Double stranded cDNA was synthesized using the Invitrogen Superscript Double-Stranded cDNA synthesis kit and was subsequently labeled using Nimblegen one color DNA labeling kit (Roche Nimblegen Inc. WI). 4 µg of Cy3-labeled cDNA from each group was hybridized onto Musmusculus 4×72 Nimblegen microarray using Nimblegen Hybridization system 4 according to manufacturer's instructions (Roche).
These data express the importance of these pathways in response of S. cerevisiae to propolis and confirm a high degree of overlapping in gene function among the microarray hybridization and the system biology analysis.
Following dye incorporation and sample cleanup, sample cDNA and control cDNA were mixed together with spiked controls that aid in hybridization calibration, and then the mixed sample was hybridized to the microarray surface using a Maui Hybridization System (Biomicro, Salt Lake City, UT).
For mRNA expression experiments, microarrays were hybridized using the MAUI hybridization system (BioMicro), which promotes active mixing during hybridization.
Double stranded cDNA was synthesized and followed by an in vitro transcription reaction to amplify aRNA incorporated with biotin labeling system for the microarray hybridization in triplicate.
The microarray hybridization results were analyzed using AlmaZen system v.2.1 software (Bioalma, Madrid, Spain) and normalized by applying the locally weighted scatter plot smoothing (LOWESS) algorithm [ 55].
Labeled cDNAs were purified using Nucleospin Extract II columns (Macherey Nagel, Düren, Germany) and hybridized in a Discovery station using ChipHybe 80 hybridization buffer at 42°C for 12 h without any final stringency washes (hybridization automate, reagents and microarray hybridization procedure are from Ventana Medical System, Tucson, AZ, USA).
The microarrays were hybridized and washed in a NimbleGen Hybridization System 4 (NimbleGen Systems, Inc., Madison, WI).
The microarray hybridization was carried out at 65°C in the automated MAUI hybridization system (BioMicro Systems, USA).
Hybridization of the microarray slides was performed using the MAUI Hybridization system (MAUI Mixer SC Hybridization Chamber mixers, Biomicro Cat. no. 02A00830) using the Pronto!Universal Hybridization Kit.
