Samples of 45 flies were ground to a fine powder were ground to a fine powder using matched glass pestle and grinder, followed by the immediate addition of 600 µl of RNeasy lysis buffer (QIAGEN, Courtaboeuf, France). After initial tissue disruption, total RNA was extracted using the RNeasy system, following the manufacturer’s instructions.
Label
biotin
Label protocol
For each experimental type, three biologically independent samples were used. Biotinylated RNA was produced and hybridized to Affymetrix Drosophila GeneChip microarrays (Part Number 510548; Affymetrix, Santa Clara, USA) according to the method published in Irving et al, 2001.
Description
Analytic steps were performed using the programs within Affymetrix Microarray Suite 5.0 (Affymetrix) or Excel (Microsoft, Redmond, WA) with a combination of built in functions and custom formulae. The raw data were sorted using the Absent/Marginal/Present flags generated by the Microarray Suite functions. Whilst an Absent might indicate that there was no mRNA of particular type present in a sample, Marginal and Absent flags may indicate problems with the hybridisation, therefore only data points marked as present were retained.
Data processing
The remaining data mass for each microarray was then normalised to itself, making the median of all the present measurements one. The subsequent analysis of results was carried out on the genes present in all three replicates of a sample type.
