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Sample GSM334191 Query DataSets for GSM334191
Status Public on Feb 19, 2009
Title Sertoli cells: DEHP-treated Sample 2 vs. Vehicle-treated Sample 3
Sample type RNA
 
Channel 1
Source name Laser capture microdissected Sertoli cells-rich areas from DEHP-treated mouse
Organism Mus musculus
Characteristics Strain: C57BL/6J
Gender: male
Age: 21 weeks
Tissue: laser-capture microdissected Sertoli-cells rich areas (intermediate layer of the seminiferous tubule epithelium)
Treatment protocol di-(2-ethylhexyl)-phthalate (DEHP) in solution into corn oil was administered at 200 mg/Kg/day for 21 days by daily gavage
Growth protocol Fourteen-week-old male C57BL/6J mice were purchased from Charles River (Les Oncins, France). Mice were acclimatized for four weeks, housed in polycarbonate cages at 22+2°C on a 12 hour light/dark cycle and allowed free access to water and food. In vivo studies were conducted under European Union Guidelines for the use and care of laboratory animals and were approved by an independent ethics committee.
Extracted molecule total RNA
Extraction protocol Immediately following euthanasia, the right testis of DEHP-treated or vehicle-treated C57BL/6J male mice were embedded in Tissue-Tek O.C.T. compound (Sankura Finetek, Torrance,CA) and snap frozen with isopentane cooled in liquid nitrogen. Sections of 10µm (Leica CM3050S cryostat) were mounted on microscope slides which were immediately fixed, stained with hematoxylin-eosin, dehydrated by sequential baths in ice-cold 70%, 95% and 100% ethanol and stored under vacuum. Laser capture microdissection of Leydig cells was performed with the Arcturus PixCell IIe (Arcturus, California, USA) using CapSure® Macro LCM Caps (Molecular Devices Corporation). To avoid contaminations, the laser power and duration was adjusted for each section (ranging from 50 to 70 mW and 0.6 to 0.8 ms respectively). Approximately 5000 laser shots were performed per tissue section. Total RNA was isolated from single caps obtained from independent mice using the PicoPure® RNA Isolation Kit (Molecular Devices Corporation) and was amplified for two rounds with RiboAmp® RNA Amplification Kit (Molecular Devices Corporation) according to the manufacturers instructions.
Label Cy5
Label protocol For each sample, 1µg of amplified RNA was labelled using the Agilent Low Input Linear Amplification Labelling Kit (Agilent Technologies) according to manufacturer’s instructions.
 
Channel 2
Source name Laser capture microdissected Sertoli cells-rich areas from vehicle-treated mouse
Organism Mus musculus
Characteristics Strain: C57BL/6J
Gender: male
Age: 21 weeks
Tissue: laser-capture microdissected Sertoli-cells rich areas (intermediate layer of the seminiferous tubule epithelium)
Treatment protocol Eighteen week-old mice were randomly divided into two groups (DEHP-treated and vehicle-treated, n=4 per group). di-(2-ethylhexyl)-phthalate (DEHP, 200 mg/Kg/day) in solution into corn oil (DEHP-treated group) or corn oil (vehicle-treated group) were administered for 21 days by daily gavage
Growth protocol Fourteen-week-old male C57BL/6J mice were purchased from Charles River (Les Oncins, France). Mice were acclimatized for four weeks, housed in polycarbonate cages at 22+2°C on a 12 hour light/dark cycle and allowed free access to water and food. In vivo studies were conducted under European Union Guidelines for the use and care of laboratory animals and were approved by an independent ethics committee.
Extracted molecule total RNA
Extraction protocol Immediately following euthanasia, the right testis of DEHP-treated or vehicle-treated C57BL/6J male mice were embedded in Tissue-Tek O.C.T. compound (Sankura Finetek, Torrance,CA) and snap frozen with isopentane cooled in liquid nitrogen. Sections of 10µm (Leica CM3050S cryostat) were mounted on microscope slides which were immediately fixed, stained with hematoxylin-eosin, dehydrated by sequential baths in ice-cold 70%, 95% and 100% ethanol and stored under vacuum. Laser capture microdissection of Leydig cells was performed with the Arcturus PixCell IIe (Arcturus, California, USA) using CapSure® Macro LCM Caps (Molecular Devices Corporation). To avoid contaminations, the laser power and duration was adjusted for each section (ranging from 50 to 70 mW and 0.6 to 0.8 ms respectively). Approximately 5000 laser shots were performed per tissue section. Total RNA was isolated from single caps obtained from independent mice using the PicoPure® RNA Isolation Kit (Molecular Devices Corporation) and was amplified for two rounds with RiboAmp® RNA Amplification Kit (Molecular Devices Corporation) according to the manufacturers instructions.
Label Cy3
Label protocol For each sample, 1µg of amplified RNA was labelled using the Agilent Low Input Linear Amplification Labelling Kit (Agilent Technologies) according to manufacturer’s instructions.
 
