title: Human 50-mer oligo library from MWGBiotech Oligo Sets
status: Public on Aug 21 2018
contact.city: Atlixco
contact.first: Verónica
contact.last: Vallejo-Ruiz
contact.email: vero.vallejoruiz@gmail.com
contact.state: Puebla
contact.address: Km 4.5 Carretera Federal Atlixco-Metepec
contact.department: Centro de Investigación Biomédica de Oriente
contact.country: Mexico
contact.institute: Instituto Mexicano del Seguro Social
contact.phone: +52 2444440122
organism: Homo sapiens
manufacture_protocol: Printing of arrays. Human 50-mer oligo library set ?A? from MWGBiotech Oligo Sets (http:/www.mwgbiotech.com) was used. It contains 10,000 gene specific oligonucleotide probes representing the best-annotated genes from human. Oligo library was resuspended to 50µM in Micro Spotting solution (ArrayIt Brand Products.). SuperAmine coated slides 25x75 mm (TeleChem International INC) were printed in duplicate, and fixed at 80°C for 4 hours. For pre-hybridization the slides were re-hydrated with water vapor at 60°C, and fixed with two cycles of UV light (1200J). After boiling for two minutes at 92°C, slides were washed with 95% ethanol for one minute and prehybridzed in 5X SSC, 0.1% SDS and 1% BSA for one hour at 42°C. The slides were washed and dried for further hybridization.
  Probe preparation and hybridization to arrays. 10 µg of total RNA were used for cDNA synthesis incorporating dUTP-Alexa555 or dUTP-Alexa647 employing the Firs-Strand cDNA labeling kit (Invitrogen). Incorporation of fluorophore was analyzed by using the absorbance at 555 nm for Alexa555 and 650 nm for Alexa647. Equal quantities of labeled cDNA were hybridized using hybridization solution UniHyb (TeleChem International INC). The arrays were incubated for 14 h at 42°C, and then washed tree times with 1X SCC, 0.05 % SDS at room temperature.
  Data acquisition and analysis of array images. Acquisition and quantification of array images was performed in GenePix 4100A with its accompanying software GenePix from Molecular Devices. All images were captured using 10µm resolution. For each spot the Alexa555 and Alexa647 density mean value and background mean value were calculated with software ArrayPro Analyzer from Media Cibernetics.