G2505b microarray scanner system

Manufactured by Agilent Technologies

The G2505B Microarray Scanner System is a compact, high-resolution microarray scanner designed for the analysis of DNA, RNA, and protein microarrays. It provides fast and accurate scanning of microarray slides with 16-bit data collection and a maximum resolution of 3 microns per pixel.

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7 protocols using g2505b microarray scanner system

Microarray Gene Expression Analysis

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Fluorescence signals of the hybridized microarrays were monitored using Agilent’s Microarray Scanner System G2505B and the Scan Control Software (Agilent Technologies). The Agilent Feature Extraction Software (FES) version 10.2.1.3 was used to read out and process the microarray image files. For the determination of differential gene expression, the FES derived output data files were further analyzed using the Rosetta Resolver^® Gene Expression Data Analysis System (Rosetta Biosoftware, Seattle, WA, USA). The local signal of each spot was measured inside a 300 μm diameter circle. The local background was determined within 40 μm wide rings approximately 40 μm distant from the signal. Then, local background was subtracted from the local signal intensity to calculate the net signal intensity and the ratio of Cy5 to Cy3. The ratios were normalized to the median of all ratios, considering only those spots with fluorescence intensities three times larger than that of the control herring sperm DNA and spotting buffer negative controls. The values represent the means of four single spots and standard deviations. Cutoff was chosen at > twofold expression with P<0.01.

Dkhil M.A., Lubbad M.Y., Al-Shaebi E.M., Delic D, & Al-Quraishy S. (2015). The antiplasmodial and spleen protective role of crude Indigofera oblongifolia leaf extract traditionally used in the treatment of malaria in Saudi Arabia. Drug Design, Development and Therapy, 9, 6235-6246.

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Comparative Transcriptional Profiling of L. salivarius Mutants

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L. salivarius UCC118 wild-type and both mutant strains were grown in MRS, and RNA was extracted in duplicate, as described above, in exponential and stationary phases. Labelling of cDNA with Cy3 and Cy5 dyes was carried out using a chemical labelling kit (Kreatech), following the manufacturer’s instructions. Microarray slides were hybridized for 16 h at 55 °C and scanned using an Agilent Microarray Scanner system (G2505B) with Agilent scan control software (version 7.0). Agilent feature extraction software (version 9.1) was used to process the image file and the extracted data were further processed using an in-house microarray transform platform, as previously described [2 (link)]. Genes were selected as being significantly changed in expression if their fold change in Cy3/Cy5 ratio was >3 and where the P value was <0.0001. Four microarray conditions were carried out in duplicate: wild-type against ΔlncRNA in exponential phase, wild-type against ΔlncRNA Δ
LSL_1884 in exponential phase, wild-type against ΔlncRNA in stationary phase and wild-type against ΔlncRNA Δ
LSL_1884 in stationary phase.

Cousin F.J., Lynch D.B., Chuat V., Bourin M.J., Casey P.G., Dalmasso M., Harris H.M., McCann A, & O’Toole P.W. (2017). A long and abundant non-coding RNA in Lactobacillus salivarius. Microbial Genomics, 3(9), e000126.

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Transcriptional Analysis of Sparus aurata

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Transcriptional analysis was done using the Aquagenomics Sparus aurata oligonucleotide microarray v2.0 (4 × 44 K) (SAQ) platform. Platform information and transcriptomic raw data are available through Gene Expression Omnibus (GEO) at NCBI (accession numbers GPL13442 and GSE144055, respectively).
Analyses were conducted using a one-color RNA labelling (Agilent One-Color RNA Spike-In kit; Agilent Technologies). Total RNA (200 ng) from each sample pool were reverse-transcribed with spike-in. Total RNA was used as template for Cyanine-3 labelled cRNA synthesis and amplification kit (Quick Amp Labelling kit). cRNA samples were purified using the RNeasy micro kit (Qiagen). Dye incorporation and cRNA yield were checked (NanoDrop ND-2000); Cy3-labeled cRNA (1.5 mg) with specific activity > 6.0 pmol Cy3/mg cRNA were fragmented (60 °C, 30 min), and then mixed with the hybridization buffer (Gene Expression Hybridization kit, Agilent Technologies), and hybridized (65 °C, 17 h) to the array (ID 025603, Agilent Technologies). Washes were conducted using Gene expression wash buffers, stabilization and drying solutions. Microarray slides were scanned (Agilent G2505B Microarray Scanner System) and spot intensities and other quality control features extracted (Agilent Feature Extraction software version 10.4.0.0).

Firmino J.P., Vallejos-Vidal E., Sarasquete C., Ortiz-Delgado J.B., Balasch J.C., Tort L., Estevez A., Reyes-López F.E, & Gisbert E. (2020). Unveiling the effect of dietary essential oils supplementation in Sparus aurata gills and its efficiency against the infestation by Sparicotyle chrysophrii. Scientific Reports, 10, 17764.

