title: MusV29K
status: Public on Nov 04 2009
contact.city: Marseille
contact.first: Isabelle
contact.last: Lesur Kupin
contact.email: lesur@tagc.univ-mrs.fr
contact.address: 163 av. de Luminy - case 928
contact.department: Bioinformatics
contact.country: France
contact.institute: INSERM U928
contact.phone: (+33)4 91 82 87 11
organism: Mus musculus
manufacture_protocol: The microarrays MUSV29K contain 7771 sequences. The following cDNA libraries were used: the NIA Mouse 15K cDNA clone set, 2NbMT (thymus), NbMLN (lymph node), and 3NbMS (spleen). Detailed descriptions of these cDNA libraries are available at the UniGene database website (2NbMT: Lib.544, 3NbMS: Lib. 553, NbMLN: Lib.567, NIA 15K: Lib. 8622). All of the libraries were cloned into pT3T7D-Pac vector, exept for the NIA 15K Mouse cDNA clone set, which was cloned into pSPORT1 vector. The NIA 15K Mouse cDNA clone set is a rearrayed and resequenced set of 15 000 bacterial clones derived from 11 embryo cDNA libraries. 2NbMT, NbMLN and 3NbMS are sequenced I.M.A.G.E libraries. These libraries contain 84127 clones. We used the Microarrays Quality Control tool to select clones matching a single region on the mouse genome and for which at least one 3' EST and one 5'EST have been identified. We selected 7771 bacterial clones matching 6622 mouse genes. 73% (4833) of the genes are represented by a single cDNA clone and about 27% (1789) of the genes included in this gene set are represented by two or more different cDNA clones, providing internal controls to assess the reproducibility of gene expression measurements. We also added 8 positive controls (poly-A LBP2S and Cot1, a mix of DNA fragments containing repeated sequences) and 2205 negative controls (empty spots and CG03, an Arabidopsis thaliana cDNA sequence). In summary, the MUSV29K mouse microarray we designed contains 9984 spots, identifying 6622 mouse genes.
  PCR amplifications were performed in 96-well microliters plates using the following primers: 5'-CCAGTCACGACGTTGTAAAACGAC-3' and 5'-GTGTGGAATTGTGAGCGGATAACAA-3'. The reactions were performed as described in Diehl et al. by transferring few Escherichia coli from a growth culture with a plastic 96pins replicator (Proteigene, #X5055) to 100µl of PCR mix pH8,5 containing 1,5mM MgCl2, 1M Bétaine, 375µM dATP, dTTP, dGTP, dCTP, and 5U of GoTaq DNA polymerase (Promega, #M3171). The plates were incubated for 6 min at 94°C, before 38 cycles of 94°C for 30s, 65°C for 45s and 72°C for 3min30s, followed by a final elongation phase at 72°C for 10 min. Amplification products were not quantitated, but their quality was systematically checked on 1% agarose gels. 11,03% of the bacterial clones were estimated to be nonamplified and 4,38% showed multiple bands after PCR amplification. Unpurified PCR products were then transfered to 384-well microplates using the TECAN Genesis workstation 150, before being evaporated, taken up to 30µl of distilled water, and spotted onto nylon membranes (Hybond-N+; Amersham Bioscience). This step was conducted using a MicroGrid-II arrayer (Apogent Discoveries) equipped with a 64-pin biorobotics printhead. Microarrays were hybridized with  ƔdATP 33P-labelled probe, whose oligonucleotide sequence is common to all spotted PCR product (LBP2S 5'-TCACACAGGAAACAGCTATGAC-3'). It showed uniform signal intensities across individual membranes.