8 protocols
AccessionType
bioassay_data_transformation
ID_REF = VALUE = log ratio KDX1/control
feature_extraction
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).
image_aquisition
The hybridization images were analyzed by GenePix Pro 6.0 (Axon Instruments, CA).
hybridization
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
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).
grow
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.
nucleic_acid_extraction
Total RNA was isolated using TRIzol reagent (Life Technologies). RNA concentrations were determined by measuring absorbance at 260nm.
specified_biomaterial_action
KDX1 was subcloned into yeast multicopy vector ( pRS426 )