Identification and validation of endogenous reference genes for expressionprofiling of T helper cell differentiation by quantitative real-time RT-PCR

Real-time RT-PCR method was exploited to identify endogenous reference gene s in differentiating human T helper cells. When using this technology in ou r experimental system, finding a set of genes whose mRNA expression levels would not change appeared to be very challenging. Our initial plan to use t he expression level of GAPDH in normalizing the results failed, because the mRNA expression of GAPDH underwent significant changes during the cell cul ture. Additional studies on the transcription of several other classical ho usekeeping genes led to similar results. Our second approach was to use res ults from an extensive survey of gene expression done by oligonucleotide mi corarrays and to select another panel of genes for testing. This resulted i n the identification of three genes whose expression was relatively stable in our experimental system and, therefore, suitable as endogenous reference genes in these cells. The results indicate that the expression level of a constitutively expressed gene may change during the cell culture in vitro, which emphasizes again the importance of carefully validating endogenous co ntrol genes for comparative quantification. (C) 2001 Elsevier Science.