Gene Expression Profiling in Cells with Enhanced ?-Secretase Activity

Magold, Alexandra I.; Cacquevel, Matthias; Fraering, Patrick C.

Reversed transcription and labeling: was performed for 2 hrs at 42uC in a final reaction volume of 40 ml containing 1X SuperScript II buffer (Invitrogen), 40 units RNasin (Promega, Madison, USA), 10 mM DTT, 0.5 mM dATP, dGTP, dTTP, 0.2 mM dCTP, 0.1 mM of either Cy3-dCTP or Cy5-dCTP (GE Healthcare, Uppsala, Sweden) and 400 units of SuperScript II reverse transcriptase (Invitrogen). The RNA strand was hydrolyzed by adding 2 ml 500 mM EDTA and 4.5 ml 1 M NaOH and heating at 65uC for 15 minutes; the solution was then neutralized by adding 2.5 ml 1 M Tris (pH 6.8) and 4.5 ml 1 M HCl. The labeled cDNA was purified using the Qiagen MiniElute PCR Purification Kit (Cologne, Germany), eluting in 50 ml of EB buffer according to the manufacturer's instructions. The Cy3 and Cy5 labeled targets were combined and mixed with 400 ml of TE, 20 mg Cot 1 DNA (Invitrogen), 10 mg polyadenylic acid (Sigma, St. Louis, USA) and 10 mg yeast tRNA (Sigma). This mixture was concentrated to a final volume of 19.4 ml using a Microcon YM-30 filter (Millipore, Billerica, USA) according to the manufacturer's instructions. 20X SSC and 10% SDS were added to final concentrations of 3X and 0.4%, respectively, in a final volume of 24 ml. This mixture was heated for 2 minutes at 98uC, pipetted immediately onto the cDNA microarray and, after covering with a glass cover slip (Erie Scientific, Portsmouth, USA), placed in a humidified chamber (Telechem, Sunnyvale, USA) and allowed to hybridize at 64uC for 20 hrs. Slides were then washed at room temperature twice for 5 minutes in 2X SSC, 0.1% SDS, twice for 1 minute in 0.2X SSC, once for 1 minute in 0.1X SSC and once for 5 minutes in 0.1X SSC, 0.1% Triton X-100. After drying, slides were scanned on a microarray scanner (Agilent Technologies) and the resulting TIFF images were analyzed using the GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, USA). The mouse cDNA microarrays used in this study consisted of approximately 17,000 PCR products generated from cDNA clones and control DNAs spotted onto Nexterion AL slides (Schott, Mainz, Germany). A complete description of the slides and their content can be obtained from the Lausanne DNA Array Facility (http://www.unil.ch/dafl). The microarray data set discussed in this publication has been deposited in the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and is accessible through GEO Series accession number GSE16379. Note that Hamster genomic sequence information is not yet sufficiently available to the research community. Consequently no commercial hamsterspecific microarrays were available at the time of the experiment. However, the strategy to use a microarray from a closely related species is not new and has proven successful before [81] .