Presenilin-1 detection in brain neurons and FOXp3 in peripheral blood mononuclear cells: Normalizer gene selection for real time reverse transcriptase PCR using the ΔΔCt method

Abstract

Quantification of gene expression is important to confirm changes in levels of gene expression in disease. Prior quantification methods include standard curves, absolute quantification, and relative quantification. This paper describes an analytic method for the relative quantification of Presenilin-1 (PS-1) in neurons and Forkhead-box (FOX) p3 in PBMNCs using real-time PCR analytic techniques. A comparative Ct method (ΔΔCt) is described in which the quantity of target normalized to a normalizer gene reference is given by 2-ΔΔCt where ΔΔCt = [Ct of the gene of interest in the unknown specimen - Ct normalizer gene in the unknown specimen] - [Ct of the gene of interest in the calibrator specimen - Ct normalizer gene in the calibrator specimen]. The calibrator specimen is ideally from a non-treated control specimen and is analyzed on every assay plate with the unknown specimens of interest. The use of the ΔΔCt methodology allows for a higher throughput and a more economical approach to investigate gene expression. We applied this methodology to the quantification of PS-1 and FOXp3 genes and compare the levels of expression by normalizing to different normalizer genes using the ΔΔCt methodology. We find that use of GAPDH is the optimum normalizer gene for the genes analyzed in neurons from human brain and in PBMNCs.