title: Silkworm 23K oligo array version 2.0
status: Public on Nov 16 2010
contact.city: Chongqing
contact.first: Ling
contact.last: Gan
contact.email: gl9089@gmail.com
contact.address: 160#Xue Yuan Road,RongchangCounty
contact.department: 1The Department of Veterinary Medicine
contact.country: China
contact.institute: Southwest University
contact.phone: +8613983416663
organism: Bombyx mori
manufacture_protocol: The 23 K oligonucleotide microarrays were constructed in CapitalBio Corp. (Beijing, China), which consist of 23,134 5'-amino-modified 70-mer probes. In addition to internal controls, eight intergenic sequences from the yeast used as external controls were synthesized. The oligonucleotide probes were printed on amino silaned glass slides using a SmartArray microarrayer (CapitalBio Corp.). An aliquot of 5 µg total RNA was used to produce fluorescent dye-labeled cDNA with Eberwine’s linear RNA amplification method and subsequent enzymatic reaction according to a previous published protocol (Guo et al., 2005). The labeled DNA was purified with PCR Clean-up NucleoSpin kits (Macherey-Nagel), resuspended in Elution buffer, and quantified by UV spectrophotometry. Labeled control and test samples were quantitatively adjusted based on the efficiency of Cy-dye incorporation and mixed into 80 µL hybridization solution (3×SSC, 0.2% SDS, 25% formamide and 5×Denhart’s). DNA in hybridization solution was denatured at 95oC for 3 min prior loading on a microarray. Hybridization was performed under LifterSlipTM (Erie Company), which allows for even dispersal of hybridization solutions between the microarray and coverslip. The hybridization chamber was placed on a 3D-Tilting Agitator (CapitalBio Corp.) to facilitate the microfluidic circulation under the coverslip. The array was hybridized at 42oC overnight and washed with two consecutive washing solutions (0.2% SDS, 2×SSC at 42℃ for 5 min, and 0.2% SSC for 5 min at room temperature). After hybridization, microarrays were scanned with a confocal LuxScanTM scanner (CapitalBio Corp.), and the data of obtained images were extracted with SpotData Software (CapitalBio Corp.). The raw data was normalized using a space and intensity-dependant LOWESS program (Yang et al., 2002).