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Sample GSM492930 Query DataSets for GSM492930
Status Public on Oct 05, 2011
Title whole blood-control-LPS-± 6 h-CON14
Sample type RNA
 
Source name peripheral blood, LPS, ±6 h
Organism Homo sapiens
Characteristics tissue: whole blood
gender: male
age: 57
smoking status: non-smoking
disease: healthy
sample type: control
treatment: lipopolysaccharide (LPS)
Treatment protocol Serial venous whole blood samples were obtained between 8:00 a.m. and 10:00 a.m., after overnight fasting, in one 7 ml heparin-coated tube (Greiner). Between 10-60 minutes after blood draw, 2.5 ml of blood was transferred into a PAXgene tube (Qiagen) and used as the basal (non-LPS stimulated) sample. This tube was kept at room temperature for a minimum of 2 h, and subsequently stored at –20 °C. The remaining blood (4.5 ml) was stimulated by addition of LPS (10 ng/ml blood; E. coli, Sigma). LPS-stimulated samples were laid flat and incubated at a slow rotation for 5-6 h at 37° C before a 2.5 ml sample of this LPS-stimulated blood was transferred into a PAXgene tube (Qiagen), and treated as described for the basal sample.

Patients were arbitrarily selected from all patients to serve as primary cohort (nMDD = 21 (#MDD01-MDD21); nControls = 21 (#Con01-Con21)), or replication cohort (nMDD = 12 (#MDD22-35); nControls = 13 (#Con22-37)) using microarrays.
Growth protocol NA.
Extracted molecule total RNA
Extraction protocol RNA was prepared using PAX (PreAnalytiX; Qiagen) gene blood collection tubes following the manufacturer's recommendations (after thawing of the PAX tubes for 2 h at room temperature). The protocol includes lysis of blood cells, digestion of proteins (prot K treatment), and an on-column DNaseI digestion. The purity of RNA was first determined by spectrophotometry (NanoDrop ND–1000 UV-Vis Spectrophotometer, Nanodrop Technologies). Because of contaminants due to the isolation procedure, visible in the spectrophotogram at 200-230 nm, RNA samples were precipitated using ethanol, and NaAc with addition of 0.1 µg linear acryl-amide. Subsequently, RNA quality was determined by spectrophotometry and by using the RNA 6000 NanoChip kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Only RNA passing quality control criteria (RIN values > 8) was used for further analysis.
Label Cy5
Label protocol Labeling of RNA (1 µg) was according to the Agilent protocol (Agilent technologies, Palo Alto, USA). Using spectrophotometry, cRNA concentration and labeling efficiency were determined (Nanodrop ND-1000 UV-Vis Spectrophotometer, Nanodrop Technologies). For all subjects, the same set-up has been used with the basal sample labeled with Cy3 (green) and the LPS-stimulated sample labeled with Cy5 (red), except for one subject (MDD31). This excludes the possibility that differences between subjects are due to a color bias for which two-color arrays are known.
 
Hybridization protocol From each subject, labeled cRNA (1.5 µg Cy3-labeled basal blood sample; 1.25 µg Cy-5 labeled LPS-stimulated blood sample) was hybridized for 15-17 h at 65 °C onto the 44K Human Agilent whole genome arrays according to the manufacturer’s protocol. After washing, the arrays were quick-dried using acetonitrile.
Scan protocol Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using two color scan setting for 1x44k array slides. Scan resolution 10 µm. PMT set to 100%.
Description LPS-stimulated gene expression in healthy control.
CON14_LPS
Data processing Signal intensities were extracted using Agilent Feature Extraction (v8.0). Data were analyzed with Limma (Bioconductor) using median signals and median background. Control spots were normalized, but were set not to influence normalization. Non-uniform signals were removed prior to further analysis. After background subtraction (Edwards, offset 30), within normalization (Loess) and between array normalization (Aquantile) data were exported to SPSS. Further data selection for inclusion consisted of the following criteria (in this order): 1) Signal intensities for any gene should be 1.3x background (> 6.8 (log2)), and have non-saturated levels (<15.7 (log2)); 2) In addition, for any gene the signal should be present in >80% of the control as well as in >80% of the MDD samples to avoid type-1 errors due to a limited number of participants in each group. Genes present in <80 % of MDD or control samples were given an absent-call. From the total of 41,675 spots, filtering resulted in 22,789 spots for the basal sample and 22,935 spots in the LPS-stimulated sample, with an overlap of 21,107 spots (>92%).
 
Submission date Jan 04, 2010
Last update date Oct 05, 2011
Contact name sabine Spijker
Organization name Neuroscience Campus Amsterdam
Department Center for Neurogenomics and Cognitive Research
Street address De Boelelaan 1085
City Amsterdam
ZIP/Postal code 1081HV
Country Netherlands
 
Platform ID GPL6848
Series (1)
GSE19738 Major Depressive Disorder blood gene expression

Data table header descriptions
ID_REF
VALUE Loess and Aquantile normalized log2 signal intensity (Limma)

Data table
ID_REF VALUE
A_24_P475014 null
A_24_P456043 null
A_32_P148948 null
A_24_P772004 null
A_32_P234391 null
A_32_P98162 null
A_24_P407439 null
A_32_P37704 null
A_32_P115162 null
A_32_P53212 null
A_32_P7521 null
A_24_P889962 null
A_32_P85684 null
A_32_P198868 null
A_32_P107441 7.836
A_32_P68736 null
A_32_P77178 6.856
A_32_P76441 8.231
A_32_P42426 7.060
A_32_P35106 null

Total number of rows: 41000

Table truncated, full table size 732 Kbytes.




Supplementary file Size Download File type/resource
GSM492930_Con14_251239149501.txt.gz 11.8 Mb (ftp)(http) TXT
Processed data included within Sample table

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