNA Copy Number Analysis
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