Extraction of ribonucleic acid (RNA), integrity assessment, and quantification of RNA were conducted following the methods of Ishimaru et al. (2007 ). Microarray experiment was conducted based on the method of Takehisa et al. (2012 (link)). Total RNA (2.5 ng) was amplified to obtain complementary RNA (cRNA) using a Quick Amp Labeling kit and labelled using One-color (Agilent technologies) cyanine-3 (Cy3)-CTP, according to the modified manufacturer’s instruction. The Cy3-labeled cRNA was purified by Rneasy Mini Kit (Qiagen). Concentration of cRNA was quantified by a NanoDrop ND-1000 UV–VIS spectrophotometer (NanoDrop Technologies). A total of 900 ng Cy3-labeled cRNAs were fragmented and hybridized on a slide glass of rice 4 × 44 K microarray RAP-DB (G2519F#15241; Agilent Technologies). Hybridization and washing of the hybridized slides were performed according to the manufacturer’s instructions. Slides were scanned on an Agilent G2505B DNA microarray scanner, and background of the Cy3 raw signals was corrected using the Feature Extraction (ver. 10.5.1.1, Agilent Technologies).
G2505b dna microarray scanner
Manufactured by Agilent Technologies
Sourced in United States, Belgium
The G2505B DNA microarray scanner from Agilent Technologies is a high-performance instrument designed for the detection and analysis of DNA microarray samples. It features a sensitive fluorescence detection system and advanced image processing capabilities to capture and analyze microarray data.
Automatically generated - may contain errors
Lab products found in correlation
Quick amp labeling kit, by Agilent Technologies (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Nanodrop nd 1000 uv vis, by Thermo Fisher Scientific (1 mentions)
Bio prime array cgh kit, by Thermo Fisher Scientific (1 mentions)
Mouse ribopure blood rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Illustra mrna purification kit, by GE Healthcare (1 mentions)
Agilent feature extraction software v 9.5.1, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Feature extraction v9, by Agilent Technologies (1 mentions)
Agilent genomic dna labeling kit plus, by Agilent Technologies (1 mentions)
Rice 4 44 k microarray rap db, by Agilent Technologies (1 mentions)
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Cy3 dctp, by GE Healthcare (1 mentions)
Cy5 dctp, by GE Healthcare (1 mentions)
Feature extraction software 9, by Agilent Technologies (1 mentions)
244k array, by Agilent Technologies (1 mentions)
Bioprime array cgh genomic labeling system, by Thermo Fisher Scientific (1 mentions)
Rneasy column, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
9 protocols using g2505b dna microarray scanner
1
Microarray Analysis of Rice Transcriptome
2
Methylation-Specific DNA Immunoprecipitation Assay
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Briefly, 10 μg of whole blood genomic DNA was sonicated by using a 2 mm probe at amplitude 40 for 30 cycles with 15 seconds on and off, to obtain fragment sizes of 100–800 base pairs. About 3 μg of sheared DNA (INPUT) was immunoprecipitated overnight with anti-5-mathyl cytosine antibody (Diagenode, Belgium) along with positive and negative control DNA provided in the Diagenode kit. Enriched methylated fragments were purified by standard phenol-chloroform-ethanol precipitation [26 (link)]. The quality analysis of enriched immunoprecipitated (IP) methylated fragments was evaluated by real time PCR with positive, negative, GAPDH and AX1 primers (Diagenode, Belgium) [27 (link)]. INPUT and IP were differentially labeled with cyanine3 and cyanine5 (Amersham Biosciences, USA) by indirect method using Bio-prime Array CGH kit (Invitrogen, USA) [28 (link),29 (link)]. Equal concentrations of labeled INPUT and IP DNA were co-hybridized onto the Agilent 244K Human CpG Island, high density oligonucleotide array at 65°C for 40 hours with the continuous rotation at 18 rpm as per the Agilent MeDIP protocol version 1.1 (Agilent Technology, USA). The slide was washed using wash buffers (Agilent Technologies, Santa Clara, CA, USA), dried and scanned using G2505B DNA microarray scanner (Agilent Technology, USA) with Sure Scan High resolution technology (Additional file 1 ).