 
Hybridization protocol Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min), Wash buffer 2 (Agilent Technologies, 37°C, 1 min) and Stabilization and Drying Solution (Agilent Technologies, 30 sec)
Scan protocol Scanned on an Agilent G2565AA scanner.
Images were quantified using Agilent Feature Extraction Software (version 9.5.1.1).
Description none
Data processing Data were analyzed under R (www.r-project.org) using the limma package of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function with the following weight function (assigning a weight of 1 or 0 to each spot): myfunw<-function(x) { okType<-x[,ControlType]==0 okFoundRed<-x[,rIsFound]==1 okFoundGreen<-x[,gIsFound]==1 okPixRed<-((x[,rNumPixOLHi]+x[,rNumPixOLLo])/x[,rNumPix])<=0.1 okPixGreen<-((x[,gNumPixOLHi]+x[,gNumPixOLLo])/x[,gNumPix])<=0.1 okBGRed1<-(x[,rMedianSignal]/x[,rBGMedianSignal])>=1.5 okBGRed2<-(x[,rMeanSignal]/x[,rBGMeanSignal])>=1.5 okBGRed3<-(x[,rMedianSignal]-x[,rBGMedianSignal])/x[,rBGPixSDev]>=2 okBGRed4<-(x[,rMeanSignal]-x[,rBGMeanSignal])/x[,rBGPixSDev]>=2 okBGGreen1<-(x[,gMedianSignal]/x[,gBGMedianSignal])>=1.5 okBGGreen2<-(x[,gMeanSignal]/x[,gBGMeanSignal])>=1.5 okBGGreen3<-(x[,gMedianSignal]-x[,gBGMedianSignal])/x[,gBGPixSDev]>=2 okBGGreen4<-(x[,gMeanSignal]-x[,gBGMeanSignal])/x[,gBGPixSDev]>=2 okSatRed<-x$rIsSaturated==0 okSatGreen<- x$gIsSaturated==0 okUnifRed<-x$rIsFeatNonUnifOL==0 okUnifGreen<-x$gIsFeatNonUnifOL==0 okUnifBGRed<-x$rIsBGNonUnifOL==0 okUnifBGGreen<-x$gIsBGNonUnifOL==0 okPopRed<-x$rIsFeatPopnOL==0 okPopGreen<-x$gIsFeatPopnOL==0 okPopBGRed<-x$rIsBGPopnOL==0 okPopBGGreen<-x$gIsBGPopnOL==0 okManualFlag<-x$IsManualFlag==0 okAbBGRed<-x$rIsWellAboveBG==1 okAbBGGreen<-x$gIsWellAboveBG==1 as.numeric(okType & okFoundRed & okFoundGreen & okPixRed & okPixGreen & ((okBGRed1 & okBGRed2 & okBGRed3 & okBGRed4) | (okBGGreen1 & okBGGreen2 & okBGGreen3 & okBGGreen4)) & okSatRed & okSatGreen & okUnifRed & okUnifGreen & okUnifBGRed & okUnifBGGreen & okPopRed & okPopGreen & okPopBGRed & okPopBGGreen & okManualFlag & okAbBGRed & okAbBGGreen) }. Only the spots that had a weight of 1 on at least 6 microarrays corresponding to 3 dye-swap experiments were further analyzed. Local background was substracted on Cy3 and Cy5 data and a lowess normalization was applied to the log(Cy5/Cy3) data using the normalizeWithinArrays function.
 
Submission date Oct 16, 2008
Last update date Feb 19, 2009
Contact name Pascal GP Martin
E-mail(s) pascal.martin@inrae.fr
Organization name INRAE
Department UMR1332 BFP
Lab FDFE
Street address 71 avenue Edouard Bourlaux
City Villenave d'Ornon
ZIP/Postal code 33140
Country France
 
Platform ID GPL4134
Series (2)
GSE13237 Effect of DEHP on adult mouse Sertoli cells rich areas (SCRA)
GSE13241 Effect of DEHP on adult mouse

Data table header descriptions
ID_REF
VALUE Lowess normalized log2(DEHP-treated sample/vehicle-treated sample) representing Cy5/Cy3 or Cy3/Cy5 depending on the sample

Data table
ID_REF VALUE
120 0.013978043
190 -0.286658649
192 0.075127767
202 -0.078474746
208 -0.108604212
216 -0.172476344
221 -0.551694401
228 -0.124003261
235 1.434483401
263 0.085661134
267 -0.082599404
270 -0.013112998
275 -0.463610706
278 0.44939163
280 -0.066156922
283 0.191264164
285 -0.418431435
286 0.020277307
287 0.275968026
292 -0.342573017

Total number of rows: 16095

Table truncated, full table size 280 Kbytes.




Supplementary file Size Download File type/resource
GSM334191.txt.gz 15.0 Mb (ftp)(http) TXT
Processed data included within Sample table

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