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Status |
Public on Feb 13, 2010 |
Title |
MDCK-Late Passage-biological replicate3 |
Sample type |
RNA |
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Source name |
Total RNA from late passage MDCK were plated on tissue culture flasks and allowed to polarize for four days
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Organism |
Canis lupus familiaris |
Characteristics |
species: Canine gender: Female organ: Kidney cell line: Madin Darby Canine Kidney
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Biomaterial provider |
Total RNA was received from Erik Young at the Department of Surgery, Hackensack University Medical Center, Hackensack, NJ
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Treatment protocol |
Early and late passage MDCK from T25 flasks that had been seeded with 8 x 105 cells and allowed to become confluent and polarize over 4 days in culture cells
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Growth protocol |
MDCK cells were obtained from the ATCC at passage number 55 (CCL-34) and were subcultured in MEM (ATCC) supplemented with 10% fetal bovine serum (Sigma) and an antibiotic/antimycotic (Invitrogen). Cells were cultured for 4-7 days before being re-seeded at 4-12 x 103 cells/cm2 in T25 culture flasks. Cells were maintained in humidified incubator at 37oC and 5% CO2 and were fed three times per week.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated from biological triplicates of early and late passage MDCK from T25 flasks using a Ribopure kit (Ambion, Austin, TX). Acquired RNA was precipitated with EtOH and subsequently purified employing columns, procedures and reagents from an RNEasy kit (Qiagen, Germantown, MD) and resuspended in RNAse-free H2O provided.
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Label |
Biotin
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Label protocol |
Complementary DNA and complemetary RNA synthesis were performed according to Affymetrix Expression Analysis protocols (see www.affymetrix.com). Briefly, double-stranded cDNA was synthesized from 5ug of total RNA using the Superscript double-stranded cDNA synthesis kit (Invitrogen). Following phenol/chloroform extraction and ethanol precipitation, a biotin-labeled in-vitro transcription reaction was carried out using the cDNA template (Enzo Life Sciences, Farmingdale, NY).
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Hybridization protocol |
Fifteen micrograms of cRNA was fragmented for hybridization to Affymetrix Canine Genome 2.0 Array GeneChips (Santa Clara, CA), which contains approximately 18,000 C. familiaris mRNA/EST-based transcripts and over 20,000 non-redundant predicted genes. Hybridization was done overnight at 45°C for 16 hours using the Genechip Hybridization Oven 640 (Affymetrix). Washing and staining (Streptavidin Phycoerythrin) was performed with the Genechip Fluidics Station 450 (Affymetrix) using the EukGE-WS2v4_450 protocol
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Scan protocol |
Images were acquired using the Affymetrix GeneArray Scanner 3000. Data was extracted using Affymetrix GeneChip Operating Software version 1.0.
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Description |
Young_L3_08-01-07
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Data processing |
Expression intensity values were extracted from the raw image files using the Robust Multi-Array Average algorithm with the Affymetrix Expression ConsoleTM. Quantile normalization was performed to normalize the distribution of probe intensities for all the arrays in a given set
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Submission date |
Feb 13, 2009 |
Last update date |
Feb 17, 2009 |
Contact name |
Saleena Ghanny |
E-mail(s) |
ghannysa@njms.rutgers.edu
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Organization name |
Rutgers University
|
Street address |
185 South Orange Ave
|
City |
Newark |
ZIP/Postal code |
07103 |
Country |
USA |
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Platform ID |
GPL3738 |
Series (1) |
GSE14837 |
Madin Darby Canine Kidney (MDCK) Cells: Early & Late Passage |
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