title: Multi-species (Bovine, Mouse, Frog) oocyte 14k array
status: Public on Dec 15 2010
contact.city: Quebec
contact.first: Eve-Lyne
contact.last: Sylvestre
contact.email: eve-lyne.sylvestre.1@ulaval.ca
contact.state: Quebec
contact.address: 2440 Boul Hochelaga
contact.country: Canada
contact.institute: Université Laval
organism: Xenopus laevis
  Bos taurus
  Mus musculus
description: Bos taurus (clones 1-1249), Mus musculus (clones 1250-2399), Xenopus Laevis (clones 2400-3552).
manufacture_protocol: Pools of total RNA (1 μg) from bovine, mouse and xenopus oocytes and somatic tissues were used for cDNA production. The mRNAs were reverse transcribed, and the cDNAs were amplified using the BD SMART PCR cDNA Synthesis Kit (BD Biosciences, Mississauga, ON, Canada) according to the manufacturer's instructions. The SSH was performed with the PCR Select cDNA Subtraction Kit (BD Biosciences) as described by the manufacturer. Briefly, the pools consisted of oocytes for the tester and  somatic tissues for the driver. The PCR was performed in a ThermoHybaid Hybrid MultiBlock System (Bio-Rad, Mississauga, ON, Canada). The subtracted PCR products generated by SSH were cloned into the TOPO Cloning 5-min PCR Cloning Kit (Invitrogen) and then transformed into DH5-α-T1 Max Efficiency cells (Invitrogen).Purified PCR products were speedvac-evaporated (SPD SpeedVac ThermoSavant), resuspended in a mixture of equal parts 3× SSC (1× SSC: 0.15 M sodium chloride and 0.015 M sodium citrate with dimethyl sulfoxide), and spotted in four replicates on GAPS II glass slides (Corning, Corning, NY) using a VersArray Chip WriterPro robot (Bio-Rad). (bovine: 1076 clones, mouse: 1086 clones, frog:1086 clones). Full detailed procedure is described in : Vallee M,Gravel C,Palin MF,Reghenas H,Stothard P,et al. (2005) Identification of novel and known oocyte-specific genes using complementary DNA subtraction and microarray analysis in three different species. Biol Reprod 73: 63-71.