corn oil was 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 1500 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 Leydig cells from DEHP-treated mouse
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 1500 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. Additionally, we deleted the spots that were called present only on the 3 dye-swaps corresponding to a single DEHP-treated biological replicates. 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.
