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Accurate and precise transcriptional profiles from 50 pg of total RNA or 100 flow-sorted primary lymphocytes

ABSTRACT: 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

ORGANISM(S): Mus musculus

PROVIDER: GSE5130 | GEO | 2006/06/23

SECONDARY ACCESSION(S): PRJNA96635

REPOSITORIES: GEO

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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

2008-06-13 | E-GEOD-5130 | biostudies-arrayexpress

Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling

Project description:For more than a decade, microarrays have been a powerful and widely used tool to explore the transcriptome of biological systems. However, the amount of biological material from cell sorting or laser capture microdissection is much too small to perform microarray studies. To address this issue, RNA amplification methods have been developed to generate sufficient targets from picogram amounts of total RNA to perform microarray hybridisation. In this study, four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. The quantitative and qualitative performances of the methods were assessed. Microarrays were hybridised with the amplified targets and the amplification protocols were compared with respect to the quality of expression profiles, reproducibility within a concentration range of input RNA, and sensitivity. Four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. For each protocol, one RNA amplification was performed from 250 pg, and one from 500 pg of human universal RNA by two operators in two independent laboratories and compared to the amplified aRNA obtained from 2 µg and 100 ng RNA inputs following the standard protocol proposed by Affymetrix. A negative control (amplification without total RNA) and a positive control (if available) were included in each experimental batch. Samples indicating 50, 100, and 1000 pg RNA inputs correspond to 3 additional quantities of total RNA used to synthesise the cDNA target using the nugen protocol for comparison (250, 500 pg + 50, 100, 1000 pg).

2009-06-02 | E-GEOD-15398 | biostudies-arrayexpress

gene activation by IFNs in wild type and Pias1 null BMMs

Project description:Microarray analysis was designed to study the effect of PIAS1 on interferon (IFN) signaling pathway by comparing the gene activation profiles of wild type and Pias1 null cells in response to IFN treatments. Microarray analysis was performed essentially following the manufacturer’s instructions (Affymetrix). Briefly, bone marrow-derived macrophages (BMMs) from wile type or Pias1 null littermates were either untreated, or treated with IFN-beta (500 U/ml) or IFN-gamma (10 ng/ml) for 2h or 4h. Total RNA was prepared with RNA-STAT60 (TEL-TEST) and purified with the RNeasy kit (Qiagen). Double stranded complementary DNA was synthesized from 20 ug of total RNA according to Affymetrix methodology, and purified with Phase Lock Gels (Eppendorf). Biotin-labeled RNA was synthesized with the BioArray High Yield RNA Transcript Labeling Kit (Enzo). Samples were cleaned, fragmentated and hybridized to murine genome (MGU74Av2) Genechips (Affymetrix) as instructed. GeneChips were stained with phycoerythrin-streptavidin (Molecular Probes) and scanned with a GeneChip scanner (Affymetrix). Keywords: parallel sample

2004-07-16 | GSE1552 | GEO

Dorsal root ganglion (katz-affy-mouse-210210)

Project description:The molecular identity of touch sensation is still largely unknown. We choose an extrem and very specific example, touch sensation of pennis glan, to study this problem. It's known from previous retro-grade labeling result that only one DRG in rat innervate the pennis glan, which makes it idea for microarry analysis. To clone the mechanosensory receptor specifically or highly expressed in the primary sensory neurons innervating pennis glan. There is a specific mechanosensory receptor in the primary sensory neurons innervating pennis glan. 1. Retrograde labeling the innervating nerve by DiI 2. Dissect DRGs, dissociate neurons and pick up the fluorescent cells. 3. Pool around 20 positive cells and 50 control cells (big diameter neuron and small to medium diameter neuron respectively), purify total RNA. 4. Two rounds of amplification 5. Affymetrix analysis Keywords: dorsal root ganglion, touch sensatiion

2008-04-19 | GSE11207 | GEO

Seed development in LEAFY COTYLEDON1 mutants

Project description:Analysis of gene expression in Ws-0 lec1 (LEAFY COTYLEDON1) mutant Arabidopsis thaliana. Developmental stages studied includes 24-Hr post-fertilization, globular stage, cotyledon stage, mature green stage, post-mature green stage, and seedlings. Microarray Methods: Total RNA was extracted using the Hot Borate Method [Stones et. al. PNAS vol 98 no 20: 11806-11811 (2001)]. Biotinylated cRNA were prepared using the ENZO BioArray High Yield RNA Transcript Labeling Kit (Farmingdale, NY). cRNA was subsequently hybridized to Affymetrix ATH1 Arabidopsis GeneChips. The scanned array images were analyzed using Affymetrix Microarray Suite 4.0 (MAS 4.0) with a global scaling intensity set at 500. Keywords: other

