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Reference gene stability in peripheral blood mononuclear cells determined by qPCR and NanoString

Title:
Reference gene stability in peripheral blood mononuclear cells determined by qPCR and NanoString
Abstract:
Quantitative real-time PCR (qPCR) is commonly used for gene expression analyses with defined documentation guidelines to compare published results. To minimize the impact of variances from qPCR performance, sample handling and processing reference genes are used. Their selection process cannot be completely aligned due to variations in experimental conditions. Furthermore, the named sources of error are also present when determining the stability of the reference genes themselves. Even software applications that are used to identify the best reference genes rarely coincide on their rankings and can be misleading under certain conditions. In previous experiments, peripheral blood mononuclear cells (PBMC) were analyzed to identify the most stable reference gene(s). Twelve of the 13 investigated genes showed sample type specific differences in the expression. Direct mRNA measurement was performed in the form of a NanoString analysis, a multiplexed absolute quantification method. The external validation showed a high concordance of the reference gene expression levels. However, it identified the same sample type specific expression pattern for only some of the tested reference genes. By comparing various combinations of reference genes with both methods we are able to suggest a set of well-performing reference genes.
Year:
2014
Publication type:
Journal article
Journal:
Microchimica Acta
ISSN:
0026-3672
Number:
13-14
Volume:
181
Pages:
1733-1742
Language:
English
Document status:
Published
PubListerURL:
https://publister.bib.th-wildau.de/publister/public/publication/619
Keywords:
Reference genes; qPCR; NanoString; PBMC; Validation
Radke, L., Giese, C., Lubitz, A., Hinderlich, S., Sandig, G., Hummel, M., et al. (2014). Reference gene stability in peripheral blood mononuclear cells determined by qPCR and NanoString Microchimica Acta. 181 (13-14), 1733-1742.
@article{619,
    author           = {Radke, Lars and Giese, Christoph and Lubitz, Annika and Hinderlich, Stephan and Sandig, Grit and Hummel, Michael and Frohme, Marcus},
    title            = {Reference gene stability in peripheral blood mononuclear cells determined by qPCR and NanoString},
    journal          = {Microchimica Acta},
    year             = {2014},
    volume           = {181},
    number           = {13-14},
    pages            = {1733-1742},
    doi              = {10.1007/s00604-014-1221-x},
}


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