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In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling: Part 3

ABSTRACT: E12.5 mouse whole embryos and placentas were pooled within 3 litters and total RNA was extracted. Fluorescently-labeled, linearly-amplified cRNA targets were prepared from the RNA samples at various input levels. A "full-scale" reference was prepared using the standard input level of 6.0 ug total RNA, and one round of amplification was performed with 250 ng and 50 ng total RNA inputs. For 10 ng and 2 ng inputs, two rounds of amplification were used. Targets were compared on a microarray platform designed for this test using a "dye-swapped" experimental design, and sets of significantly differential genes were compared to the full scale reference's list to determine specificity and sensitivity in terms of detection of significant expression differences. Keywords: dye swap design,quality control testing design

ORGANISM(S): Mus musculus

PROVIDER: GSE3507 | GEO | 2005/10/28

SECONDARY ACCESSION(S): PRJNA93545

REPOSITORIES: GEO

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In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling: Part 3

Project description:E12.5 mouse whole embryos and placentas were pooled within 3 litters and total RNA was extracted. Fluorescently-labeled, linearly-amplified cRNA targets were prepared from the RNA samples at various input levels. A "full-scale" reference was prepared using the standard input level of 6.0 ug total RNA, and one round of amplification was performed with 250 ng and 50 ng total RNA inputs. For 10 ng and 2 ng inputs, two rounds of amplification were used. Targets were compared on a microarray platform designed for this test using a "dye-swapped" experimental design, and sets of significantly differential genes were compared to the full scale reference's list to determine specificity and sensitivity in terms of detection of significant expression differences. Keywords: dye swap design,quality control testing design E12.5 mouse whole embryos and placentas were pooled within 3 litters and total RNA was extracted. Fluorescently-labeled, linearly-amplified cRNA targets were prepared from the RNA samples at various input levels. A "full-scale" reference was prepared using the standard input level of 6.0 ug total RNA, and one round of amplification was performed with 250 ng and 50 ng total RNA inputs. For 10 ng and 2 ng inputs, two rounds of amplification were used. Targets were compared on a microarray platform designed for this test using a "dye-swapped" experimental design, and sets of significantly differential genes were compared to the full scale reference's list to determine specificity and sensitivity in terms of detection of significant expression differences.

2010-06-30 | E-GEOD-3507 | biostudies-arrayexpress

Transcription profiling of human treated cord blood-derived human mast cells control and TGF-beta treated

Project description:Cord blood-derived stem cells were in vitro cultured in the presence of 40 ng/ml SCF, 20 ng/ml IL6 and 2 microM lysophosphatidic acid for 5 week. Then mast cells were magnetically isolated and further cultured for 4 days in the presence or absence of 2 ng/ml TGF-betaI. Total RNA was isolated and processed according to the Agilent Low-input RNA Linear Amplification Kit and microarray hybridization protocol. Experiment Overall Design: 5 biological replicates from 5 donors and one technical replicate (dye-swap)

2008-06-13 | E-GEOD-4906 | biostudies-arrayexpress

In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling: Part1

Project description:E12.5 mouse whole embryos and placentas were pooled within 3 litters and total RNA was extracted. Fluorescently-labeled, linearly-amplified cRNA targets were prepared from the RNA samples and compared on a novel microarray design to validate the system. A "dye-swapped" experimental design was used to assess the accuracy and precision of the system. The in situ-synthesized 60-mer oligonucleotide microarray platform contains approximately 22,000 DNA features, and was designed to detect transcripts from the entire NIA cDNA clone collection. Keywords: dye swap design,quality control testing design

2005-10-28 | GSE3516 | GEO

Highly Parallel Genome-Wide Expression Analysis of Single Mammalian Cells (Optimization)

Project description:Pre-Amplification Assay Optimization for Low RNA Inputs and Single Cells Low Input Total RNA

2012-02-27 | E-GEOD-34364 | 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

The impact of PCR duplication on RNAseq data generated using NovaSeq 6000, NovaSeq X, AVITI and G4 sequencers

Project description:Gene expression profiles generated with RNA sequencing can be biased by RNA amount and methods utilised for cDNA library generation. Polymerase chain reaction (PCR) amplification can generate high PCR duplicate proportions and introduce bias in transcript counts. In this study, we investigate the impact of input amount and PCR cycle number on the PCR duplication rate and on the RNA-seq data quality. We used a range of inputs (1 ng - 1,000 ng) and assessed the PCR duplication rate using unique molecular identifiers (UMIs). For broader applicability, we sequenced the data on four different short-read sequencing platforms: Illumina NovaSeq 6000, Illumina NovaSeq X, Element Biosciences AVITI, and Singular Genomics G4. We highlight the limitations of using input amounts below 125 ng and the advantages for using UMIs for deduplication. We contrast the data obtained from different sequencers and discuss the benefits and drawbacks of using Illumina library conversion kits.

2024-03-13 | GSE261432 | GEO

Comparing control and TGF-beta treated cord blood-derived human mast cells

Project description:Cord blood-derived stem cells were in vitro cultured in the presence of 40 ng/ml SCF, 20 ng/ml IL6 and 2 microM lysophosphatidic acid for 5 week. Then mast cells were magnetically isolated and further cultured for 4 days in the presence or absence of 2 ng/ml TGF-betaI. Total RNA was isolated and processed according to the Agilent Low-input RNA Linear Amplification Kit and microarray hybridization protocol. Keywords: Agilent Whole Human Genome, dual channel, LogRatio values are log(10) values

2006-06-23 | GSE4906 | GEO

RNA sequencing of primary isolated unstimulated murine splenic DC1 and DC2 from specific pathogen free (SPF), Germ free (GF) and Interferon alpha/beta receptor 1 (Ifnar) knock out mice

Project description:The transcriptomes of primary isolated unstimulated murine splenic cDC1 and cDC2 from specific pathogen free (SPF) were compared to cDC1 and cDC2 from Germ Free (GF) and Interferon alpha/beta receptor 1 (Ifnar) knock out mice. Total RNA was prepared from sort-purified cDC1 and cDC2 (for each sample 3 individual mice were pooled) using Trizol reagent (Invitrogen) in combination with the miRNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions. For poly-A-dependent cDNA synthesis and a first amplification step 10 ng of total RNA was used as input in the Smart-Seq v4 mRNA Ultra Low Input RNA Kit (Clontech) and processed according to the manufacturer’s instructions. 1 ng of the purified cDNA was used for tagmentation and library completion with the Nextera XT library preparation kit (Illumina). In the following, 2x75nt paired-end sequencing was performed on a NextSeq 500.

2020-03-30 | E-MTAB-8883 | biostudies-arrayexpress

Mesangial Cell Study

Project description:mesangical cells exposed to glycated albumin with and without aminoguanidine compared in a loop design with dye swaps and one total replication (total of 12 slides) Keywords: loop

2007-05-14 | GSE4839 | GEO

BASIS miRNA data

Project description:399 tumors profiled using Agilent miRNA microarrays (Product Number G4872A, design ID 046064). The arrays are based on miRBase release 19.0 and 2006 human miRNAs are represented. 150 ng total RNA was used as input.

| EGAD00010000917 | EGA

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