3
Mouse Blood RNA Isolation and Microarray Analysis
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RNA was isolated from blood samples using a Mouse RiboPure™‐Blood RNA Isolation Kit (Life Technologies) and then amplified and labeled with Cy3 using a Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA, USA) as per the manufacturers’ protocols. The cRNA was hybridized on a Whole Mouse Genome 4 × 44K Array and readings were performed using a G2505B DNA microarray scanner (Agilent Technologies). The software BRB‐ArrayTools v.4.6.0 (http://linus.nci.nih.gov/BRB-ArrayTools.html ) was used for gene expression analysis. Using this software, quantile normalization was applied and 27 144 genes passed the preliminary filtering criteria. Furthermore, gene set enrichment analysis was performed using class comparison by Gene Sets in the BRB tool software, which allowed analysis of gene sets showing differential expression among classes. The gene sets are pre‐included as a module of BRB‐ArrayTools, using publicly available Gene Ontology tools, lymphoid signatures or BioCarta Pathways domains. The Bioconductor GO package, in combination with SOURCE annotation, was also used for the analysis. The resulting gene sets passed either the LS/KS permutation test or the Efron‐Tibshirani test (P < 0.005).
4
Maize Gene Expression Analysis
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Microarray analyses were conducted as described previously [44 (link)]. Maize internode sections were ground under liquid nitrogen and total RNA extracted with the TRIZOL reagent (Invitrogen, Sydney) according to the manufacturer’s instructions. Polyadenylated mRNA was purified using the Illustra mRNA Purification Kit (GE Biosciences). Sample quality and RNA concentration were assessed using an Agilent Bioanalyzer. The mRNA was reverse-transcribed into double-stranded cDNA, which was labelled with Cy3 or Cy5 fluorescent dye, using the Agilent Low RNA Linear Amp kit. Biological replicates were labelled alternately using Cy3 or Cy5 to guard against dye-bias. The cDNA was hybridized to Agilent 4 × 44 k maize gene microarrays [70 (link)] and the microarrays were washed according to Agilent standard protocols. The microarray chips were scanned with an Agilent G2505B DNA Microarray Scanner at two laser power settings (100% and 10%). The images were inspected visually for image artefacts, and feature intensities were extracted, filtered and normalized with Agilent Feature Extraction Software (v 9.5.1). The data were normalized using quantile normalization (BOLSTAD http://www.ncbi.nlm.nih.gov/pubmed/12538238 ). The normalized data were used to compare expression levels of genes related to cell wall compositions.
5
DNA Microarray Scanning Protocol
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Hybridized slides were scanned at 5 µm resolution using an Agilent G2505B DNA microarray scanner. Default settings were modified to scan the same slide twice at two different sensitivity levels (XDR Hi 100% and XDR Lo 10%). The two linked images generated were analyzed together and data were extracted and background subtracted using the standard procedures contained in the Agilent Feature Extraction (FE) Software version 9.5.1. The software returns a series of spot quality measures to assess the reproducibility and the reliability of spot intensity estimates. These parameters are summarized in a quality control report and were evaluated in order to support the high quality of the data acquired.
6
Genome-wide DNA Copy Number Analysis
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The DNA was labeled according to the Agilent Technologies protocol. For each tumor sample and gender-matched pooled normal control DNA from Promega (Human Genomic DNA), 500 ng of DNA was fragmented by heating. The tumor DNA and the control DNA were enzymatically labeled using an Agilent Genomic DNA Labeling Kit PLUS (Agilent, 5188–5309). The tumor samples were labeled with ULS-Cy5 and the control DNA with ULS-Cy3, and then hybridized to 244 K Human Whole-Genome arrays (G441B) from Agilent at 65°C for 48 h in a rotating chamber at 20 rpm.
After washing, the slides were scanned with an Agilent G2505B DNA Microarray Scanner at a resolution of 5 µm, using default parameters. The acquisition of signals from the resulting scanned images and normalization were performed with Feature Extraction v9.5 software (Agilent Technologies), using default parameters. The normalized data were recentralized using a custom in-house script and then analyzed with CGH Analytics v3.4.40 software, using the ADM-2 segmentation method with a threshold setting of 6. The probes were mapped on the human genome build 36 (UCSC hg18, March 2006).
