Dataset Information

Fluorescence intensity normalisation: correcting for time effects in large-scale flow cytometric analysis.

ABSTRACT: A next step to interpret the findings generated by genome-wide association studies is to associate molecular quantitative traits with disease-associated alleles. To this end, researchers are linking disease risk alleles with gene expression quantitative trait loci (eQTL). However, gene expression at the mRNA level is only an intermediate trait and flow cytometry analysis can provide more downstream and biologically valuable protein level information in multiple cell subsets simultaneously using freshly obtained samples. Because the throughput of flow cytometry is currently limited, experiments may need to span over several weeks or months to obtain a sufficient sample size to demonstrate genetic association. Therefore, normalisation methods are needed to control for technical variability and compare flow cytometry data over an extended period of time. We show how the use of normalising fluorospheres improves the repeatability of a cell surface CD25-APC mean fluorescence intensity phenotype on CD4(+) memory T cells. We investigate two types of normalising beads: broad spectrum and spectrum matched. Lastly, we propose two alternative normalisation procedures that are usable in the absence of normalising beads.

SUBMITTER: Dendrou CA

PROVIDER: S-EPMC2798117 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Fluorescence intensity normalisation: correcting for time effects in large-scale flow cytometric analysis.

Dendrou Calliope A CA Fung Erik E Esposito Laura L Todd John A JA Wicker Linda S LS Plagnol Vincent V

Advances in bioinformatics 20091117

A next step to interpret the findings generated by genome-wide association studies is to associate molecular quantitative traits with disease-associated alleles. To this end, researchers are linking disease risk alleles with gene expression quantitative trait loci (eQTL). However, gene expression at the mRNA level is only an intermediate trait and flow cytometry analysis can provide more downstream and biologically valuable protein level information in multiple cell subsets simultaneously using ...[more]

PMID: 20049162

Similar Datasets

Project description:To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) "Candidatus Accumulibacter phosphatis" (henceforth referred to as "Ca. Accumulibacter"), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different "Ca. Accumulibacter" strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of "Ca. Accumulibacter" 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that "Ca. Accumulibacter" 16S rRNA genes of the EBPR sludge were clearly differentiated into four "Ca. Accumulibacter" clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different "Ca. Accumulibacter" cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 mum, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 mum in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for "Ca. Accumulibacter." The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that "Ca. Accumulibacter" strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.

Dataset Information

Fluorescence intensity normalisation: correcting for time effects in large-scale flow cytometric analysis.

Publications

Fluorescence intensity normalisation: correcting for time effects in large-scale flow cytometric analysis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets