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

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 (deltadelatCt) is described in which the quantity of target normalized to a normalizer gene reference is given by 2-deltadelatCt where deltadelatCt = [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 deltadelatCt 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 deltadelatCt methodology. We find that use of GAPDH is the optimum normalizer gene for the genes analyzed in neurons from human brain and in PBMNCs.

Regulatory expression of components in the BMP pathway in white adipose tissues of cattle

“…The cDNA synthesized from 5 or 10 ng of total RNA was subjected to RT-qPCR analyses as the template. Ct values were determined, and the abundance of gene transcripts was analyzed by the ▵▵Ct method using Hprt1 as the corrected gene (Duran et al, 2005). Gene expression in the roughage diet group (Exp 1) or control group (Exp 2) in each fat depot was set to 100.…”

Gene expression for HIV-associated dementia and HIV encephalitis in microdissected neurons 1: preliminary analysis

“…As previously described, 39 autopsied cryopreserved brain tissue was obtained from the National Institutes of Health-sponsored National NeuroAIDS Tissue Consortium sites 40,41 (Table 1). At each of the National NeuroAIDS Tissue Consortium sites, the diagnosis of HIV-1-positive individuals with and without HAD and HIVE was made based on premortem neurological and clinical neuropsychological examination of the patients and at postmortem by neuropathological examination.…”

“…39 A Leica laser microdissection microscope (Leica Corporation, Bannockburn, IL) was used for laser capture microdissection using standardized settings and the laser beam precisely followed the neuron's outer membrane. Only neurons with nucleoli were microdissected.…”

“…The quality of the cRNA is paramount to ensure nonbiased representation of labeled transcripts containing the complement of the probe sequences deposited on the array. 39 Ten micrograms of purified cRNA were fragmented to uniform size and applied to CodeLink Human Whole Genome Bioarrays (GE Healthcare, manufacturer instructions) in hybridization buffer. The specifications, use, and descriptions of the GenUS BIOSYSTEMS CodeLink human CHIPS were as described previously.…”

“…31 Laser capture microdissection has been used successfully in the study of several human brain diseases, including the HIV-1-infected brain, subacute sclerosing panencephalitis, Parkinson's disease, and Huntington's disease. [32][33][34][35][36][37][38][39] In the current study, only cases with HAD and HIVE (as well as HIV-1-positive controls) were used. Thus, work in NeuroAIDS has progressed to the point where cell-specific studies will be able to elucidate additional information using novel approaches.…”