DNA methylation titration data from two samples with known methylation differences
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Pan Du
PROVIDER: E-GEOD-23789 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Two samples with known methylation differences. Sample A is NA 10923 from Coriell Institute for Medical Research. It is a B-Lymphocyte sample from a male donor. Sample B is HTB-38 cell line from ATCC (http://www.atcc.org). It is a colon cancer sample from a female donor. Sample A and B were normalized into the same concentration, and then mixed in five different titration ratios: 100:0, 90:10, 75:25, 50:50 and 0:100
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Project description:We have developed a total RNA amplification and labeling strategy for use with Affymetrix GeneChips. Our protocol, which we denote BIIB, employs two rounds of linear T7 amplification followed by Klenow labeling to generate a biotinylated cDNA. In benchmarking studies using a titration of mouse universal total RNA, BIIB outperformed commercially available kits in terms of sensitivity, accuracy, and amplified target length, while providing equivalent results for technical reproducibility. BIIB maintained 50 and 44% present calls from 100 and 50 pg of total RNA, respectively. Inter- and intrasample precision studies indicated that BIIB produces an unbiased and complete expression profile within a range of 5 ng to 50 pg of starting total RNA. From a panel of spiked exogenous transcripts, we established the BIIB linear detection limit to be 20 absolute copies. Additionally, we demonstrate that BIIB is sensitive enough to detect the stochastic events inherent in a highly diluted sample. Using RNA isolated from whole tissues, we further validated BIIB accuracy and precision by comparison of 224 expression ratios generated by quantitative real-time PCR. The utility of our method is ultimately illustrated by the detection of biologically expected trends in a T cell/B cell titration of 100 primary cells flow sorted from a healthy mouse spleen. Keywords: Development and validation of an amplification and labeling protocol for transcriptional profiling on Affymetrix GeneChips, cDNA target on Affymetrix Genechip, small or limiting total RNA samples
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Project description:This data is part of a large-scale platform comparison experiment. Whole mouse and adult mouse colon RNA was mixed at different concentrations (100:0, 75:25, 50:50, 25:75, and 0:100) and distributed across multiple centers for analysis. Eight microarray platforms and RT-PCR were tested for different labeling protocols, amplification protocols, and data preprocessing approaches in order to maximize data intercomparability. The role of universal reference RNA was also examined. Probes of different platforms were matched using gene symbols and/or RefSeq/GenBank accession numbers. Several different normalization procedures were applied. Evaluation criteria included linearity, sensitivity/specificity, and reproducibility within and between platforms, labeling methods, and laboratories. Experiment Overall Design: Whole mouse (sacrificed on day 1, strain: C57BL/6) and adult mouse (8 weeks, strain: C57BL/6 ) colon RNA from multiple animals was pooled respectively. RNA was purified following the manufacturerâs recommendations and integrity was analyzed using an Agilent BioAnalyzer 2100 according to manufacturer instructions. Five RNA samples were created by mixing the two original samples at different concentrations (100:0, 75:25, 50:50, 25:75, and 0:100). The five master mix samples were then divided into duplicate sets of the five sample RNAs, distributed to individual laboratories, and hybridized to eight different microarray platforms at several different genome centers. The five samples included here were prepared from 10ng of total RNA using a T7 double amplification protocol as described in Schwab et al., 2003 and hybridized to Affymetrix MOE430 arrays as recommended by the manufacturer using the standard protocol. No replicates were produced.
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