Genotype : wild-type Stage : 9 d.p.c., 16-18 somite stage Tissue : forebrain & surrounding tissue Treatments applied Agent : oxygenation Route : whole embryo culture Dose : 5% oxygen Time : 2.0 hrs in culture Intervention : none Risk: >60% malformed at 24h Conditional biomarker: p53 protein induction Technical extraction & labeling Tissue procurement : conventional microdissection Sampling unit : 6-8 forebrains pooled Hybridization extract preparation : 2 ug total cellular RNA (Qiagen RNeasy kit Cat. 74103) Labeling (cDNA) : fluorescein-12-dCTP, biotinyl-11-dCTP Hybridization condition : 56-59oC, overnight, stringency washes 0.5X SSC, 0.1% SDS, 0.06X SSC, 0.01% SDS, and 0.06X SSC Detection & raw data organization Immunodetection : anti-FL with Cy3-tyramide, peroxidase-conjugated streptavidin with Cy5-tyramide Scanning : Micromax Custom Scanning and Data Processing Service (PerkinElmer Life Sciences, Cat.MPS401), Cy3 channel 532 nm and Cy5 channel 635 nm Spot measurement : ImaGene Method 1: Calculates average intensity of all pixels within the circle with intensity values falling in the predetermined range (e.g., 50% - 95% of maximal intensity in a grid square of a spot). Background correction : The background is calculated from the background range of pixels (e.g., 5-15% of maximal intensities) between the background rings. Local background is the subtracted from signal from every spot and every channel separately. Gene expression matrix : 2400 element array less 12 control spots and 6 spots from incomplete printing resulting in 2382 spots
