5 protocols
Title: Affymetrix CEL analysis. Description:
hybridization protocol
Affymetrix Generic Hybridization
RNA was isolated using the RNeasy Plant Mini Protocol (QIAGEN, Clifton Hill, Australia) and the quality of the RNA was assessed using the Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) where spectrophotometric analysis was carried out to determine the A260 to A280 ratio and the concentration. For qRT-PCR, cDNA synthesis was carried out using the Biorad (Perth) iScript cDNA synthesis kit according to manufacturers instructions. For arrays, the cRNA was prepared from 3 ug of total RNA almost exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual.
To prepare total RNA for microarray analysis, we used retinas from 4 month-old Wt and TgIGF-I mice. We used the recommended Affymetrix protocol for RNA extraction (Expression Analysis Technical Manual; http://www.affymetrix.com). Briefly, each pair of retinas were homogenised with Trizol (Invitrogen) before precipitation and column purification (Qiagen). On-column DNase I digestion was carried out (Qiagen).
(Parameters: Extracted product = total RNA, Amplification = none)