Quantification of DNA methylation for COMT, RELN and HTR2C in schizophrenia using methylight assay

Epigenetic is an interaction between the environment and gene and has been attributed with pathogenesis of many complex diseases such as Schizophrenia (Sz) and also has been debated for future pharmacotherapeutic approach. The purpose of this study is to optimize the MethyLight assay method for quan...

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Main Authors: Abd. Rahim, Nour El Huda, Rahim, Mohd Nabil Fikri, Ku Zaifah, Norsidah, Talib, Norlelawati A.
Format: Conference or Workshop Item
Language:English
Published: 2015
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Online Access:http://irep.iium.edu.my/44477/1/44477.pdf
http://irep.iium.edu.my/44477/
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Institution: Universiti Islam Antarabangsa Malaysia
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spelling my.iium.irep.444772018-10-31T03:28:38Z http://irep.iium.edu.my/44477/ Quantification of DNA methylation for COMT, RELN and HTR2C in schizophrenia using methylight assay Abd. Rahim, Nour El Huda Rahim, Mohd Nabil Fikri Ku Zaifah, Norsidah Talib, Norlelawati A. QH426 Genetics Epigenetic is an interaction between the environment and gene and has been attributed with pathogenesis of many complex diseases such as Schizophrenia (Sz) and also has been debated for future pharmacotherapeutic approach. The purpose of this study is to optimize the MethyLight assay method for quantification of DNA methylation for Catechol-O-methyltransferase (COMT), Reelin (RELN) and 5-hydroxytryptamine (serotonin) 2C receptor (HTR2C) genes and assessing 2 reference genes, (β-actin) ACTB and ALU for its use. To assess the efficiency of MethyLight assay at different percentage of DNA methylation, DNA of universal human methylated and unmethylated samples were made into serial methylation percentage and subjected to bisulfite treatment. The primers and probes of the genes were designed to cover CpG rich sites while reference genes based on suggested literature. To assess the sensitivity of the assay at different DNA concentration, the universal human methylated DNA was diluted into 1:3 serial dilutions. Both assessments were subjected to real-time PCR assay. Amplification curves for target and reference genes were plotted at acceptable Cq values. However, the assay for HTR2C, ALU and ACTB were unable to differentiate the Cq values based on the percentage of DNA methylation, whilst the amplification assay and Cq values of RELN and COMT were proportionate and able to differentiate the percentage of DNA methylation. Serial dilution of the samples showed an acceptable standard curves with R2 range from 0.80 to 1.00. In conclusion, ALU and ACTB can be used for reference genes as both assays showed no differences of Cq values against various percentage of DNA methylation. However ALU was more preferable as the dilution series showed a better assay efficiency especially at lower concentration of DNA. The assay for COMT and RELN were sensitive and specific for detection and quantification of DNA methylation. 2015 Conference or Workshop Item NonPeerReviewed application/pdf en http://irep.iium.edu.my/44477/1/44477.pdf Abd. Rahim, Nour El Huda and Rahim, Mohd Nabil Fikri and Ku Zaifah, Norsidah and Talib, Norlelawati A. (2015) Quantification of DNA methylation for COMT, RELN and HTR2C in schizophrenia using methylight assay. In: 29th Scientific Meeting of Malaysian Society of Pharmacology and Physiolgy (MSPP), 24-25 August 2015, Setia City Convention Centre, Shah Alam, Malaysia. (Unpublished)
institution Universiti Islam Antarabangsa Malaysia
building IIUM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider International Islamic University Malaysia
content_source IIUM Repository (IREP)
url_provider http://irep.iium.edu.my/
language English
topic QH426 Genetics
spellingShingle QH426 Genetics
Abd. Rahim, Nour El Huda
Rahim, Mohd Nabil Fikri
Ku Zaifah, Norsidah
Talib, Norlelawati A.
Quantification of DNA methylation for COMT, RELN and HTR2C in schizophrenia using methylight assay
description Epigenetic is an interaction between the environment and gene and has been attributed with pathogenesis of many complex diseases such as Schizophrenia (Sz) and also has been debated for future pharmacotherapeutic approach. The purpose of this study is to optimize the MethyLight assay method for quantification of DNA methylation for Catechol-O-methyltransferase (COMT), Reelin (RELN) and 5-hydroxytryptamine (serotonin) 2C receptor (HTR2C) genes and assessing 2 reference genes, (β-actin) ACTB and ALU for its use. To assess the efficiency of MethyLight assay at different percentage of DNA methylation, DNA of universal human methylated and unmethylated samples were made into serial methylation percentage and subjected to bisulfite treatment. The primers and probes of the genes were designed to cover CpG rich sites while reference genes based on suggested literature. To assess the sensitivity of the assay at different DNA concentration, the universal human methylated DNA was diluted into 1:3 serial dilutions. Both assessments were subjected to real-time PCR assay. Amplification curves for target and reference genes were plotted at acceptable Cq values. However, the assay for HTR2C, ALU and ACTB were unable to differentiate the Cq values based on the percentage of DNA methylation, whilst the amplification assay and Cq values of RELN and COMT were proportionate and able to differentiate the percentage of DNA methylation. Serial dilution of the samples showed an acceptable standard curves with R2 range from 0.80 to 1.00. In conclusion, ALU and ACTB can be used for reference genes as both assays showed no differences of Cq values against various percentage of DNA methylation. However ALU was more preferable as the dilution series showed a better assay efficiency especially at lower concentration of DNA. The assay for COMT and RELN were sensitive and specific for detection and quantification of DNA methylation.
format Conference or Workshop Item
author Abd. Rahim, Nour El Huda
Rahim, Mohd Nabil Fikri
Ku Zaifah, Norsidah
Talib, Norlelawati A.
author_facet Abd. Rahim, Nour El Huda
Rahim, Mohd Nabil Fikri
Ku Zaifah, Norsidah
Talib, Norlelawati A.
author_sort Abd. Rahim, Nour El Huda
title Quantification of DNA methylation for COMT, RELN and HTR2C in schizophrenia using methylight assay
title_short Quantification of DNA methylation for COMT, RELN and HTR2C in schizophrenia using methylight assay
title_full Quantification of DNA methylation for COMT, RELN and HTR2C in schizophrenia using methylight assay
title_fullStr Quantification of DNA methylation for COMT, RELN and HTR2C in schizophrenia using methylight assay
title_full_unstemmed Quantification of DNA methylation for COMT, RELN and HTR2C in schizophrenia using methylight assay
title_sort quantification of dna methylation for comt, reln and htr2c in schizophrenia using methylight assay
publishDate 2015
url http://irep.iium.edu.my/44477/1/44477.pdf
http://irep.iium.edu.my/44477/
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