Fluorescence Intensity Normalisation: Correcting for Time Effects in Large-Scale Flow Cytometric Analysis

[摘要] A next step to interpret the findings generated by genome-wide association studies is to associatemolecular quantitative traits with disease-associated alleles. To this end, researchers are linking diseaserisk alleles with gene expression quantitative trait loci (eQTL). However, gene expression at themRNA level is only an intermediate trait and flow cytometry analysis can provide more downstreamand biologically valuable protein level information in multiple cell subsets simultaneously using freshlyobtained samples. Because the throughput of flow cytometry is currently limited, experiments mayneed to span over several weeks or months to obtain a sufficient sample size to demonstrate geneticassociation. Therefore, normalisation methods are needed to control for technical variability and compareflow cytometry data over an extended period of time. We show how the use of normalisingfluorospheres improves the repeatability of a cell surface CD25-APC mean fluorescence intensity phenotypeonCD4+memory T cells. We investigate two types of normalising beads: broad spectrum andspectrum matched. Lastly, we propose two alternative normalisation procedures that are usable in theabsence of normalising beads.

[学科分类] 生物技术