Title: Affymetrix Generic Hybridization. Description:
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.
array scanning protocol
Standard Affymetrix Scanning protocol. (Parameters: Scanning hardware = 418 [Affymetrix], Scanning software = MAS/GCOS/GREX [Affymetrix])
nucleic acid extraction protocol
Single cell suspensions were lysed in buffer RLT or Trizol and the RNA extracted following the RNeasy micro kit (Qiagen). The integrity of RNA was ensured by analysis of ribosomal 18S and 28S RNA intensity using an Agilent 2100 bioanalyser (Agilent Technologies). (Parameters: Extracted product = total_RNA, Amplification = none)