Total RNA isolation from tissue: Cell culture is treated with 500μl of TriZol (Invitrogen,Carlsbad, CA) in a 1.5ml eppendorf and centrifuged for 20 min (4oC) at 12,000 x g. The supernatant is transferred to a fresh eppendorf and precipitated by the addition of 100ul chloroform and vortexed vigorously for 15 seconds. The sample is incubated for 3 minutes at room temperature and centrifuged for 15 minutes (4oC) at 10,000 x g. The aqueous phase is transferred to a fresh eppendorf and precipitated by the addition of 250μl isopropanol. The precipitate is incubated for 10 minutes at room temperature and centrifuged for 20 minutes (4oC) at 10,000 x g. The sample is washed once with 70% ethanol and allowed the pellet to air dry prior to resuspension in 100μl of DEPC treated water. The RNA is purified by Rneasy Clean up column (Qiagen, Valencia, CA).
Label
biotin
Label protocol
Prior to hybridization, 5.5μg of ssDNA is fragmented by incubation for 1hr at 37oC, follows by 2 min at 93oC. The 45μl mixture is added with 12μl of 5X TdT buffer, 2μl of TdT, 1μl of 5mM DNA labeling reagent, and is incubated at 37oC for 1hr, and follows by 70oC for 10 min.
Hybridization protocol
Hybridization to the Affymetrix GeneChip Gene 1.0 ST Array is performed according to standard Affymetrix protocols. 5.5μg of fragmented and labeled DNA is added to 3.7μl of control oligonucleotide B2 (3nM), 11μl of 20x eukaryotic hybridization controls (bioB, bioC, bioD, and cre), 15.4μl of DMSO, 110μl of 2x hybridization buffer, and H2O to a final volume of 220μl. The hybridization cocktail is heated to 99oC for 5 min. An Affymetrix Gene 1.0 ST array is prepared by wetting for 10 min at 45oC with 1x hybridization buffer. The hybridization cocktail is transferred to a 45oC heat block for 5 min, and is clarified by centrifugation. The buffer solution is removed from the array and the clarified hybridization cocktail is added to the array cartridge.4 The array is hybridized for 16 hours in a 45oC rotisserie oven (60 rpm).
Scan protocol
The hybridized array is washed, labeled with phycoerythrin conjugated streptavidin (Molecular Probes, Eugene, OR), scanned in the Affymetrix scanner signal detected and registered by Expression Console software (Affymetrix).
Description
Mouse ES cells sample1
Data processing
Extracted the expression data, performed normalization and transformation (Threshold all values to 5, Median Shift normalization to the 75 percentile, Baseline transformation using the median of all Samples) and Found Significant Differentially Expressed Genes. Unpaired t-test and Benjamini-Hochberg Correction were performed to produce table consisting of Probe Names, p-values, corrected p-values, Fold change (Absolute) and regulation. Fold change gives the absolute ratio of normalized intensities (no log scale) between the average intensities of the samples grouped.
