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Sample GSM227700 Query DataSets for GSM227700
Status Public on Aug 12, 2008
Title Striatum GABAergic cells replicate 2
Sample type RNA
 
Channel 1
Source name amplified cDNA from murine P1 striatal GABAergic neurons labeled with Cyanine-5 (red).
Organism Mus musculus
Characteristics postnatal day 1 old (C57Bl6, GAD67-eGFP) mice, GABAergic cells from striatal tissue
Treatment protocol GABAergic eGFP+ cells from whole brains were isolated from four GAD67-eGFP mice at P1. For the isolation of cells from different areas, brains of six P1 old mice were dissected into olfactory bulb, cortex, striatum, and cerebellum. Brain tissue was dissociated to single cell suspensions using the Neuronal Tissue Dissociation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Cells were resuspended in PBS (Gibco) and sorted on a FACS Vantage SE cell sorter (Becton Dickinson).
Growth protocol Animals were kept under standard conditions on a C57Bl/6 background.
Extracted molecule polyA RNA
Extraction protocol Flow-sorted cells were immediately subjected to SuperAmpTM lysis buffer. mRNA was isolated via magnetic bead technology, reversely transcribed to cDNA, and amplified in a global PCR using the SuperAmpTM Technology (Miltenyi Biotec). For the common reference, RNA was extracted from whole brain of four age-matched mice using the NucleoSpin® RNA Clean-up Kit (Macherey Nagel) and amplified equally to the flow-sorted cells.
Label Cy-5
Label protocol 250 ng of each of the cDNAs was labeled with Cy5- (sample) or Cy3-dCTP (common reference) in a Klenow Fragment (10 units per sample) reaction for 2 h at 37°C before heat inactivating at 70°C for 5 min. Labeled cDNA was purified using the CyScribe GFX Purification Kit (GE Healthcare) and quantified by OD measurement.
 
Channel 2
Source name amplified cDNA from murine P1 whole brain RNA labeled with Cyanine-3 (red).
Organism Mus musculus
Characteristics postnatal day 1 old (C57Bl6) mice
Treatment protocol GABAergic eGFP+ cells from whole brains were isolated from four GAD67-eGFP mice at P1. For the isolation of cells from different areas, brains of six P1 old mice were dissected into olfactory bulb, cortex, striatum, and cerebellum. Brain tissue was dissociated to single cell suspensions using the Neuronal Tissue Dissociation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Cells were resuspended in PBS (Gibco) and sorted on a FACS Vantage SE cell sorter (Becton Dickinson).
Growth protocol Animals were kept under standard conditions on a C57Bl/6 background.
Extracted molecule polyA RNA
Extraction protocol Flow-sorted cells were immediately subjected to SuperAmpTM lysis buffer. mRNA was isolated via magnetic bead technology, reversely transcribed to cDNA, and amplified in a global PCR using the SuperAmpTM Technology (Miltenyi Biotec). For the common reference, RNA was extracted from whole brain of four age-matched mice using the NucleoSpin® RNA Clean-up Kit (Macherey Nagel) and amplified equally to the flow-sorted cells.
Label Cy-3
Label protocol 250 ng of each of the cDNAs was labeled with Cy5- (sample) or Cy3-dCTP (common reference) in a Klenow Fragment (10 units per sample) reaction for 2 h at 37°C before heat inactivating at 70°C for 5 min. Labeled cDNA was purified using the CyScribe GFX Purification Kit (GE Healthcare) and quantified by OD measurement.
 
 
Hybridization protocol Agilent whole mouse genome 44k microarrays were hybridized according to the manufactures instructions (http://www.chem.agilent.com/scripts). Briefly, 2.5 µg of combined Cy3- and Cy5-labeled and purified cDNAs were adjusted to a volume of 200 µl and denatured 5 min at 95°C. After adding of 50 µl control targets (Agilent) and 250 µl 2x Hybridization Buffer (Agilent), samples were incubated on the microarrays at 65°C for >16 h. Afterwards microarrays were washed with Wash Buffer I (Agilent) for 1 min at 37°C, with Wasch Buffer II (Agilent) for 1 min at 25°C, and dried after 30 s incubation in Acetonitrile.
Scan protocol Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies, Palo Alto, USA).
Description Biological replicate 2 of 3. P1 GABAergic neurons from striatum vs. total brain reference.
Data processing Scanned images were analyzed using the Agilent Feature Extraction software (Version 8.1.1.1) by which the local background was subtracted and a rank consistency based probe selection for Lowess normalization was done. After filtering the data with respect to signal significance a two-tailed t test was used to determine the signal versus background significance.
 
Submission date Sep 07, 2007
Last update date Aug 14, 2011
Contact name Olaf Hardt
E-mail(s) olaf.hardt@miltenyibiotec.de
Phone 0049-221-95048212
Organization name Miltenyi Biotec GmbH
Department MACSmolecular Business Unit, Research and Development
Street address Stöckheimer Weg 1
City Cologne
State/province NRW
ZIP/Postal code 50829
Country Germany
 
Platform ID GPL2872
Series (1)
GSE8984 Gene expression profiling of GABAergic neurons isolated from different brain regions

Data table header descriptions
ID_REF
VALUE processed log10 ratio (Cy5/Cy3); the signal left after all the Feature extraction processing steps have been completed (e.g. background substraction)

Data table
ID_REF VALUE
1 -1.86E+00
2 0.00E+00
3 -2.86E-02
4 3.24E-01
5 -1.68E-01
6 2.95E-01
7 -1.49E+00
8 -8.26E-01
9 9.67E-01
10 -4.90E-02
11 1.63E-01
12 8.56E-02
13 0.00E+00
14 -1.49E+00
15 0.00E+00
16 -3.28E-01
17 1.11E-02
18 8.72E-03
19 -1.42E-01
20 -2.85E-01

Total number of rows: 43790

Table truncated, full table size 650 Kbytes.




Supplementary file Size Download File type/resource
GSM227700.txt.gz 11.6 Mb (ftp)(http) TXT
Processed data included within Sample table

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