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DNA methylation titration data from two samples with known methylation differences

ABSTRACT: 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

ORGANISM(S): Homo sapiens

PROVIDER: GSE23789 | GEO | 2010/12/01

SECONDARY ACCESSION(S): PRJNA130615

REPOSITORIES: GEO

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DNA methylation titration data from two samples with known methylation differences

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 Sample A and Sample B were mixed in five different titration ratios: 100:0, 90:10, 75:25, 50:50 and 0:100

2010-12-01 | E-GEOD-23789 | biostudies-arrayexpress

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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

2006-06-23 | GSE5130 | GEO

2h dose response_AFB treatment

Project description:RNA isolated from the 0, 10, 25, 50 and 100 micromolar AFB1 cultures at 120 min treatment was used for cDNA microarray experiments. For each array hybridization experiment, RNAs from the treated sample and its corresponding time-matched control were co-hybridized to arrays and respectively quantified in different channels. A dye swap strategy was used to eliminate the dye bias. Keywords: dose response

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Transcription profiling of mouse tissue samples to assess an RNA amplification and labelling methodology

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. Experiment Overall Design: Various experimental designs were used as part of the validation process for this total RNA amplification and labeling protocol. 103 samples were analyzed on Affymetrix GeneChips as part of this GEO series. Details of the BIIB protocol and supporting experiments are published in Genomics. 2006 Jul;88(1):111-21

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Titration-based normalization of antibody amount improves consistency of ChIP-seq experiment

Project description:Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is most frequently utilized in research areas of chromatin biology and epigenetics. The ENCODE Consortium provided guidelines about antibody but the fundamental aspect regarding antibody titer was not addressed. Here, we introduced a simple and quick DNA-based quantification of chromatin input, allowing to normalize the antibody amount in individual ChIP reaction to the optimal titer. We further demonstrated the titration-based normalization of antibody amount generates improved assay outcome with high consistency among samples in an experiment or recurrent experiments for a broad range of chromatin input amount. Chromatin immunoprecipitation-sequencing (ChIP-seq) was performed for histone H3K27ac mark in the conditions of various titer of antibody in ChIP reactions..

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