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Measurement of altered A?PP isoform expression in frontal cortex of patients with Alzheimer's disease by absolute quantification real-time PCR.

ABSTRACT: Enzymatic cleavage of amyloid-? protein precursor (A?PP) produces amyloid-? (A?) peptides which form the insoluble cortical plaques characteristic of Alzheimer's disease (AD). A?PP is post-transcriptionally processed into three major isoforms with differential cellular and tissue expression patterns. Changes in A?PP isoform expression may be indicative of disease pathogenesis in AD, but accurately measuring A?PP gene isoforms has been difficult to standardize, reproduce, and interpret. In light of this, we developed a set of isoform specific absolute quantification real time PCR standards that allow for quantification of transcript copy numbers for total A?PP and all three major isoforms (A?PP695, A?PP751, and A?PP770) in addition to glyceraldehyde-3-dehydrogenase (GAPDH) and examined expression patterns in superior frontal gyrus (SFG) and cerebellar samples from patients with (n = 12) and without AD (n = 10). Both total A?PP and A?PP695 transcripts were significantly decreased in SFG of patients with AD compared to control (p = 0.037 and p = 0.034, respectively). A?PP751 and A?PP770 transcripts numbers were not significantly different between AD and control (p > 0.15). There was trend for decreased percentage A?PP695 (p = 0.051) and increased percentage A?PP770 (p = 0.013) expression in SFG of patients with AD. GAPDH transcripts levels were also decreased significantly in the SFG of patients with AD compared to control (p = 0.005). Decreasing total A?PP and A?PP695 copy number was associated with increased plaque burden and decreased cognitive function. In this study we describe a simple procedure for measuring A?PP isoform transcripts by real-time PCR and confirm previous studies showing altered A?PP isoform expression patterns in AD.

SUBMITTER: Tharp WG

PROVIDER: S-EPMC3325610 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Measurement of altered AβPP isoform expression in frontal cortex of patients with Alzheimer's disease by absolute quantification real-time PCR.

Tharp William G WG Lee Yong-Ho YH Greene Shane M SM Vincellete Elise E Beach Thomas G TG Pratley Richard E RE

Journal of Alzheimer's disease : JAD 20120101 2

Enzymatic cleavage of amyloid-β protein precursor (AβPP) produces amyloid-β (Aβ) peptides which form the insoluble cortical plaques characteristic of Alzheimer's disease (AD). AβPP is post-transcriptionally processed into three major isoforms with differential cellular and tissue expression patterns. Changes in AβPP isoform expression may be indicative of disease pathogenesis in AD, but accurately measuring AβPP gene isoforms has been difficult to standardize, reproduce, and interpret. In light ...[more]

PMID: 22258516

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Project description:BACKGROUND: Digital polymerase chain reaction (dPCR) is an increasingly popular technology for detecting and quantifying target nucleic acids. Its advertised strength is high precision absolute quantification without needing reference curves. The standard data analytic approach follows a seemingly straightforward theoretical framework but ignores sources of variation in the data generating process. These stem from both technical and biological factors, where we distinguish features that are 1) hard-wired in the equipment, 2) user-dependent and 3) provided by manufacturers but may be adapted by the user. The impact of the corresponding variance components on the accuracy and precision of target concentration estimators presented in the literature is studied through simulation. RESULTS: We reveal how system-specific technical factors influence accuracy as well as precision of concentration estimates. We find that a well-chosen sample dilution level and modifiable settings such as the fluorescence cut-off for target copy detection have a substantial impact on reliability and can be adapted to the sample analysed in ways that matter. User-dependent technical variation, including pipette inaccuracy and specific sources of sample heterogeneity, leads to a steep increase in uncertainty of estimated concentrations. Users can discover this through replicate experiments and derived variance estimation. Finally, the detection performance can be improved by optimizing the fluorescence intensity cut point as suboptimal thresholds reduce the accuracy of concentration estimates considerably. CONCLUSIONS: Like any other technology, dPCR is subject to variation induced by natural perturbations, systematic settings as well as user-dependent protocols. Corresponding uncertainty may be controlled with an adapted experimental design. Our findings point to modifiable key sources of uncertainty that form an important starting point for the development of guidelines on dPCR design and data analysis with correct precision bounds. Besides clever choices of sample dilution levels, experiment-specific tuning of machine settings can greatly improve results. Well-chosen data-driven fluorescence intensity thresholds in particular result in major improvements in target presence detection. We call on manufacturers to provide sufficiently detailed output data that allows users to maximize the potential of the method in their setting and obtain high precision and accuracy for their experiments.

Dataset Information

Measurement of altered A?PP isoform expression in frontal cortex of patients with Alzheimer's disease by absolute quantification real-time PCR.

Publications

Measurement of altered AβPP isoform expression in frontal cortex of patients with Alzheimer's disease by absolute quantification real-time PCR.

Similar Datasets

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