The Challenge of Stability in High-Throughput Gene Expression Analysis: Comprehensive Selection and Evaluation of Reference Genes for BALB/c Mice Spleen Samples in the Leishmania infantum Infection Model.. Mus musculus

The interaction of Leishmania with BALB/c mice induces dramatic changes in transcriptome patterns in the parasite, but also in the target organs (spleen, liver…) due to its response against infection. Real-time quantitative PCR (qPCR) is an interesting approach to analyze these changes and understand the immunological pathways that lead to protection or progression of disease. However, qPCR results need to be normalized against one or more reference genes (RG) to correct for non-specific experimental variation. The development of technical platforms for high-throughput qPCR analysis, and powerful software for analysis of qPCR data, have acknowledged the problem that some reference genes widely used due to their known or suspected "housekeeping" roles, should be avoided due to high expression variability across different tissues or experimental conditions. In this paper we evaluated the stability of 112 genes using three different algorithms: geNorm, NormFinder and RefFinder in spleen samples from BALB/c mice under different experimental conditions (control and Leishmania infantum-infected mice). Despite minor discrepancies in the stability ranking shown by the three methods, most genes show very similar performance as RG (either good or poor) across this massive data set. Our results show that some of the genes traditionally used as RG in this model (i.e. B2m, Polr2a and Tbp) are clearly outperformed by others. In particular, the combination of Il2rg + Itgb2 was identified among the best scoring candidate RG for every group of mice and every algorithm used in this experimental model. Finally, we have demonstrated that using "traditional" vs rationally-selected RG for normalization of gene expression data may lead to loss of statistical significance of gene expression changes when using large-scale platforms, and therefore misinterpretation of results. Taken together, our results highlight the need for a comprehensive, high-throughput search for the most stable reference genes in each particular experimental model Overall design: 47 BALB/c mice (14-15 weeks old) were used in this study. Mice were randomly separated in two groups: (i) 23 control mice and (ii) 24 mice that were infected with 10^6 stationary-phase L. infantum promastigotes via tail vein. Mice were euthanized by cervical dislocation and spleens were removed and immediately stored in RNAlater at -70C. After RNA extraction and reverse transcription, Real-time PCR was performed using QuantStudio 12K Flex Real-Time PCR System following manufacturer’s instructions, using Custom TaqMan OpenArray Real-Time PCR Plates. Ct values obtained from RT-qPCR were analized by three different algorithms (geNorm, NormFinder and RefFinder) in order to evaluate the stability of the 112 genes and identify the most suitable for normalization of gene expression. Please note that the three algorithms were used for the identification of the best Reference genes in all samples. Once identified, those RG were used to normalize gene expression using geNorm only. The sample data table includes the normalized data using Il2rg+Itgb2 as reference genes, as identified and validated in the associated publication. The 'geNorm_Polr2a_Tbp_normalized.txt' includes the data normalized using Polr2a+Tbp as reference genes, two reference genes traditionally used in the literature for this model.