Microarray analysis filtering
RNA was harvested using the Qiagen RNeasy Plant mini kit extraction protocol. 10 mg of total RNA was sent for hybridisation to Affymetrix chips. Data received from the facilities was analysed using the Affymetrix microarray software suite. Initially GeneChip data was normalised and scaled to a value of 500. Following comparison of experiment to baseline GeneChips, filtering analysis was carried out to identify differentially regulated genes.
Firstly detection values were filtered: all probe Ids whose detection was deemed significantly absent by the software, or had a signal level <50 were eliminated from the data set. The second microarray analysis filter eliminated all probe Ids that were deemed to have no significant change in expression by the software. Finally all probe Ids with a SLR between –0.999 and 0.999 were eliminated. The datasets obtained were exported to Microsoft excel and compared to update annotation data available from the Sheen and Schroeder labs using Microsoft Access (http://genetics.mgh.Harvard.edu/sheenlab; www.biology.ucsd.edu/labs/schroeder/genechips.htlm).
This resulted in statistical significant datasets containing genes differentially regulated to a SLR ≥ 1 or ≤ -1 with correct TIGR annotations.