An absolute expression analysis examines the cell intensity file (*.CEL) from one experiment. For each transcript represented on the probe array, the expression algorithm computes the 1) detection call [present, absent, or marginal (unable to call the transcript present or absent), or no call]; 2) detection p-value; 3) signal (background subtracted and adjusted for noise); 4) stat pairs; 5) stat pairs used. Parameter settings: Alpha1=0.05; Alpha2=0.065; SF=1.000; Tau=0.015.
The Cell Analysis algorithm calculates a single intensity value for each probe cell on an array. This intensity value, representative of the hybridization level of its target, is derived as follows. The bordering pixels of the probe cell are excluded. The remaining pixel intensity distribution is calculated, and the intensity value associated with 75% of the distribution is used as the probe cell intensity. The intensities for all probe cells are saved in the .CEL file.
Scanner parameter settings: Pixel size=3; Filter=570; Number of Scans=2.
nucleic acid hybridization to array protocol
The fragmented cRNA is mixed with the hybridization cocktail, and heat shocked at 99C for 5 minutes. In the meanwhile, the probe array is filled with hybridization buffer and incubated at 45C for 10 minutes with rotation. The heated hybridization cocktail is then transferred to a 45C heat block for 5 minutes, and spun at maximum speed in a microcentrifuge for 5 minutes. Fill the probe array cartridge with the clarified hybridization cocktail and place probe array in rotisserie box in 45C oven. Hybridize for 16 hours.
nucleic acid labeling protocol
See pages 2.1.10-2.1.17 of the GeneChip Expression Analysis Technical Manual for all details. First-strand cDNA is synthesized from (5.0 to 20.0 ug) high-quality total RNA, using the GeneChip T7-Oligo(dT) Promoter Primer Kit or random primers provided with the SuperScript Choice Kit. Second-strand synthesis is carried out in: 1X second-strand reaction buffer, 200uM each dNTP mix, 10U E. coli DNA ligase, 40U E. coli DNA polymerase I, 2U E. coli RNase H in a final volume of 150 uL.The double-stranded cDNA is cleaned-up using the components supplied with the GeneChip Sample Cleanup Module. Enzo BioArray HighYield RNA Transcript Labeling Kit (Affymetrix, P/N 900182) is used to synthesize biotin-labeled cRNA target. The latter is cleaned up using the components supplied with the GeneChip Sample Cleanup Module.
nucleic acid extraction protocol
This protocol utilizes the Qiagen RNeasy Mini kit and protocol (cat #74104) with a few exceptions. Briefly, one T150 flask of high-density hFLPP was washed 2X in PBS and harvested by the addition of 2mL RLT lysis buffer. Obtained samples were snap-froze in Liquid Nitrogen and stored at -80C until all desired study samples were obtained. Next, the resultant extracts were homogenized using a QIAshredder spin column. To the samples, an equal volume of 70% ethanol was added. The samples were than applied to 4xRNeasy Columns and the standard RNeasy Mini protocol was followed without DNase treatment. For elution, 2x50ul volumes of RNase-free H20 were used. The resultant RNA was stored at -80C until needed. Before use, RNA was quantified by 260/280 absorbance readings and total RNA quality was determined using the RNA Nano LabChip (Agilent Technologies, http://we.home.agilent.com, cat#5065-4476) and the Agilent 2100 bioanalyzer (cat#G2938C) per the manufacturer's protocol (cat#G2941-90126). Satisfactory samples were used in a subsequent array analysis.
10,000-15,000 cells/cm2 are grown in treated T150 culture flasks, in a base media composed of DMEM and MCDB, and supplemented with 10% human serum,albumin (1mg/ml), 2 mercaptoethanol (100nM), and 10mM Nicotinamide. The media is buffered at a pH of 7.40.
Monolayer hFLPP are collected at approximately passage 5 and plated (4x10e6 cells) on 10cm2 ultra-low binding plates (costar) in media composed of DMEM and MCDB, and supplemented with 10% human serum, albumin (1mg/ml), 2-mercaptoethanol (100nM), and 10mM Nicotinamide. The media is buffered at a pH of 7.40. The cells are allowed to differentiate for 48h in suspension clusters before RNA is then extracted.