After washing, the slides were scanned with an Agilent G2505B DNA Microarray Scanner at a resolution of 5 µm, using default parameters. The acquisition of signals from the resulting scanned images and normalization were performed with Feature Extraction v9.5 software (Agilent Technologies), using default parameters. The normalized data were recentralized using a custom in-house script and then analyzed with CGH Analytics v3.4.40 software, using the ADM-2 segmentation method with a threshold setting of 6. The probes were mapped on the human genome build 36 (UCSC hg18, March 2006).
7
RNA Extraction and Microarray Analysis
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Extraction of ribonucleic acid (RNA), integrity assessment, and quanti cation of RNA were conducted following the methods of Ishimaru et al. (2007) . Microarray experiment was conducted based on the method of Takehisa et al. (2012) . Total RNA (2.5 ng) was ampli ed to obtain complementary RNA (cRNA) using a Quick Amp Labeling kit and labelled using One-color (Agilent technologies) cyanine-3 (Cy3)-CTP, according to the modi ed manufacturer's instruction. The Cy3-labeled cRNA was puri ed by Rneasy Mini Kit (Qiagen). Concentration of cRNA was quanti ed by a NanoDrop ND-1000 UV-VIS spectrophotometer (NanoDrop Technologies). A total of 900 ng Cy3-labeled cRNAs were fragmented and hybridized on a slide glass of rice 4 44K microarray RAP-DB (G2519F#15241; Agilent Technologies). Hybridization and washing of the hybridized slides were performed according to the manufacturer's instructions. Slides were scanned on an Agilent G2505B DNA microarray scanner, and background of the Cy3 raw signals was corrected using the Feature Extraction (ver. 10.5.1.1, Agilent Technologies).
8
Array CGH Analysis of DNA Samples
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Array CGH analysis was performed using standard methods described [5 (link)]. In brief, 300 ng of genomic DNA was labeled with Cy3-dCTP or Cy5-dCTP (GE Healthcare, Belgium) using Bioprime array CGH genomic labeling system (Invitrogen, Belgium). For the labeling, we used the “triangle method”: DNA samples from patients and controls were labeled and hybridized using a dye swap in trios consisting of at least one control per triangle. Samples were hybridized on 244K arrays (design ID 014693, Agilent, Belgium) for 40 h at 65°C. After washing, the samples were scanned at 5 μm resolution using a DNA microarray scanner G2505B (Agilent, Belgium). The scan images were analyzed using the feature extraction software 9.5.3.1 (Agilent) and further analyzed with “arrayCGHbase” [6 (link)]. Copy number variations were taken into consideration when two or more flanking probes were exceeding a value of the intensity ratios ± four times the standard deviation of log2 of all intensity ratios for that experiment. Always two experiments investigating the same sample with a dye swap were compared and only when an alteration is present in both experiments was the region included for further analysis. Inconsistencies were inspected manually.
9
Transcriptome Analysis of Primary HSCs
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Total RNA from primary HSCs was isolated using RNeasy columns (Qiagen GmbH). Three cell samples were used for each siRNA. Samples quantity and quality were measured with a capillary 2100 Bioanalyzer and mRNA was labeled using the LRILAK PLUS two-color kit (Agilent Technologies). The Cy3-ctp and Cy5-ctp labeled cDNAs were isolated by the RNeasy Mini kit (Qiagen GmbH). These samples were hybridized using Custom Microarray GE, 4 × 44 k according to the manufacturer´s instructions. Slides were scanned using the DNA microarray scanner G2505B (Agilent Technologies) and the images were analyzed with the Agilent Scan Software Feature Extraction v9.5 for normalization and data analysis. Those genes which were 2 fold up-regulated or down-regulated in the hybridization were introduced into the AmiGO 2 Gen ontology database. Bonferroni-corrected p values less than 0.05 were regarded as a statistically significant association. The p-value denotes the significance of GO term enrichment in the differentially expressed gene list.
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