IMR Press / FBL / Volume 10 / Issue 3 / DOI: 10.2741/1751

Frontiers in Bioscience-Landmark (FBL) is published by IMR Press from Volume 26 Issue 5 (2021). Previous articles were published by another publisher on a subscription basis, and they are hosted by IMR Press on imrpress.com as a courtesy and upon agreement with Frontiers in Bioscience.

Article

Presenilin-1 detection in brain neurons and FOXp3 in peripheral blood mononuclear cells: normalizer gene selection for real time reverse transcriptase pcr using the ∆∆Ct method

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1 Department of Psychiatry and Behavioral Sciences, University of Miami School of Medicine, Miami, FL 33136, USA
2 Department of Neurology, University of Miami School of Medicine, Miami, FL 33136
3 Department of Pathology, University of Miami School of Medicine, Miami, FL 33136
4 Comprehensive Drug Research Center, University of Miami School of Medicine, Miami, FL 33136
5 Pediatrics McDonald Foundation GeneTeam, University of Miami School of Medicine, Miami, FL 33136
6 Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33136
7 Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA
8 Miami VA Medical Center, Miami, FL 33136
Front. Biosci. (Landmark Ed) 2005, 10(3), 2955–2965; https://doi.org/10.2741/1751
Published: 1 September 2005
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 (∆∆Ct) is described in which the quantity of target normalized to a normalizer gene reference is given by 2-∆∆Ct where ∆∆Ct = [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 ∆∆Ct 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 ∆∆Ct methodology. We find that use of GAPDH is the optimum normalizer gene for the genes analyzed in neurons from human brain and in PBMNCs.

Keywords
Real Time Reverse Transcriptase PCR
Taqman
Presenilin-1 (PS-1)
neurons
PBMNCs
Forkhead-box (FOX) p3
GADPH
normalizer genes
∆∆Ct methodology
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