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Sample GSM991045 Query DataSets for GSM991045
Status Public on Dec 31, 2012
Title Glioblastoma FFPE-GBM
Sample type genomic
 
Channel 1
Source name FFPE tissue, glioblastoma
Organism Homo sapiens
Characteristics tissue: glioblastoma
sample type: formalin-fixed paraffin-embedded tissue
Treatment protocol Excess paraffin was removed with a scalpel from either side of 40-60um scrolls. Sectioning samples reduces accumulation of debris during the sorting process. Each sectioned piece was collected into individual microcentrifuge tubes, then washed three times with 1 ml Xylene for 5 minutes to remove remaining paraffin. Each sample was rehydrated in sequential ethanol washes (100% 5 minutes x2, then 95%, 70%, 50% and 30% ethanol) and washed 2 times in 1ml 1 mM EDTA pH 8.0. A 1 ml aliquot of 1 mM EDTA pH 8.0 was added to the samples and incubated at 95oC for 80 minutes to facilitate the removal of protein cross-links present in FFPE tissue. Samples were then cooled to room temperature for > 5 minutes, followed by addition of 300 ul PBS pH 7.4 and gentle centrifugation for 2 minutes at 3.6 x g. The supernatant was carefully removed and the pellet washed three times with 1 ml PBS pH 7.4/0.5mM CaCl2 to remove EDTA. Each sample was digested overnight (6-17 hours) in 1ml of a freshly prepared enzymatic cocktail containing 50 units/ml of collagenase type 3, 80 units/ml of purified collagenase, and 100 units/ml of hyaluronidase in PBS pH 7.4/0.5mM CaCl2 buffer. Each enzyme was rehydrated with PBS pH 7.4/0.5mM CaCl2 buffer, then stored at -200C immediately prior to addition to the cocktail mixture. Following overnight digestion, 500 ul NST was added to each sample to facilitate pelleting. Samples were centrifuged for 5 minutes at 3000 x g, after which pellets were resuspended in 750 ul of NST/10% fetal bovine serum and then passed through a 25 G needle 10-20 times. The samples were filtered through a 35 um mesh and collected into a 5 ml Polypropylene round bottom tube. The mesh was rinsed with an additional 750 ul of NST/10% fetal bovine serum and placed on ice while processing remaining samples. The total volume in the tube for each sample was approximately 1.5 ml. An equal volume of 20ug/ml DAPI was added to each tube to achieve a final concentration of 10ug/ml DAPI prior to flow sorting with a BD Influx cytometer with ultraviolet excitation (Becton-Dickinson, San Jose, CA). The optimal settings for sorting FFPE samples with the Influx sorter were as follows: Drop formation was achieved with piezzo amplitude of 6-10 volts and a drop frequency of 30 khertz. The sort mode was set to purity yield with a drop delay of 31.5-32. Sheath fluid pressure was typically 17-18 psi with a 100 um nozzle. For single parameter DNA content assays, DAPI emission was collected at >450nm. DNA content and cell cycle were then analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA).
Growth protocol None.
Extracted molecule genomic DNA
Extraction protocol DNA from sorted nuclei was extracted using an amended protocol from the QIAamp® DNA Micro Kit from Qiagen (Valencia, CA). Briefly, each sorted sample was resuspended in 180ul buffer ATL and 20ul proteinase K, then incubated for 3 hours at 56°C for complete lysis. Samples were bound and washed according to QIAamp® DNA Micro Kit instructions, eluted into 50ul of H20, then precipitated overnight with 5ul 3 M sodium acetate and 180 ul 100% EtOH. Each sample was then centrifuged for 30 minutes at 20,000 x g, and washed in 1 ml of 70% EtOH for 30 minutes at 20,000 x g. The samples were carefully decanted and the DNA pellet was dried by speed vacuum then resuspended in a small volume (e.g. 10-50ul) of H20 for final concentrations suitable for accurate quantification.
Label Cy5
Label protocol Genomic DNAs from sorted FFPE samples were amplified using the Ovation® WGA FFPE System from NuGEN® Technologies (San Carlos, CA). DNA was processed in accordance with the Ovation® WGA FFPE standard protocol with an alternate T7 endonuclease fragmentation step. Resulting amplified product was either used as template for aCGH analysis or processed with the Nugen Encore ds-DNA module according to the supplier's instructions in order to generate double-stranded end repaired DNA as input for library suitable for next-generation sequencing. Extracted fresh frozen sourced genomic DNA was amplified using the phi29 based Illustra GenomiPhi V2 Amplification kit from GE Healthcare Bio-sciences Corp (Piscataway, NJ) according to our published protocols. A 100 ng aliquot of pooled 46,XX DNA (Promega, Madison, WI) was amplified with the matching amplification protocol to generate a suitable reference for each aCGH experiment using amplified DNA template. In all cases, the quality of the amplification product was assessed by gel electrophoresis. Fresh frozen phi29 amplified and FFPE non-amplified DNAs were treated with DNAse 1 prior to Klenow-based labeling. High molecular weight phi29 templates were digested for 30 minutes while the smaller fragmented FFPE samples were digested for only 1 minute. In each case, 1ul of 10x DNase 1 reaction buffer and 2ul of DNase 1 dilution buffer were added to 7ul of DNA sample and incubated at room temperature, then transferred to 70°C for 30 minutes to deactivate DNase 1. In contrast, the amplified FFPE sourced DNAs do not require DNase 1 treatment prior to Klenow-based labeling. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP, respectively, using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols.
 
