Abstract
BackgroundThe quantification of gene expression is an important tool in the evaluation of the immune response to vaccines. Reliable reference genes for gene expression studies in mouse draining lymph nodes after vaccination have not been reported.ResultsThe utility of seven potential reference genes was investigated using commercially available Taq-man primer/probe mixes. Results were evaluated with RefFinder, a web-based program including multiple algorithm methods such as geNorm, NormFinder, BestKeeper and the comparative delta-Ct. Further assessment was done by applying the candidate reference genes in relative expression calculations with genes related to the magnitude and longevity of the humoral immune responses. The ubiquitin C gene, Ubc, was found to be the most reliable reference gene when validated with well-known genes that are expressed at relatively low levels after vaccination. The optimal time of sample collection varied depending on the function of the target genes.ConclusionsThis study identified Ubc as the most reliable reference gene and provides useful information for studies examining immunological gene expression in the draining lymph nodes after vaccination in mice.
Highlights
The quantification of gene expression is an important tool in the evaluation of the immune response to vaccines
We examined the expression of the aforementioned genes and determined their suitability as reference genes for draining and non-draining lymph nodes in mice after vaccination with a diphtheria– tetanus–acellular pertussis vaccine (DTaP)
RNA quality and RT controls The draining lymph nodes in vaccinated mice were larger than the non-draining lymph nodes and lymph nodes in unvaccinated mice and this was reflected in the RNA concentrations
Summary
The quantification of gene expression is an important tool in the evaluation of the immune response to vaccines. Reliable reference genes for gene expression studies in mouse draining lymph nodes after vaccination have not been reported. Relative quantification of mRNA levels is widely used to determine the expression levels of target genes [1]. The accuracy of this assay relies heavily on the choice of reference genes for normalization [2, 3]. Ideal reference genes must have a relatively high but stable expression with minimal variation between individual samples and without disturbance from the experimental conditions [2, 3]. It is critical to select appropriate reference gene(s) for each circumstance including particular tissues and specific experimental designs [6, 7]
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