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Sample GSM503626 Query DataSets for GSM503626
Status Public on Jul 19, 2010
Title WJ1_passage20_replicate1
Sample type RNA
 
Source name WJ1, passage 20
Organism Homo sapiens
Characteristics cell type: Wharton's jelly stem cell line
Growth protocol Institutional Review Board (DSRB, National Healthcare Group) approval was given for collection of umbilical cords from human subjects by informed patient consent. hWJSCs were derived from 6 different umbilical cords using a method previously described by our group (Fong et al. 2007. Reprod Biomed Online 15:708-718). Primary cultures of hWJSCs were established in sterile plastic tissue culture flasks (BD, USA) in the presence of a culture medium comprised of DMEM (high glucose), 20% fetal bovine serum (FBS), glutamine, insulin-transferrin-selenium (ITS), bovine fibroblast growth factor (bFGF) and antimycotic-antibiotic solution (Invitrogen, USA). Once confluent, the hWJSCs were passaged by detachment and disassociation with trypsin, washed and re-seeded into new flasks in the presence of the same medium above. A karyotypically normal human embryonic stem cell line (hESC) (HES3, ESI Singapore Ltd.) and a variant hESC line (BG01V, SCRC-2002, ATCC) were cultured on mitomycin-C treated mouse embryonic fibroblasts (MEFs) in bulk culture medium (BCM) composed of KnockOut DMEM medium (Invitrogen) supplemented with 20% KO serum replacement, 0.1 mM ß mercaptoethanol, 1 mM glutamine, 0.1 nM nonessential amino acids, 50 units /ml penicillin, 50 µg/ml streptomycin, 1:200 dilution of ITS (insulin-transferrin-selenium) (Invitrogen) and 16 ng/ml bFGF (Chemicon). Three human embryonic carcinoma cell lines (hECC) (NCCIT, CRL-2073, ATCC), (NTERA-2 cl.D1, NT2D1; CRL-1973, ATCC), and GCT27C4 (kind gift from Dr Martin Pera) were cultured respectively in the following basal media RPMI 1640, High Glucose DMEM, and DMEM/F12 (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone) and L-glutamine (Invitrogen). A human fetal fibroblast cell line (hFC) (Detroit 551, D551; CCL-110, ATCC) was grown in DMEM supplemented with 10% fetal bovine serum (Hyclone) and L-glutamine (Invitrogen).
Extracted molecule total RNA
Extraction protocol Total RNA from the hESC and hWJSC cell lines were extracted with RNeasy Mini Kit (Qiagen). Total RNA from the hECC and human fibroblast cell lines was prepared according to the TRIzol reagent protocol (Invitrogen) and purified with the RNeasy MinElute Cleanup Kit (Qiagen). Total RNA yield was determined using Nanodrop™ 1000 (Thermo Fisher Scientific) while RNA integrity was evaluated with Bioanalyzer 2100 (Agilent Technologies).
Label biotin
Label protocol The microarray experiment was carried out according to the manufacturer’s recommendations for the Affymetrix GeneChip Human Genome U133 plus 2.0 Array. The GeneChip One-Cycle cDNA Synthesis Kit (Affymetrix) was used to perform the first-strand and second-strand synthesis reactions. First-strand synthesis was carried out with 3.5 µg sample RNA, poly-A RNA controls (Eukaryotic Poly-A RNA Control Kit, Affymetrix), and T7-Oligo(dT) Primer at 42 oC for 1 hr. The second-strand reaction was performed at 16 oC for 2 hrs. The double-stranded cDNA was purified with the GeneChip Sample Cleanup Module (Affymetrix). In vitro transcription to produce biotin-labeled cRNA was carried out with the GeneChip IVT Labeling Kit (Affymetrix) at 37 oC for 16 hrs. The adjusted cRNA yield was determined by the difference between the amount of cRNA measured after in vitro transcription and the starting amount of total RNA. 20 µg of purified biotin-labeled cRNA was then fragmented at 94oC for 35 min.
 
Hybridization protocol The GeneChip Hybridization, Wash and Stain Kit (Affymetrix) was used for the hybridization, wash and scan steps. 15 µg of fragmented cRNA probes were mixed with the Control Oligonucleotide B2 and Eukaryotic Hybridization Controls (bioB, bioC, bioD, cre) before application to the GeneChip Array. Hybridization was carried at 45 oC, 60 rpm for 16 hrs in the Hybridization Oven 640 (Affymetrix). Washing of the GeneChip Arrays was done on the Fluidics Station 450 (Affymetrix) with the Fluidics Script FS450_0001.
Scan protocol Scanning was carried out with the GeneChip Scanner 3000. The scanned data was retrieved using the Affymetrix GeneChip Operating Software (GCOS) and inspected with the quality control parameters for image quality, presence of artifacts, grid alignment, border staining and hybridization controls.
Description WJ1_P20_R1
Data processing The Affymetrix GeneChip Human Genome U133 plus 2.0 Array CEL files were then imported into Expression Console (Affymetrix) for data processing. Quality control parameters were checked for the tabular report, signal distribution, and array comparisons. The performance report and signal detection calls for each GeneChip Array were analysed with the MAS 5.0 analysis. The expression summary file was generated with the RMA algorithm (parameters were RMA Background Correction to PM-only probes and quantile normalization). Two datasets analyses were performed. The first dataset contained the hESCs, hECCs, hWJSCs and hFC samples that were generated by our group. The second dataset contained a meta-analysis of the hWJSCs and hFC that were generated by our group and selected samples available from the public GEO database (human mesenchymal stem cells, umbilical vein endothelial cells, normal stroma, tumour stroma, fibroblast ).
 
Submission date Feb 01, 2010
Last update date Jul 19, 2010
Contact name Woon-Khiong Chan
E-mail(s) dbscwk@nus.edu.sg, g0500139@nus.edu.sg
Organization name National University of Singapore
Department Biological Sciences
Lab Molecular Genetics Laboratory
Street address S2-05-19, 14 Science Drive 4
City Singapore
ZIP/Postal code 117543
Country Singapore
 
Platform ID GPL570
Series (2)
GSE20125 Transcriptome analysis of human Wharton’s jelly stem cells: meta-analysis
GSE20126 Transcriptome analysis of human Wharton’s jelly stem cells
Relations
Reanalysis of GSM503608

Data table header descriptions
ID_REF
VALUE RMA signal intensity

Data table
ID_REF VALUE
AFFX-BioB-5_at 7.780544
AFFX-BioB-M_at 8.05667
AFFX-BioB-3_at 7.585989
AFFX-BioC-5_at 9.004041
AFFX-BioC-3_at 9.228559
AFFX-BioDn-5_at 10.04029
AFFX-BioDn-3_at 11.37579
AFFX-CreX-5_at 12.51552
AFFX-CreX-3_at 12.80689
AFFX-DapX-5_at 9.341465
AFFX-DapX-M_at 10.47245
AFFX-DapX-3_at 10.46777
AFFX-LysX-5_at 5.830568
AFFX-LysX-M_at 6.594488
AFFX-LysX-3_at 7.718092
AFFX-PheX-5_at 7.074605
AFFX-PheX-M_at 7.688879
AFFX-PheX-3_at 7.548898
AFFX-ThrX-5_at 7.818195
AFFX-ThrX-M_at 8.163538

Total number of rows: 54675

Table truncated, full table size 1052 Kbytes.




Supplementary file Size Download File type/resource
GSM503626_WJ1P20_R1.CEL.gz 4.1 Mb (ftp)(http) CEL
Processed data included within Sample table

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