Comprehensive selection of reference genes for quantitative RT-PCR analysis of murine extramedullary hematopoiesis during development

The purpose of this study was to perform a comprehensive evaluation and selection of reference genes for the study of extramedullary hematopoiesis during development and the early post-natal period. A total of six candidate reference genes (ACTB, GAPDH, HPRT1, PPID, TBP, TUBB3) in four organs (heart, liver, spleen, and thymus) over five perinatal time points (Embryonic days 14.5, 16.5, 18.5, Post-natal days 0, 21) were evaluated by quantitative real-time PCR. The expression stability of the candidate reference genes were analyzed using geNorm, NormFinder, Bestkeeper, Delta CT method, and RefFinder software packages. Detailed methodology for isolation of high quality/purity RNA and analysis is presented. Detailed analysis demonstrated that TBP is the best single reference gene for embryonic samples and HPRT1 is the best single reference gene for post-natal and pooled embryonic and post-natal samples. Organ-level analysis demonstrated that HPRT1 was the most suitable reference gene for heart, liver and thymus samples, while TBP was the best candidate for spleen samples. In general, TUBB3 was consistently the least stable gene for normalization. This is the first study to describe a systematic comprehensive selection of reference genes for murine extramedullary hematopoietic tissues over a developmental time course. We provide suggested reference genes for individual tissues and developmental stages and propose that a combination of reference genes affords flexibility in experimental design and analysis.