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miRNA Profiling of A549 Cells

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miRNA profiling of A549 and A549-T24 cells were done from Exiqon (Vedbaek, Denmark). Total RNA including miRNA was extracted from A549 and A549-T24 cells using Qiagen miRNeasy mini kit following the manufacturer protocol. The total RNA samples having adequate quality for analysis by miRCURY LNA miRNA microarray platform were labeled using the miRCURY LNA microRNA Hi-Power Labelling Kit, Hy3/Hy5 and pairs of sample were hybridized on the miRCURY LNA microRNA Array (6th gen - hsa, mmu & rno) and the array was read in Agilent G2505B Micro array scanner system in an ozone free environment. Only those miRNAs which were dysregulated by at least two fold (ΔLMR ≥2) were taken into consideration.

Chatterjee A., Chattopadhyay D, & Chakrabarti G. (2014). miR-17-5p Downregulation Contributes to Paclitaxel Resistance of Lung Cancer Cells through Altering Beclin1 Expression. PLoS ONE, 9(4), e95716.

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Genome-Wide Microarray Analysis of RNA

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Following RNA concentration and integrity analysis, the 44 samples were analyzed using Agilent 4×44K Whole Human Genome Oligo Microarrays at the Cancer Institute and Hospital, Chinese Academy of Medical Sciences, according to the manufacturer's specifications. In brief, 500 ng purified total RNA was reversed transcribed in vitro using the Low RNA Input Linear Amplification Kit PLUS (Agilent) and then transcribed into cRNA labeled with Cy3. In total, 1.65 μg cRNA was hybridized to each microarray. After hybridization, the slides were washed and scanned with an Agilent G2505B Microarray Scanner System. The fluorescence intensities of the scanned images were extracted and preprocessed using Agilent Feature Extraction Software (v9.1). The raw data were normalized using the GeneSpring GX software program, version 11.5 (Silicon Genetics, Redwood City, CA, USA). The raw and processed data are publicly available on the Gene Expression Omnibus (GEO) website under the accession number GSE93520.

Zhang B., Feng L., Guo H., Li H., Wang Y., Zhang K., Yu X, & Cheng S. (2017). Villi-specific Gene Expression Reveals Novel Prognostic Biomarkers in Multiple Human Cancers. Journal of Cancer, 8(14), 2793-2801.

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Whole Human Genome Microarray Analysis

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All sample-labeling, hybridization, washing and scanning steps were conducted at the Cancer Institute and Hospital, Chinese Academy of Medical Sciences, according to the manufacturer’s specifications. In brief, 1.65 µg of Cy3-labeled cRNA was generated from 500 ng of total RNA by in vitro transcription using Low RNA Input Linear Amplification Kit PLUS (Agilent) and hybridized to the Whole Human Genome Oligo Microarray (Agilent). After hybridization, the slides were washed and then scanned with the Agilent G2505B Microarray Scanner System. The fluorescence intensities on scanned images were extracted and preprocessed using Agilent Feature Extraction Software (v9.1). The raw data were normalized by the median scale method using the R package “limma” (www.r-project.org). Probes representing the same gene were further screened and only the probe exhibiting the largest mean intensity was retained. Consequently, an expression matrix containing 19,503 unique genes (listed in Table S2) was obtained and used in the subsequent analysis. The raw and processed data are publicly available on the Gene Expression Omnibus (GEO) website under the accession number GSE43767.

Feng L., Wang J., Cao B., Zhang Y., Wu B., Di X., Jiang W., An N., Lu D., Gao S., Zhao Y., Chen Z., Mao Y., Gao Y., Zhou D., Jen J., Liu X., Zhang Y., Li X., Zhang K., He J, & Cheng S. (2014). Gene Expression Profiling in Human Lung Development: An Abundant Resource for Lung Adenocarcinoma Prognosis. PLoS ONE, 9(8), e105639.

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Transcriptome Profiling of Head and Neck Squamous Cell Carcinoma

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Clinical specimens of a panel of 22 HNSCCs and corresponding macroscopically normal mucosa, adjacent to the tumor, were collected as described [40 (link)]. In short, 13 males and 9 females were included with an average age of 59.6 ±10.2 years (range 33-82). Specimens were obtained from the oral cavity (17 of 22, 77%) and the oropharynx (5 of 22, 23%) and all mucosa samples were judged free from dysplasia. All oropharyngeal tumors were HPV negative. RNA was isolated with TRIzol, analyzed on an Agilent 2100 Bioanalyzer (Agilent) and complementary DNA (cDNA) synthesis was performed (Agilent). Expression profiles were determined by microarray (4×44K Whole Human Genome Arrays G4112F) hybridization and readout was performed on a G2505B microarray scanner system (Agilent). Data analysis was performed as described previously [41 ]. Data have been uploaded to GEO with accession number GSE83519.

de Boer D.V., Martens-de Kemp S.R., Buijze M., Stigter-van Walsum M., Bloemena E., Dietrich R., Leemans C.R., van Beusechem V.W., Braakhuis B.J, & Brakenhoff R.H. (2017). Targeting PLK1 as a novel chemopreventive approach to eradicate preneoplastic mucosal changes in the head and neck. Oncotarget, 8(58), 97928-97940.

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