8 protocols
ID_REF = VALUE = log ratio KDX1/control
The average fluorescence intensity for each spot was calculated and local background was subtracted. All data normalization and selection of fold-changed genes were performed using GeneSpring 7.3.1 (Agilent Technologies, USA).
The hybridization images were analyzed by GenePix Pro 6.0 (Axon Instruments, CA).
Labeled cDNAs were mixed with hybridization solution (MYcroarray.com), and the hybridization mixtures were heated at 65℃ for 5 min and then immediately cooled on ice for 5 min. Hybridization mixtures were directly loaded onto assembled MYcroarray.com microarray. The arrays hybridized at 50℃ for 16 hr using Agilent Hybridization oven (Agilent Technology, USA).
Labeling reactions were performed with a Bioprime labeling kit (Invitrogen) in a volume of 50 μl with a modified dNTP pool containing 120 μM each of dATP, dGTP, and dCTP; 60 μM dTTP; and 60 μM Cy5-dUTP (for CosR-knockdown) or Cy3-dUTP (for the wild type).
Yeast cell was grown on SD-ura medium. Yeast cell was grown overnight at 30°C . The culture was refreshed to 0.2 O.D and grown at 30°C for 5h 30min. Cells were collected.
Total RNA was isolated using TRIzol reagent (Life Technologies). RNA concentrations were determined by measuring absorbance at 260nm.
KDX1 was subcloned into yeast multicopy vector ( pRS426 )