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Sample GSM399000 Query DataSets for GSM399000
Status Public on May 27, 2010
Title PHN_R2
Sample type RNA
 
Source name Primary Human Neurons
Organism Homo sapiens
Characteristics cell type: Primary Human Neurons from 21-week fetuses
Biomaterial provider ScienCell Research Laboratories, Carlsbad, CA
Growth protocol Fresh primary human neurons (PH-N) from 21-week fetuses were obtained from ScienCell Research Laboratories (Carlsbad, CA) and were directly pelleted 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
E-mail(s) brandon.smith@nrc-cnrc.gc.ca
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
ID_REF
VALUE Median-centered, log2 signal intensity

Data table
ID_REF VALUE
b_let-7a 4.349456731
b_let-7b 5.089454293
b_let-7c 4.074968859
b_let-7d 5.022916437
b_let-7e 4.582765954
b_let-7f 5.029120096
b_let-7g 3.188625993
b_let-7i 2.745847731
b_miR-1 -0.423896177
b_miR-100 1.883555161
b_miR-103 1.536106896
b_miR-106b 2.094843029
b_miR-107 1.936311179
b_miR-10a 1.993751155
b_miR-122a 0.957426266
b_miR-124a 6.056820762
b_miR-125a 5.195944103
b_miR-125b 5.8272934
b_miR-126 1.048784078
b_miR-126* 0.834554354

Total number of rows: 422

Table truncated, full table size 10 Kbytes.




Supplementary file Size Download File type/resource
GSM399000.txt.gz 178.2 Kb (ftp)(http) TXT
Processed data included within Sample table

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