5 protocols
The arrays were scanned on the Illumina BeadArray Reader
Hybridization of labeled cRNA to the BeadChip, and washing and scanning were performed according to the Illumina GeneExpression Direct Hybridization Manual manual. Essentially, the amplified, biotin-labeled human cRNA samples were resuspended in a solution of Hyb E1 buffer (Illumina) and 25% (v/v) formamide at a final concentration of 25 ng/µL. 1.5 µg of each cRNA were hybridized. Hybridization was allowed to proceed at 58 C for 16 hours, after which, the bead array matrix was washed for 10 minutes with 1X High temperature buffer (Illumina), followed by a subsequent 10 minute wash in Wash E1BC buffer. The arrays were then washed with 100% ethanol for 10 min to strip off any remaining adhesive on the chip. A 2 minute E1BC wash was performed to remove residual ethanol. The array signal was developed via 10 minute incubation in Block E1 buffer, containing Streptavidin-Cy3 at a final concentration of 1µg/mL. The BeadChip was washed a final time in Wash E1BC buffer for five minutes and subsequently dried via centrifugation for 4 minutes at a setting of 275 rcf at room temperature.
Amplification and labelling of RNA was performed using the Illumina TotalPrep 96 RNA kit, as per manufacturer's instructions
tissue was extracted into RNAlater (Ambion) and RNA was isolated usign miRneasy kit (Qiagen) as per manufacturers instructions
oral infection with 1 x 109 CFU C. rodentium