2005-01-01 | GSE1051 | GEO

Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray (22k Linearity)

Project description:E12.5 mouse whole embryo and E12.5 placenta total RNA were pooled to create 25:75, 50:50, and 75:25 ratio mixtures, based on Bioanalyzer quantitation. These samples, along with the original unmixed RNAs, were used as templates for duplicate linear amplification labeling reactions. cRNA target mixtures were hybridized against a Universal Mouse Reference (Stratagene). Pairwise comparison using the NIA Microarray Analysis (ANOVA) software produced log ratios, which were compared to the expected log ratios for genes showing statistically significant (FDR<0.05) differential expression between unmixed embryo and placenta. Keywords: cell type comparison design,development or differentiation design,normalization testing design,reference design

2005-10-28 | GSE3508 | GEO

Pico profiling from 10 cells

Project description:Current expression profiling methods use RNA from hundreds of thousands or thousands cells. Many fields of biology can not use microarrays due to the nature of the biological systems used that are formed by hundreds or dozens of cells. Here we present a method that can handle RNA amount limitation and gives gene expression profiles from as little as 10 cells. We first validate the method hybridizing amplified RNA from MAQC samples A and B. To do that, 25 ng or 100 pg were used and expression profiles obtained as good as when compared to Affymetrix's chemistry for amplification and labeling. The same experiment was done but using sorted cells from two comercial cell lines (SW620 and SW480) obtaining the same differential expression profiling from 2000 cells or 10 cells. The central step of the method is Whole Transcriptome Amplification (WTA) from Sigma that allows the amplification of very small amounts of RNA as starting material. MAQC samples A and B where amplified using Whole Transcriptome Amplification (WTA) kit from Sigma starting from 25 ng (standard conditions) or 100 pg in triplicates. Amplified cDNA was the fragmented and labeled using the 10kv2 SNP chemistry from Affymetrix and hybridized onto Affymetrix GeneST arrays. The same method was used for RNA isolated from 2000 and 10 cells from cell lines SW620 and SW480. RNA was isolated by magnetic bead purification after treating sorted cells with high DTT concentration and 65ºC to inactivate RNAses.

2010-10-01 | E-GEOD-20595 | biostudies-arrayexpress

MAQC-III/SEQC Main Study ABCD titration experiment, ERCC spike-in profiles and gene-level estimates for a selected test set

Project description:Comparison to RNA-Seq showed different strengths and weaknesses for different regimes of expression strength. At sufficient read-depth, both platforms seemed comparable for gene-level expression profiling. Comparison to RNA-Seq showed comparable accuracy and precision with microarrays for ERCC spike-ins at medium to high expression levels. At low expression levels, the array showed signal attenuation but better precision, while RNA-Seq maintained accuracy albeit with highly inflated variance. In summary, both platforms can be meaningfully applied in a similar range of expression strength. Assessment of (precision, accuracy, reproducibility, mutual information, titration order consistency, and known mixing ratio recovery) by measurement of known-ratio mixtures of two RNA reference samples. The array probes 776 complex genes representative of the AceView gene model annotation, as well as 92 ERCC spike-in controls.

2015-04-03 | E-GEOD-48016 | biostudies-arrayexpress

Mus musculus

Project description:Accurate and precise transcriptional profiles from 50 pg of total RNA or 100 flow-sorted primary lymphocytes

| PRJNA96635 | ENA

NT2 DMSO vs. RA

Project description:NT2 cells were treated with DMSO (control) or 10uM Retinoic Acid (RA). Total RNA was isolated using TriReagent, and subsequent cleanup with RNeasy columns (Qiagen) according to the manufacturer's directions. Hybridizations were performed according to Affymetrix guidelines using the Human HG-U133A chip containing 22,500 unique genetic elements. Double-stranded cDNA was synthesized using Superscript II RT (Invitrogen). In vitro transcription labeling was performed with biotin labeled nucleotides using the Bioarray High Yield RNA Labeling Kit (ENZO). 20 ul of labeled RNA per sample was added to the hybridization cocktail, with a total of 15 ug hybridized to the chip (16 hours, 45C). RMA analysis was performed using Gene Traffic software (Iobion), where raw data for each hybridization was normalized, background corrected and filtered for signal intensity of 50 or greater in at least one comparison. Keywords: other

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