title: INRArray 01.4
status: Public on Feb 19 2009
contact.city: Villenave d'Ornon
contact.first: Pascal
contact.middle: GP
contact.last: Martin
contact.email: pascal.martin@inrae.fr
contact.address: 71 avenue Edouard Bourlaux
contact.department: UMR1332 BFP
contact.country: France
contact.institute: INRAE
organism: Mus musculus
manufacture_protocol: Genes were identified from  the literature and from microarray screenings of liver RNA extracted from mice treated with RXR (LG100268) or PPARalpha (fenofibrate) pharmacological activators. PCR primers were selected to amplify a specific fragment of 295 selected cDNAs. The amplified cDNAs were cloned into pCR 2.1 TOPO vector (Invitrogen). The list of all cDNA fragments and their sizes are available at www.inra.fr/internet/Centres/toulouse/pharmacologie/lpt.htm. In addition, a set of 13 yeast cDNAs (spikes) were identified by hybridizing mouse liver, kidney and intestine total RNAs to a yeast macroarray and selecting spotted yeast cDNAs that did not yielded a significant signal. These yeast cDNAs were cloned into pCR 2.1 TOPO to produce the spotted probes and into pSP64(polyA) vector (Promega) to produce the corresponding mRNAs to be spiked into the samples before labeling. Twelve of the Yeast mRNAs (one was used as negative control) were produced by in vitro transcription, assayed and controlled on a Bioanalyzer 2100 (Agilent) before being mixed together. The absence of cross-hybridization of the spiked-in yeast mRNAs with the spots corresponding to mouse genes on INRArray was also controlled. All cDNAs (mouse and yeast) were then amplified using common PCR primers targeting the pCR 2.1 cloning vector at both sides of the inserts. PCR amplicons were assayed and diluted at 200 ng/µL in 50% DMSO before being spotted onto nylon membranes (Millipore) using a VersArray Chipwriter Pro (Biorad) at the GenoToul biochip facility (http://biopuces.genotoul.fr/). During spotting, wells containing 50% DMSO without amplicons were systematically inserted between wells containing amplicons to assess background signal on the whole surface of the arrays. The final macroarrays contained 768 spots (4 x 12 pins each spotting a 4x4 subgrid).