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Sample GSM64047 Query DataSets for GSM64047
Status Public on Jul 15, 2005
Title Aldosterone-responsive genes in kidney cortical collecting duct cell mpkCCDc14 (6 hr treatment)-3
Sample type RNA
 
Channel 1
Source name Kidney cortical collecting duct cell line (mpkCCDc14)
Organism Mus musculus
Characteristics Kidney cortical collecting duct cell line (mpkCCDc14)
Biomaterial provider Dr. Alain Vandewalle lab (Institut National de la Sante et de la Recherche Medicale, France)
Treatment protocol Cells were treated with 10-6M aldosteron for 6 hr.
Growth protocol mpkCCD cells were cultured on 30 mm collagen-coated 6-transwell plates at a density of 1.5-2.0 x 106 cells/well in a modified medium described previously with 1% Pen-strep. Fresh medium was changed every other day. 2-3 days later, while transepithelial cell resistance reach ~2 KΩ, cell medium was replaced with dexamethasone-free and charcoal-treated steroid-free FBS for another 2-3 days. Fresh medium was changed everyday.
Extracted molecule total RNA
Extraction protocol Total RNAs were isolated with RNA STAT-60 (Tel-Test, Inc). The contaminated genomic DNA in the total RNA was removed with DNase treatment & removal reagents (Ambion).
Label Cy5
Label protocol Microarray analysis was performed following the protocol from UCSF genomics core laboratories (original document can be found at www.arrays.ucsf.edu). Briefly, the first strand cDNA was synthesized with 20 µg total RNA, 1.0 µg oligo dT, 830 µM each dATP, dCTP, dGTP, 166 µM dTTP, 666 µM 5-(3-aminoallyl)- dUTP (Ambion),0.1 m DTT, 1 µl RNase inhibitor ( Fisher), 600 U RT, 10x RT buffer and H2O to final 30 ul for 2 hours at 42oC. After hydrolysis with 10 µl 1N NaOH and 10 µl 0.5 M EDTA, cDNA was purified and recovered with Microcon 30. The Cy5 (Amersham Biosciences) was then coupled with cDNA in 0.05 M NaBiocarbonate (pH 9.0) in the dark at the room temperature for more than 1.5 hours.
 
Channel 2
Source name Kidney cortical collecting duct cell line (mpkCCDc14)
Organism Mus musculus
Characteristics Kidney cortical collecting duct cell line (mpkCCDc14)
Biomaterial provider Dr. Alain Vandewalle lab (Institut National de la Sante et de la Recherche Medicale, France)
Treatment protocol Cells were treated with vehicle (ethanol)for 6 hr.
Growth protocol mpkCCD cells were cultured on 30 mm collagen-coated 6-transwell plates at a density of 1.5-2.0 x 106 cells/well in a modified medium described previously with 1% Pen-strep. Fresh medium was changed every other day. 2-3 days later, while transepithelial cell resistance reach ~2 KΩ, cell medium was replaced with dexamethasone-free and charcoal-treated steroid-free FBS for another 2-3 days. Fresh medium was changed everyday.
Extracted molecule total RNA
Extraction protocol Total RNAs were isolated with RNA STAT-60 (Tel-Test, Inc). The contaminated genomic DNA in the total RNA was removed with DNase treatment & removal reagents (Ambion).
Label Cy3
Label protocol Microarray analysis was performed following the protocol from UCSF genomics core laboratories (original document can be found at www.arrays.ucsf.edu). Briefly, the first strand cDNA was synthesized with 20 µg total RNA, 1.0 µg oligo dT, 830 µM each dATP, dCTP, dGTP, 166 µM dTTP, 666 µM 5-(3-aminoallyl)- dUTP (Ambion),0.1 m DTT, 1 µl RNase inhibitor ( Fisher), 600 U RT, 10x RT buffer and H2O to final 30 ul for 2 hours at 42oC. After hydrolysis with 10 µl 1N NaOH and 10 µl 0.5 M EDTA, cDNA was purified and recovered with Microcon 30. The Cy3 (Amersham Biosciences) was then coupled with cDNA in 0.05 M NaBiocarbonate (pH 9.0) in the dark at the room temperature for more than 1.5 hours.
 
 
Hybridization protocol After cleanup Cy3 or Cy5-labeled cDNA with CyScribe GFX purification kit (Amersham Biosciences), the labeled cDNA was hybridized with microarray slide at 50oC for >40 hours.
Scan protocol Array data were collected with Genepix 4.0 scanner. The quality of array was examined with RBioconductor.
Description Array data were collected with Genepix 4.0 scanner. The quality of array was examined with RBioconductor. First, the uniformity of spot and labeling, and the intensity of labeling were checked with MA-plot (M=log2(Cy5/Cy3), A=log2((Cy5*Cy3)/2). Quantitative assessment such as signal to noise ratio and the median signal intensity were also performed. Only these arrays which have the reasonable qualities would be used for further analysis. The significantly different expressed genes are selected with SAM using the one class response with data (log2 Cy5/Cy3) normalized in RBioconductor. Hierarchical clustering is performed with Acuity 4.0 (Axon Instruments).
Data processing data (log2 Cy5/Cy3) is normalized in RBioconductor
 
Submission date Jul 13, 2005
Last update date Jul 14, 2005
Contact name Ting Ting Zhang
E-mail(s) tizst0@yahoo.com
Phone 617-855-2179
Fax 617-855-2179
Organization name Harvard University
Department Mclean Hospital
Lab bioorgnic and natural product
Street address 115 Millman st
City Belmont
State/province MA
ZIP/Postal code 02478
Country USA
 
Platform ID GPL2623
Series (1)
GSE2933 Aldosterone-responsive genes in kidney cortical collecting duct cell line, mpkCCDc14

Data table header descriptions
ID_REF
VALUE Log2(Cy5/Cy3) from array slide 3

Data table
ID_REF VALUE
M200000001 -0.25
M200000002 0.02
M200000003 0.23
M200000004 -0.59
M200000005 -0.09
M200000006 0.03
M200000007 -0.29
M200000008 -0.08
M200000009 0.17
M200000010 0.29
M200000011 0.17
M200000012 -0.18
M200000013 0.06
M200000014 -0.25
M200000015 -5.85e-03
M200000016 0.28
M200000017 0.18
M200000018 -0.21
M200000019 -0.04
M200000020 -0.30

Total number of rows: 16159

Table truncated, full table size 262 Kbytes.




Supplementary data files not provided

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