Analysis of denominators of molecular biology assays; β-actin and gapdh housekeeping gene expression differs between asthmatics and normals

The rapid development and exploitation of RT-PCR to quantitate in vivo mRNA levels, has led to the common use of, ß-Actin and GAPDH as denominators to compare samples which differ in their cellular and activation profiles. Expression of these genes is assumed to remain constant, allowing their levels to control for variation in processing from sample extraction to signal quantitation. Hypothesis: Samples which differ in their cellular profiles and activation status, such as bronchoalveolar lavage (BAL) and endobronchial biopsies (Bx), have different levels of expression of ß-Actin and GAPDH. Methods: ß-Actin and GAPDH mRNAs were quantitated by comoetitive RT-PCR in a cross-sectional studv of asthmatics and normals (98 BAL, 87 Bx). Results: Both β-Actin and GAPDH mRNA expression differed between asthmatics and normals in BAL and Bx (p<0.01).The results in the graphic were mirrored in BAL GAPDH and BAL β-Actin analyses. Conclusion: β- Actin and GAPDH in BAL and Bx are not suitable denominators of gene expression quantitation between normals and asthmatics with differing steroid treatment.