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IDLN-MSP: Idiolocal normalization of real-time methylation-specific PCR for genetic imbalanced DNA specimens


Santourlidis, Simeon; Ghanjati, Foued; Beermann, Agnes; Hermanns, Thomas; Poyet, Cédric (2016). IDLN-MSP: Idiolocal normalization of real-time methylation-specific PCR for genetic imbalanced DNA specimens. BioTechniques, 60(2):84-87.

Abstract

Sensitive, accurate, and reliable measurements of tumor cell-specific DNA methylation changes are of fundamental importance in cancer diagnosis, prognosis, and monitoring. Real-time methylation-specific PCR (MSP) using intercalating dyes is an established method of choice for this purpose. Here we present a simple but crucial adaptation of this widely applied method that overcomes a major obstacle: genetic abnormalities in the DNA samples, such as aneuploidy or copy number variations, that could result in inaccurate results due to improper normalization if the copy numbers of the target and reference sequences are not the same. In our idiolocal normalization (IDLN) method, the locus for the normalizing, methylation-independent reference amplification is chosen close to the locus of the methylation-dependent target amplification. This ensures that the copy numbers of both the target and reference sequences will be identical in most cases if they are close enough to each other, resulting in accurate normalization and reliable comparative measurements of DNA methylation in clinical samples when using real-time MSP.

Abstract

Sensitive, accurate, and reliable measurements of tumor cell-specific DNA methylation changes are of fundamental importance in cancer diagnosis, prognosis, and monitoring. Real-time methylation-specific PCR (MSP) using intercalating dyes is an established method of choice for this purpose. Here we present a simple but crucial adaptation of this widely applied method that overcomes a major obstacle: genetic abnormalities in the DNA samples, such as aneuploidy or copy number variations, that could result in inaccurate results due to improper normalization if the copy numbers of the target and reference sequences are not the same. In our idiolocal normalization (IDLN) method, the locus for the normalizing, methylation-independent reference amplification is chosen close to the locus of the methylation-dependent target amplification. This ensures that the copy numbers of both the target and reference sequences will be identical in most cases if they are close enough to each other, resulting in accurate normalization and reliable comparative measurements of DNA methylation in clinical samples when using real-time MSP.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Urological Clinic
Dewey Decimal Classification:610 Medicine & health
Date:2016
Deposited On:15 Apr 2016 17:46
Last Modified:08 Dec 2017 19:22
Publisher:Informa Healthcare
ISSN:0736-6205
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.2144/000114379
Official URL:http://www.biotechniques.com/BiotechniquesJournal/2016/February/IDLN-MSP-Idiolocal-normalization-of-real-time-methylation-specific-PCR-for-genetic-imbalanced-DNA-specimens/biotechniques-362951.html?autnID=342202
PubMed ID:26842353

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