Comment[ArrayExpressAccession]	E-GEOD-51957
MAGE-TAB Version	1.1											
Public Release Date	2014-06-01
Investigation Title	Array comparative hybridization (array-CGH) for detection of gene dose alterations in VACTERL association [4x180k Array v.025990]											
Comment[Submitted Name]	Array comparative hybridization (array-CGH) for detection of gene dose alterations in VACTERL association [4x180k Array v.025990]
Experiment Description	The aim of the study was to screen for pathogenic gene dose alterations in patients and fetal cases with VACTERL association using a 180K oligonucleotide microarray. The experiment was performed by hybridization of genomic DNA from patients, labelled with Cy3 fluorophore, with a mix of DNA from healthy sex-matched controls labelled with Cy5 fluorophore (fetal case FC17 was analysed with sex-mismatch for the control DNA). 36 patients/fetal cases were analyzed using a Oxford Gene Technology (OGT) 180K oligonucleotide platform with whole genome coverage (27 patients were analyzed on version 025990 and 9 patients on version 031035). The raw microarray image files were initially analyzed using the Feature Extraction software from Agilent Technologies. Further data analysis was performed using the CytoSure Interpret software from OGT.											
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl										
Person Last Name	Winberg	Winberg	Gustavsson	Papadogiannakis	Sahlin	Bradley	NordenskjM-CM-6ld	Svensson	AnnerM-CM-)n	Iwarsson	Nordgren	NordenskjM-CM-6ld
Person First Name	Johanna	Johanna	Peter	Nikos	Ellika	Frideborg	Edvard	PM-CM-$r-Johan	GM-CM-6ran	Erik	Ann	Agneta
Person Email	johanna.winberg@ki.se											
Person Affiliation	Karolinska Institutet											
Person Address	Molecular Medicine and Surgery, Karolinska Institutet, CMM L8:02, Karolinska University Hospital, Stockholm, Sweden											
Person Roles	submitter											
Protocol Name	P-GSE51957-1	P-GSE51957-4	P-GSE51957-5	P-GSE51957-2	P-GSE51957-3	P-GSE51957-6						
Protocol Description	Agilent Feature Extraction Software was used for background subtraction and LOWESS normalization. ID_REF =  VALUE = Normalized log 2 ratio (Cy3/Cy5) representing test/reference.	1.1 M-5g of genomic patient and control DNA (Promega) was diluted in water to a total volume of 19 M-5l. 20 M-5l of primer buffer (CGH labelling kit for oligo arrays, Enzo Life Sciences) was added on ice. The samples were incubated at 99M-bM-^AM-0 for 10 min and then placed on ice for 5 mins. 10 M-5l of Cy3 or Cy5 were added to each sample and subsequently 1 M-5l of Klenow polymerase was added. The samples were incubated at 37M-bM-^AM-0 for 13.5 hours and then heated to 65M-bM-^AM-0 for 10 mins before cooling to 4M-bM-^AM-0. Purification of labelled samples was performed by adding 250 M-5l of PB buffer to a spin column and adding 50 M-5l of labelled sample (the total volume). The samples were centrifuged at 14000 rpm for 1 min, and the flow-through discarded. 750 M-5l of PE wash buffer was added to each column, and the samples centrifuged at 14000 rpm for 1 min. The flow-through was discarded and the samples centrifuged once more at 14000 rpm for 1 min. DNA was eluated with 22 M-5l TE buffer and incubated at 2 min in room temperature before centrifugation at 14000 rpm for 1 min. Concentration of labelled DNA was measured using NanoDrop.	Corresponding labelled patient and reference samples were pooled and 5 M-5l CotDNA, 11 M-5l blocking buffer and 55 M-5l hybridization buffer from the Oligo aCGH/CHp-on chip hybridization kit (Agilent Technologies).Denaturation was performed at 95M-bM-^AM-0 for 5 mins followed by pre-hybridization at 37M-bM-^AM-0 for 45 min. 100 M-5l per sample were dispensed in each well on a 4x180K oligonucleotide array glass slide and a gasket slide added to close. The glass was hybridized for 24 hours in 65M-bM-^AM-0 in a rotation oven. After 24 hours, each glass was put in wash solution B1 (Agilent Technologies) for 5 min followed by 1 min in 37M-bM-^AM-0 wash solution B2 (Agilent Technologies) and subsequently air-dried and stored in a light-proof box.	No cell culture was performed.	Genomic DNA was isolated from either peripheral blood or tissue samples according to standard procedures. For fetal cases, DNA was isolated from routinely preserved fresh frozen heart, liver, lung, or spleen tissue using the Gentra Puregene Blood Kit (QIAGEN Sciences, Maryland, USA) in combination with Proteinase K (Finnzymes, Espoo, Finland) with slight modifications to the manufacturerM-bM-^@M-^Ys protocol.	The glasses were scanned on a DNA Microarray Scanner from Agilent Technologies with 3 M-5m resolution.						
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	nucleic acid extraction protocol	array scanning protocol						
Experimental Factor Name	ORGANISM PART											
Experimental Factor Type	organism part											
Comment[SecondaryAccession]	GSE51957											
Comment[GEOReleaseDate]	2014-06-01											
Comment[ArrayExpressSubmissionDate]	2013-10-31											
Comment[GEOLastUpdateDate]	2014-06-03											
Comment[AEExperimentType]	comparative genomic hybridization by array											
SDRF File	E-GEOD-51957.sdrf.txt