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Sample GSM398989 Query DataSets for GSM398989
Status Public on May 27, 2010
Title PHAf_R6
Sample type RNA
Source name Primary Human Fetal Astrocytes
Organism Homo sapiens
Characteristics cell type: Primary Human Astrocytes from 20-21-week fetuses
Biomaterial provider ALLCELLS (Emeryville, CA)
Growth protocol Frozen primary human fetal astrocytes (PH-Af) from 20-21 week fetuses were received from ALLCELLS (Emeryville, CA) and cultured in Astrocyte Medium provided by the company. Cells were then harvested for RNA extraction.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated from the cells using TRI-Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. High molecular-weight RNAs were removed by precipitation with 12.5% PEG-8000 and 1.25 M NaCl.
Label Cy3
Label protocol RNA labelling and hybridization to the microarrays was carried out using a method adapted from Thomson et al. 2004 (Thomson JM, Parker J, Perou CM, Hammond SM. 2004. A custom microarray platform for analysis of microRNA gene expression. Nat Methods 1: 47-53). Briefly, 10 or 25 µg total RNA was PEG-precipitated (as above), resuspended in 6 µl water, and mixed into a labelling reaction (final volume 10 µl) consisting of 500 ng 5’-phophate-cytidyl-uridyl-Cy3-3’ (Dharmacon, Chicago, IL), 10% DMSO, 0.1 mM ATP, 50 mM HEPES (pH 7.8), 3.5 mM DTT, 20 mM MgCl2, 0.1 µg BSA, and 20 U T4 RNA ligase (NEB, Ipswitch, MA). Reactions were incubated on ice in the dark for 2 hours before precipitation with 0.27 M sodium acetate, 20 µg glycogen, and 2.7 volumes ethanol in a 200 µl final volume. Following 10 min incubation on ice and 10 min centrifugation, the pellet was washed in 70% ethanol, centrifuged 5 min, briefly dried, and resuspended in 6 µl water.
Hybridization protocol Labelled RNA samples were hybridized to microarrays in a 35 µl final volume composed of 400 mM Na2HPO4 (pH 7.0), 5% SDS, 0.8% BSA, and 12% formamide. Hybridisation mixtures were denatured at 95°C for 4 minutes and immediately pipetted onto glass slides covered with 22×25 mm LifterSlips (Erie Scientific Company, Portsmouth, NH). Hybridizations were performed in custom-made sealed chambers submerged in a 37°C water bath overnight (18 hours). Slide washes were 3 min long: once in 2X SSC/0.025% SDS, three times in 0.8X SSC, and twice in cold 0.4X SSC. Immediately following the last wash, slides were dried by centrifugation.
Scan protocol Slides were scanned with a ScanArray 5000XL (Packard BioScience, Meriden, CT) confocal scanner at 10 µm resolution.
Description no additional information
Data processing Microarray scans were analysed with QuantArray software version 3.0 (Packard BioScience, Meriden, CT) using an adaptive quantitation method. Spots of poor quality on the arrays were flagged for removal from the data processing. Cy3 mean pixel intensities were background subtracted, log transformed, and replicate spots on the arrays were averaged. Microarray data were normalised between arrays using a standard median centering procedure. miRNAs were considered expressed if their normalised log2 hybridisation signal was ≥ 2. Differentially-expressed miRNAs were identified using a SAM (significance analysis of microarrays) threshold of 5% and a fold-change of 2.
Submission date Apr 29, 2009
Last update date May 27, 2010
Contact name Brandon Smith
Organization name National Research Council Canada
Department Institute for Biological Sciences
Lab Neurogenesis and Brain Repair Group
Street address 1200 Montreal Road
City Ottawa
State/province Ontario
ZIP/Postal code K1A 0R6
Country Canada
Platform ID GPL8415
Series (1)
GSE15888 Large-Scale Expression Analysis Reveals Distinct microRNA Profiles at Different Stages of Human Neurodevelopment

Data table header descriptions
VALUE Median-centered, log2 signal intensity

Data table
b_let-7a 1.832118683
b_let-7b 1.498734862
b_let-7c 1.143978466
b_let-7d 2.339838992
b_let-7e 2.863375083
b_let-7f 1.744337717
b_let-7g 1.730598648
b_let-7i 1.367644287
b_miR-1 -0.492871313
b_miR-100 2.521709487
b_miR-103 0.340729664
b_miR-106b 0.13069447
b_miR-107 2.383929907
b_miR-10a 0.806544895
b_miR-122a -0.074849718
b_miR-124a -0.218270274
b_miR-125a 2.511206801
b_miR-125b 0.839963597
b_miR-126 -0.378837466
b_miR-126* 0.12058132

Total number of rows: 422

Table truncated, full table size 10 Kbytes.

Supplementary file Size Download File type/resource
GSM398989.txt.gz 101.0 Kb (ftp)(http) TXT
Processed data included within Sample table

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