title: CapitalBio Operon Human MAQC (BIO and BIO1, two- and one-color)
status: Public on Sep 08 2006
contact.city: Shanghai
contact.first: Leming
contact.last: Shi
contact.email: lemingshi@fudan.edu.cn
contact.address: 2005 Songhu Road
contact.department: School of Life Sciences
contact.country: China
contact.institute: Fudan University
contact.phone: +86-18616827008
organism: Homo sapiens
description: The human genome-wide long oligonucleotide microarray was constructed inhouse at CapitalBio Corporation. Briefly, 5′-amino-modified 70-mer probes representing 21,329 H. sapiens genes from the Human Genome Oligo Set Version 2.1 (Operon/Qiagen), and internal and external controls, were printed on amino silaned glass slides using a SmartArray microarrayer (CapitalBio Corp.). Fluorescent-labeled DNA (Cy3 and Cy5-dCTP) was produced through Eberwine’s linear RNA amplification method and subsequent enzymatic reaction. This procedure has been previously described in detail (Guo, Y. et al. J. Virol. 79, 14392-14403, 2005). Briefly, double-stranded cDNA containing T7 RNA polymerase promoter sequence was synthesized with 5 µg of total RNA using Reverse Transcription System, RNase H, DNA polymerase I and T4 DNA polymerase, according to the manufacturer’s recommended protocol (Promega). The resulting labeled DNA (labeled control and test samples) was 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). Individually labeled cRNAs were not pooled before hybridization.
  Hybridization on a microarray (see hybridization configurations above) was performed under LifterSlip (Erie Company). The hybridization chamber was laid on a three-phase Tiling Agitator (CapitalBio Corp.) to facilitate the microfluidic circulation under the coverslip. The microarray was hybridized at 42°C overnight and washed with two consecutive washing solutions (0.2% SDS, 2× SSC at 42°C for 5 min, and 0.2% SSC for 5 min at 22 °C) before scanning with a confocal LuxScan scanner (CapitalBio Corp.). For two-color microarrays, the scanning setting for the Cy3 and Cy5 channels was manually balanced by visual inspection of the external control spots. The data from the obtained images were extracted with SpotData software (CapitalBio Corp.).
manufacture_protocol: CapitalBio Corporation