Channel 2
Source name Normal 46,XX reference
Organism Homo sapiens
Characteristics sample type: commercial pooled reference
Treatment protocol Excess paraffin was removed with a scalpel from either side of 40-60um scrolls. Sectioning samples reduces accumulation of debris during the sorting process. Each sectioned piece was collected into individual microcentrifuge tubes, then washed three times with 1 ml Xylene for 5 minutes to remove remaining paraffin. Each sample was rehydrated in sequential ethanol washes (100% 5 minutes x2, then 95%, 70%, 50% and 30% ethanol) and washed 2 times in 1ml 1 mM EDTA pH 8.0. A 1 ml aliquot of 1 mM EDTA pH 8.0 was added to the samples and incubated at 95oC for 80 minutes to facilitate the removal of protein cross-links present in FFPE tissue. Samples were then cooled to room temperature for > 5 minutes, followed by addition of 300 ul PBS pH 7.4 and gentle centrifugation for 2 minutes at 3.6 x g. The supernatant was carefully removed and the pellet washed three times with 1 ml PBS pH 7.4/0.5mM CaCl2 to remove EDTA. Each sample was digested overnight (6-17 hours) in 1ml of a freshly prepared enzymatic cocktail containing 50 units/ml of collagenase type 3, 80 units/ml of purified collagenase, and 100 units/ml of hyaluronidase in PBS pH 7.4/0.5mM CaCl2 buffer. Each enzyme was rehydrated with PBS pH 7.4/0.5mM CaCl2 buffer, then stored at -200C immediately prior to addition to the cocktail mixture. Following overnight digestion, 500 ul NST was added to each sample to facilitate pelleting. Samples were centrifuged for 5 minutes at 3000 x g, after which pellets were resuspended in 750 ul of NST/10% fetal bovine serum and then passed through a 25 G needle 10-20 times. The samples were filtered through a 35 um mesh and collected into a 5 ml Polypropylene round bottom tube. The mesh was rinsed with an additional 750 ul of NST/10% fetal bovine serum and placed on ice while processing remaining samples. The total volume in the tube for each sample was approximately 1.5 ml. An equal volume of 20ug/ml DAPI was added to each tube to achieve a final concentration of 10ug/ml DAPI prior to flow sorting with a BD Influx cytometer with ultraviolet excitation (Becton-Dickinson, San Jose, CA). The optimal settings for sorting FFPE samples with the Influx sorter were as follows: Drop formation was achieved with piezzo amplitude of 6-10 volts and a drop frequency of 30 khertz. The sort mode was set to purity yield with a drop delay of 31.5-32. Sheath fluid pressure was typically 17-18 psi with a 100 um nozzle. For single parameter DNA content assays, DAPI emission was collected at >450nm. DNA content and cell cycle were then analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA).
Growth protocol None.
Extracted molecule genomic DNA
Extraction protocol DNA from sorted nuclei was extracted using an amended protocol from the QIAamp® DNA Micro Kit from Qiagen (Valencia, CA). Briefly, each sorted sample was resuspended in 180ul buffer ATL and 20ul proteinase K, then incubated for 3 hours at 56°C for complete lysis. Samples were bound and washed according to QIAamp® DNA Micro Kit instructions, eluted into 50ul of H20, then precipitated overnight with 5ul 3 M sodium acetate and 180 ul 100% EtOH. Each sample was then centrifuged for 30 minutes at 20,000 x g, and washed in 1 ml of 70% EtOH for 30 minutes at 20,000 x g. The samples were carefully decanted and the DNA pellet was dried by speed vacuum then resuspended in a small volume (e.g. 10-50ul) of H20 for final concentrations suitable for accurate quantification.
Label Cy3
Label protocol Genomic DNAs from sorted FFPE samples were amplified using the Ovation® WGA FFPE System from NuGEN® Technologies (San Carlos, CA). DNA was processed in accordance with the Ovation® WGA FFPE standard protocol with an alternate T7 endonuclease fragmentation step. Resulting amplified product was either used as template for aCGH analysis or processed with the Nugen Encore ds-DNA module according to the supplier's instructions in order to generate double-stranded end repaired DNA as input for library suitable for next-generation sequencing. Extracted fresh frozen sourced genomic DNA was amplified using the phi29 based Illustra GenomiPhi V2 Amplification kit from GE Healthcare Bio-sciences Corp (Piscataway, NJ) according to our published protocols. A 100 ng aliquot of pooled 46,XX DNA (Promega, Madison, WI) was amplified with the matching amplification protocol to generate a suitable reference for each aCGH experiment using amplified DNA template. In all cases, the quality of the amplification product was assessed by gel electrophoresis. Fresh frozen phi29 amplified and FFPE non-amplified DNAs were treated with DNAse 1 prior to Klenow-based labeling. High molecular weight phi29 templates were digested for 30 minutes while the smaller fragmented FFPE samples were digested for only 1 minute. In each case, 1ul of 10x DNase 1 reaction buffer and 2ul of DNase 1 dilution buffer were added to 7ul of DNA sample and incubated at room temperature, then transferred to 70°C for 30 minutes to deactivate DNase 1. In contrast, the amplified FFPE sourced DNAs do not require DNase 1 treatment prior to Klenow-based labeling. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP, respectively, using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols.
 
