Background: Real-time polymerase chain reaction (PCR) has become an increasingly important technique for gene expression profiling because it can provide insights into complex biological and pathological processes and be used to predict disease or treatment outcomes. Although normalized data are necessary for an accurate estimation of mRNA expression levels, several pieces of evidence suggest that the expression of so-called housekeeping genes is not stable. This study aimed to validate reference genes for the normalization of real-time PCR in an N-methyl-N-nitrosourea (MNU)-induced T-cell lymphoma mouse model.
Methods: T-cell lymphomas were generated in p53-deficient mice by treatment with 37.5 mg/kg MNU. Thymus and spleen were identified as the primary target organs with the highest incidences of lymphomas. We analyzed the RNA expression levels of eight potential endogenous reference genes (Gapdh, Rn18s, Actb, Hprt, B2M, Rplp0, Gusb, Ctbp1). The expression stabilities of these reference genes were tested at different time points after MNU treatment using geNorm and NormFinder algorithms.
Results: A total of 65% of MNU-treated mice developed T-cell lymphomas, with the spleen and thymus as the major target organs. All candidate reference genes were amplified efficiently by quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Gene stability evaluation after MNU treatment and during lymphomagenesis revealed that Ctbp1 and Rplp0 were the most stably expressed genes in the thymus and spleen, respectively. RT-PCR of thymus RNA using two additional sets of primer confirmed that Ctbp1 was the most stable of all the candidate reference genes.
Conclusions: We provided suitable endogenous controls for gene expression studies in the T-cell lymphoma model.
Keywords: Lymphoma model; Quantitative real-time PCR; Reference genes; p53-deficient mouse.