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Sample GSM1317382 Query DataSets for GSM1317382
Status Public on May 01, 2014
Title Normal (H080503N)
Sample type RNA
 
Source name Liver
Organism Homo sapiens
Characteristics tissue: liver
Extracted molecule total RNA
Extraction protocol Each DNA (250ng gDNA) aliquot was 96 well plate. Then, 24 of the samples on a plate were subjected to treatment with Nsp I restriction enzyme. After inactivation of the restriction enzyme, the sticky ends of the digested DNA were ligated to Nsp I adaptor/primers using T4 ligase. Aliquots from each ligated product were added to four separate plates for PCR amplification using a universal primer. The products of all four PCR reactions for each subject were combined and then purified with purification magnetic beads in 1.5ml microtube. The yield of each amplified DNA sample was assessed by UV spectrophotometry on a microplate reader. Any DNA showing lower than expected yield (≥ 3mg/ml) or an unusual size distribution was flagged and not processed further. Subsequently, each DNA was fragmented between 25 to 125 bp using partial DNase I digestion and the size distribution of the fragmented amplicons assessed by agarose gel electrophoresis (4% agarose in Tris-Acetate-EDTA buffer).
Label biotin
Label protocol The end-labeled products were added to a hybridization solution, denatured, and added to a CytoScan™ HD Array (one array per sample).
 
Hybridization protocol Samples passing these QC steps were next end-labeled with a biotinylated nucleoitde using Terminal Deoxynucleotidyl Transferase (TdT). The end-labeled products were added to a hybridization solution, denatured, and added to a CytoScan™ HD Array (one array per sample).
Scan protocol After hybridization was complete, the DNA sample was removed from each microarray and replaced with Array Holding Buffer. Arrays were then washed on the Fluidics Station 450 using AGCC software (Affymetrix GeneChip® Command Console, Version 3.2.2) This protocol includes 6 cycles of 5 mixes/cycle with low stringency Wash Buffer A (6X SSPE, 0.01% Tween-20) at 25℃ followed by 24 cycles of 5 mixes/cycle with high stringency Wash Buffer B (0.6X SSPE, 0.01% Tween-20) at 45℃. Binding of the biotinylated targets was detected by incubating the array for 10 min in a Streptavidin–Phycoerythrin (SAPE) solution at 25℃ followed by a post stain wash comprised of 6 cycles of 5 mixes/cycle with Wash Buffer A at 25℃. A second round of staining was then completed using a 10 min incubation in Anti-Streptavidin Antibody Stain Solution at 25℃ followed by a third stain for 10 min in SAPE solution at 25℃ and a final wash consisting of 10 cycles of 6 mixes/cycle with Wash Buffer A at 30℃, filling of the array with Array Holding Buffer, and a hold at 25℃.
Data processing CNV analysis was performed using the Affymetrix Cytoscan HD platform (CEL files). CNV data were analysed with the Nexus Copy Number software (BioDiscovery, CA, ver. 6.1).
 
Submission date Jan 29, 2014
Last update date May 01, 2014
Contact name Farhan Haq
E-mail(s) farhanhaq@gmail.com
Phone +82-010-4963-8477
Organization name Gachon University
Street address 7-45, Songdo-dong, Yeonsu-gu
City Incheon
ZIP/Postal code 406-840
Country South Korea
 
Platform ID GPL16131
Series (1)
GSE54504 A Genomic Portrait of Resectable Hepatocellular Carcinomas: Implications of RB1 and FGF19 Aberrations for Patient Stratification.

Supplementary file Size Download File type/resource
GSM1317382_H080503N-CytoScanHD_633-H080503N.CEL.gz 29.2 Mb (ftp)(http) CEL
GSM1317382_H080503N-CytoScanHD_633-H080503N.cyhd.cychp.gz 80.3 Mb (ftp)(http) CYCHP
Processed data provided as supplementary file

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