The 22 K oligonucleotide microarrays were constructed in CapitalBio Corp. (Beijing, China) using the Human Genome Oligo Set Version 2.1 (Operon, Huntsville, AL), which consist of 21,329 5'-amino-modified 70-mer probes. In addition to internal controls provided by the Oligo Set vendor, eight intergenic sequences from the yeast used as external controls were synthesized. All nucleotides were dissolved in DNA Spotting Solution (CapitalBio Corp.) at 20 M and printed on amino silaned glass slide using a SmartArrayTM microarrayer (CapitalBio Corp.). After printing, the slides were baked for 1 h at 80oC and stored dry at room temperature untill use. Prior to hybridization, the slides were rehydrated over 65oC water for 10 seconds and UV cross-linked at 250 mJ/cm2 energy. The unimmobilized oligonucleotides were washed off with 0.5% SDS for 10 min at 42oC and SDS was removed by dipping the slides in anhydrous ethanol for 30 seconds. The slides were spin-dried at 1000 rpm for 1 min. 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). The data from faint spots was removed, in which the intensity was lower than the average intensity plus 2 standard deviations of the negative controls on the array. For each test and control sample, two hybridizations were performed by using a dye-swap strategy.
Support
glass
Coating
aminosilane
Description
This set includes 22k oligonucleotides, mostly 70-mers, designed based upon representative sequences in the human UniGene database.
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