Journal of Environmental Biology

Performance characteristics of bisulfite conversion and SYBR green based quantitative PCR for DNA methylation analysis

Shikha Tewari¹, Vikram Bhatia², Madhu Mati Goel², Alka Yadu³, Bhaskar Agarwal⁴,
Sandeep Kumar⁵ and Sudhir K.Goel¹*

¹Petroleum Toxicology Division, CSIR-Indian Institute of Toxicology Research, Lucknow-226 001, India

?²Department of Pathology, King George?s Medical University, Lucknow-226 003, India

³Department of Biochemistry, Saraswati Dental College, Lucknow-227 105, India

⁴Department of Prosthodontics, King George?s Medical University, Lucknow-226 003, India

⁵Department of Surgery, King George?s Medical University, Lucknow-226 003, India

*Corresponding Author email : sudhir.ji@gmail.com 

 Publication Data

?Paper received:

?08 May 2012

?Revised received:

?04 October 2012

?Re-revised received:

?15 November 2012

?Accepted:

?24 December 2012

Abstract

Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes play vital role in development of severe disease like cancer. Many techniques used for assessment of DNA methylation, bisulfite treatment followed by methylation specific polymerase reaction (MSP) are one of them, which introduce conversion of unmethylated cytosine into uracil. The significant level of bisulfite treated DNA degradation results in the failure of methylation detection. Therefore, this step is to be properly controlled to avoid the degradation of DNA. In the present study, an attempt has been made to access the incubation time of DNA with bisulfate treatment at three time points i.e. 2.5, 4 and 16 hrs to get complete conversion of cytosine to uracil. Currently, the experiments were undertaken using oral cancer tissue, with varying incubation time of bisulfite treatment and 2 representative genes viz MGMT and p16 were selected for the quantitative assessment of methylation by real time PCR. Both genes are frequently methylated at promoter region in carcinogenesis. The short term incubation for 4hrs indicated better real time threshold value for p16 and MGMT gene methylation (Ct 25.55, 27.25) and unmethylation (Ct 18.82, 25.84) in tissue whereas it was 28.16, 37.35 and 21.98, 26.19 in blood sample, respectively as compared to other incubation time which shows less degradation of full length DNA.

Key words

Epigenetic, DNA methylation, Bisulfite conversion, Methylation specific real time PCR

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