 
Hybridization protocol All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to 400k CGH arrays (Agilent Technologies, Santa Clara, CA) for 40 hours in a rotating 65°C oven.
Scan protocol All microarray slides were scanned using an Agilent 2565C DNA scanner, and the images were analyzed with Agilent Feature Extraction version 10.7 using default settings.
Data processing The aCGH data was assessed with a series of QC metrics, dye normalized and centralized, and then analyzed using an aberration detection algorithm (ADM2) which is included in Agilent Genomic Workbench 6.5 software. The latter identifies all aberrant intervals in a given sample with consistently high or low log ratios based on the statistical score derived from the average normalized log ratios of all probes in the genomic interval multiplied by the square root of the number of these probes. This score represents the deviation of the average of the normalized log ratios from its expected value of zero and is proportional to the height h (absolute average log ratio) of the genomic interval and to the square root of the number of probes in the interval.
 
Submission date Aug 22, 2012
Last update date Dec 31, 2012
Contact name Michael Thomas Barrett
E-mail(s) barrett.michael@mayo.edu
Phone 480-301-6736
Organization name Mayo Clinic Arizona
Department Molecular Pharmacology and Experimental Therapeutics
Street address 13400 East Shea Boulevard
City Scottsdale
State/province AZ
ZIP/Postal code 85259
Country USA
 
Platform ID GPL10123
Series (1)
GSE40299 Deep Clonal Profiling of Formalin-Fixed Paraffin-Embedded Clinical Samples

Data table header descriptions
ID_REF
VALUE Log2 ratio (test/reference)

Data table
ID_REF VALUE
136895 0.34448263
107619 0.15885295
68139 -1.1916621
35971 0.074856795
149692 0.207159
7554 0.39152557
38473 0.05946853
63780 0.28460637
44139 0.031863965
114499 0.28953493
167637 -0.116489075
169924 0.11922498
9391 0.3529443
178281 0.22005937
156350 1.3985265
9932 1.1326234
113431 0.28694618
51523 0.65232766
170595 0.21461573
41164 0.030408718

Total number of rows: 174207

Table truncated, full table size 3003 Kbytes.




Supplementary file Size Download File type/resource
GSM991045_US12302336_252206015710_S01_CGH_105_Dec08_1_1.txt.gz 18.6 Mb (ftp)(http) TXT
Processed data included within Sample table

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