Cytogenomics software v 2

Manufactured by Agilent Technologies

Sourced in United States

CytoGenomics software v 2.0 is a bioinformatics software tool designed for the analysis and interpretation of cytogenomic data. The software provides a comprehensive suite of tools for the visualization, analysis, and reporting of chromosomal aberrations, copy number variations, and other genomic alterations.

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6 protocols using cytogenomics software v 2

Genome-wide Copy Number Profiling

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DNA for the array CGH studies was extracted from formalin-fixed, paraffin-embedded tissues with the FormaPure kit from Agencourt. Agilent Sureprint G3 Cancer CGH + SNP 4 × 180k Microarrays were used to identify genome-wide copy number alterations. Briefly, 1µg of tumor and control DNA (normal female gDNA, Corriell Institute) were heated to 95°C for 5 minutes. Random priming was used to label DNA with CY3-dUTP (control) and CY5-dUTP (tumor) dyes from the Agilent SureTag DNA Labeling kit. The labeled DNA was then purified over columns (Agilent) and mixed in equal proportion along with Cot-1 Human DNA (Agilent) for the hybridization steps. To hybridize the DNA to the array, incubation occurred first at 95°C for 3 minutes for denaturation, followed by a 30 minute pre-hybridization step at 37°C and then a hybridization step for 35–40 hours at 65°C. Slides were then washed with Agilent Oligo ArrayCGH wash buffer 1 for 5 minutes at room temperature and wash buffer 2 for 1 minute at 37°C. Upon completion of the washes, slides were scanned using the G2505C Microarray Scanner (Agilent). The data were analyzed using the Agilent CytoGenomics software v 2.0. CGH array data are available at GEO under accession number GSE64765 (super-series GSE64766)..

Niederst M.J., Sequist L.V., Poirier J.T., Mermel C.H., Lockerman E.L., Garcia A.R., Katayama R., Costa C., Ross K.N., Moran T., Howe E., Fulton L.E., Mulvey H.E., Bernardo L.A., Mohamoud F., Miyoshi N., VanderLaan P.A., Costa D.B., Jänne P.A., Borger D.R., Ramaswamy S., Shioda T., Iafrate A.J., Getz G., Rudin C.M., Mino-Kenudson M, & Engelman J.A. (2015). RB loss in resistant EGFR mutant lung adenocarcinomas that transform to small-cell lung cancer. Nature Communications, 6, 6377.

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Array CGH Analysis of Formalin-Fixed Paraffin-Embedded Tumors

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DNA for the array CGH studies was extracted from formalin-fixed, paraffin-embedded tissues with the FormaPure kit from Agencourt. Agilent Sureprint G3 Cancer CGH+SNP 4 × 180 k Microarrays were used to identify genome-wide copy number alterations. Briefly, 1 μg of tumour and control DNA (normal female gDNA, Corriell Institute) were heated to 95 °C for 5 min. Random priming was used to label DNA with CY3-dUTP (control) and CY5-dUTP (tumour) dyes from the Agilent SureTag DNA Labeling kit. The labelled DNA was then purified over columns (Agilent) and mixed in equal proportion along with Cot-1 Human DNA (Agilent) for the hybridization steps. To hybridize the DNA to the array, incubation occurred first at 95 °C for 3 min for denaturation, followed by a 30-min pre-hybridization step at 37 °C and then a hybridization step for 35–40 h at 65 °C. Slides were then washed with Agilent Oligo ArrayCGH wash buffer 1 for 5 min at room temperature and wash buffer 2 for 1 min at 37 °C. Upon completion of the washes, slides were scanned using the G2505C Microarray Scanner (Agilent). The data were analysed using the Agilent CytoGenomics software v 2.0. CGH array data are available at GEO under accession number GSE64765 (super-series GSE64766).

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Genome-Wide Copy Number Profiling of Leukocytes

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Patients’ peripheral blood leukocytes DNA underwent aCGH as previously described [6 (link)]. Briefly, aCGH analysis used an 8×60K (G4827A-031746; Agilent Technologies, Santa Clara, CA, USA). The arrays were scanned with a G2565CA microarray scanner (Agilent Technologies, Santa Clara, CA, USA) and the images were extracted and analyzed with CytoGenomics software v2.0 (Agilent Technologies, Santa Clara, CA, USA). An ADM-2 algorithm (cut-off 6.0), followed by a filter to select regions with three or more adjacent probes and a minimum average log₂ ratio ±0.25, was used to detect copy number changes. The quality of each experiment (log ratio spread) was assessed with CytoGenomics software v2.0. Genomic positions were based on the UCSC February 2009 human reference sequence (hg19 NCBI build 37 reference sequence assembly). Filtering of copy number changes was carried out using the BENCHlab CNV software (Cartagenia, Leuven, Belgium).

