hESO-NT1 was established from human cloned blastocyst generated by SCNT of huam skin fibroblasts (HDF-f) at the Oregon National Primate Reearch Center. RNA were isolated from only nice undifferentiated colonies. hESO-7 was established from human blastocyst generated by regular IVF using oocytes from identical donor for hESO-NT1 at the Oregon National Primate Reearch Center. RNA were isolated from only nice undifferentiated colonies. A primary culture of female fetal skin fibroblasts (HDF-f) was obtained from ScienCell Research Laboratories and were cultured in DMEM/F12 supplemented with 100 IU ml-1 penicillin, 100 μg ml-1 streptomycin (Invitrogen) and 10% FBS. RNA were isolated when cells reached confluency in 75 cm3 cell culture flasks.
Growth protocol
All humanESCs were grown at 37oC, 3% CO2, 5%O2 and 92% N2 upon mitotically inactivated mouse embryonic fibroblast (MEF) feeder cells in DMEM/F12 medium (Invitrogen) with 0.1 mM nonessential amino acids, 1 mM l-glutamine, 0.1 mM β-mercaptoethanol, 5 ng/ml basic fibroblast growth factor, 10µM ROCK inhibitor (Sigma), 10% fetal bovine serum and 10% knockout serum replacement (KSR; Invitrogen). Medium was changed daily and ESC colonies were split every 5-7 days by manual disaggregation and replating collected cells onto dishes with fresh feeder layers.
Extracted molecule
total RNA
Extraction protocol
Trizol (Invitrogen) extraction of total RNA was performed according to the manufacturer's instructions, and total RNA obtained by Trizol extraction was further purified using RNeasy spin columns (Qiagen) following manufacturer instructions.
Label
Biotin
Label protocol
Microarray assays were performed in the the OHSU Gene Profiling Shared Resource. Messenger RNA is amplified and labeled from 200 ng of total RNA using the Affymetrix GeneChip 3’IVT Express Kit. In this protocol mRNA is converted to double-stranded cDNA using an oligo-dT primer linked to a T7 RNA polymerase binding site sequence and then amplified and labeled in an in vitro transcription reaction to produce biotinylated cRNA (target). Following removal of free nucleotides, target yield is measured by UV260 absorbance.
Hybridization protocol
Labeled target is fragmented at 95o C in Fragmentation Buffer (Affymetrix). The GeneChip® Hybridization, Wash, and Stain Kit (Affymetrix) is used to complete the hybridization, wash, and staining processes for GeneChip arrays following the target preparation steps. The fragmented material is combined with biotinylated hybridization control oligomer B2 and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. 8.8 μg of sample target is hybridized with the GeneChip Primeview Human array (Affymetrix) for 18 hours at 45°C, 45 rpm in an Affymetrix Hybridization Oven 645. The arrays are washed and stained on the Fluidics Station 450 (Affymetrix) in a multi-step process: stringent wash, staining with streptavidin-phycoerythrin, signal amplification with biotinylated anti-streptavidin antibody, and a final staining step. The distribution of fluorescent material on the processed array is determined using the GeneChip Scanner 3000 with the 7G upgrade (Affymetrix) and AGCC version 3.2 software (Affymetrix), yielding cell fluorescence intensity (.cel files). Image inspection is performed manually immediately following each scan.
Scan protocol
The GeneChip expression arrays contain control probe sets for both spiked and endogenous RNA transcripts (e.g., BioB, BioC, BioD, CreX and species-specific actin and GAPDH). Following image processing and absolute analysis of the array pattern with AGCC version 3.2 software (Affymetrix), arrays are normalized in Expression Console using default Affymetrix parameters for 3’ IVT Expression Robust Multichip Analyis (RMA). Sixteen values are examined to assess overall assay performance: corner, PM mean, probeset mean (all), Probeset stdev, MAD residual mean (All), MAD residual stdev RLE mean (All), RLE stdev, Actin Ave signal, Actin 3’/5’ ratio, GAPDH Ave signal, GAPDH 3’/5’ ratio, and total Signal for probe sets for BioB, BioC, BioD and CreX. Assays demonstrating poor or marginal performance are flagged.
Description
human skin gender: female,embryo: skin derived fibroblast culture Gene expression profile of HDF-f is parental human skin fibroblast for hESO-NT1 in biological replicate A.
Data processing
The processed image files (.CEL) were normalized across arrays using the robust multichip average (RMA) algorithm and log-transformed (base 2).After normalization, GeneSifter microarray expression analysis software was used to analyze the resultant data.
