2.6. Microarray Hybridization
Before use, slides were blocked by incubating the DNA chips in a blocking solution (0.02% SDS, 2× SSC) for 20 min at 50 °C and ~70 rpm in the dark. The slides were washed once in ddH2O for 10 min at 50 °C and twice always in fresh ddH2O for 15 min at RT and ~70 rpm in the dark. The slides were dried by centrifugation in a glass dish for 3 min at 900 rpm and stored in the fridge (possible for up to two month).
Labeled field samples (1 µg RNA) were mixed with 30 µL of 2× hybridization buffer, 3 µL Poly-dA (1 µM), 10 ng TBP-control and adjusted with nuclease-free water to 45 µL. Poly-dA is added to block the poly-T spacer on the probe and TBP is the TATA box gene fragment added as the positive hybridization control. The labeled RNA was then denatured for 5 min at 94 °C. After denaturation, the samples were shortly placed on ice and 15 µL of KREAblock (background blocker from KREATECH) was added. Slides were placed into an array holder; coverslips (LifterSlips, Erie Scientific, USA) were cleaned and placed onto the microarrays. Half of the hybridization mixture (30 µL) was added to one microarray. Hybridization was carried out for 1 h at 65 °C in a 50 mL Falcon tube containing a wet Whatman paper. The DNA chips were washed three times and shaken (~70 rpm) in the dark under stringent conditions. The washings were always undertaken for 10 min. The incubation in the first washing buffer (2× SSC/10 mM EDTA/0.05% SDS) and the second washing buffer (0.5× SSC/10 mM EDTA) was done at room temperature. The incubation in the third washing buffer (0.2× SSC/10 mM EDTA) was done at 50 °C.