Castinetti F., Daly A.F., Stratakis C.A., Caberg J.H., Castermans E., Trivellin G., Rostomyan L., Saveanu A., Jullien N., Reynaud R., Barlier A., Bours V., Brue T, & Beckers A. (2016). GPR101 Mutations are not a frequent cause of Congenital Isolated Growth Hormone Deficiency. Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme, 48(6), 389-393.

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Customized aCGH Microarray Design and Analysis

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The customized aCGH 8 × 60 k Agilent SurePrint G3 CGH was designed using the Agilent eArray website (https://earray.chem.agilent.com/earray/) with an average distribution of 1 probe per 150 pb.
The processing was performed according to manufacturer’s recommendations as previously described15 (link).
Results were analyzed by Agilent CytoGenomics software v.2.7 using default analysis method – CGH v2 with the ADM-2 aberration algorithm.

Perez-Carro R., Corton M., Sánchez-Navarro I., Zurita O., Sanchez-Bolivar N., Sánchez-Alcudia R., Lelieveld S.H., Aller E., Lopez-Martinez M.A., López-Molina M.I., Fernandez-San Jose P., Blanco-Kelly F., Riveiro-Alvarez R., Gilissen C., Millan J.M., Avila-Fernandez A, & Ayuso C. (2016). Panel-based NGS Reveals Novel Pathogenic Mutations in Autosomal Recessive Retinitis Pigmentosa. Scientific Reports, 6, 19531.

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Custom aCGH for SAMD11 Gene Analysis

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A custom aCGH 8×60k using the Agilent SurePrint G3 CGH technology was designed using the Agilent eArray website (https://earray.chem.agilent.com/earray/) with an average distribution of 1 probe per 150 bp in the SAMD11 gene with a total of 95 probes. Briefly, genomic DNA (200 ng) from the patient and from a sex-matched control were digested by AluI and RsaI restriction enzymes for 2 h at 37 °C and the digested products were labelled with Cy3-dUTP and Cy5-dUTP fluorochromes using the Sure Tag DNA Labeling Kit (Agilent Technologies). The labelled products were purified, hybridized and washed according to Agilent protocols. The slide was scanned on a SureScan G4900DA scanner (Agilent Technologies), and the resulting TIFF images were converted by the image conversion Feature Extraction software (Agilent Technologies). Results were analyzed by Agilent CytoGenomics software v.2.7 using default analysis method – CGH v2 with the ADM-2 aberration algorithm.

Corton M., Avila-Fernández A., Campello L., Sánchez M., Benavides B., López-Molina M.I., Fernández-Sánchez L., Sánchez-Alcudia R., da Silva L.R., Reyes N., Martín-Garrido E., Zurita O., Fernández-San José P., Pérez-Carro R., García-García F., Dopazo J., García-Sandoval B., Cuenca N, & Ayuso C. (2016). Identification of the Photoreceptor Transcriptional Co-Repressor SAMD11 as Novel Cause of Autosomal Recessive Retinitis Pigmentosa. Scientific Reports, 6, 35370.

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VOPP1 Expression and Tumor DNA Analysis

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Tumors with high levels of VOPP1 transcripts were subjected to DNA extraction. The quality of tumor DNAs was verified using a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific). Tumor DNAs and reference DNA provided in the Agilent Kit were labeled, purified, and co-hybridized in equal quantity to the Agilent Microarrays, for 21–24 h. Arrays were washed and scanned on a SureScanMicroarray Scanner according to the manufacturer’s protocols (Agilent). Images were acquired using the CytoScan Software V.2.7 (Thermo Fisher Scientific) and analyzed on CytoGenomics Software V.2.7 (Agilent).

Bonin F., Taouis K., Azorin P., Petitalot A., Tariq Z., Nola S., Bouteille N., Tury S., Vacher S., Bièche I., Rais K.A., Pierron G., Fuhrmann L., Vincent-Salomon A., Formstecher E., Camonis J., Lidereau R., Lallemand F, & Driouch K. (2018). VOPP1 promotes breast tumorigenesis by interacting with the tumor suppressor WWOX. BMC Biology, 16, 109.

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