Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging

Ruben Daum,Eva M Brauchle,Katja Schenke-Layland,Tomasz P Jurkowski,Daniel Alejandro Carvajal Berrio

doi:10.1038/s41598-019-43520-z

Abstract

DNA methylation plays a critical role in the regulation of gene expression. Global DNA methylation changes occur in carcinogenesis as well as early embryonic development. However, the current methods for studying global DNA methylation levels are invasive and require sample preparation. The present study was designed to investigate the potential of Raman microspectroscopy and Raman imaging as non-invasive, marker-independent and non-destructive tools for the detection of DNA methylation in living cells. To investigate global DNA methylation changes, human colon carcinoma HCT116 cells, which were hypomorphic for DNA methyltransferase 1, therefore showing a lower global DNA methylation (DNMT1−/− cells), were compared to HCT116 wildtype cells. As a model system for early embryogenesis, murine embryonic stem cells were adapted to serum-free 2i medium, leading to a significant decrease in DNA methylation. Subsequently, 2i medium -adapted cells were compared to cells cultured in serum-containing medium. Raman microspectroscopy and imaging revealed significant differences between high- and low-methylated cell types. Higher methylated cells demonstrated higher relative intensities of Raman peaks, which can be assigned to the nucleobases and 5-methylcytosine. Principal component analysis detected distinguishable populations of high- and low-methylated samples. Based on the provided data we conclude that Raman microspectroscopy and imaging are suitable tools for the real-time, marker-independent and artefact-free investigation of the DNA methylation states in living cells.

Highlights

DNA methylation are detected[10]
As a model system to investigate global DNA methylation changes in early embryogenesis, murine embryonic stem cells were utilized. mESCs were cultured in serum-containing medium or they were adapted to serum-free 2i medium, leading to a significant decrease in DNA methylation[31]
We demonstrated that Raman microspectroscopy and Raman imaging have the potential to detect DNA methylation states in living cells

Summary

Introduction

DNA methylation are detected[10]. Tracking of these massive changes in global DNA methylation could provide new insights about early embryogenesis. By comparing the sequences of converted and unconverted DNA, it is possible to identify methylated sites All these methods are invasive and potentially create artifacts as they require fixation and staining procedures, cell lysis or DNA isolation. There is no appropriate method established that allows the online monitoring of global DNA methylation changes in living cells. We used Raman microspectroscopy and principle component analysis (PCA) to identify Raman shifts that can indicate global DNA methylation changes in living cells. The aim of the study was to investigate if Raman microspectroscopy and imaging are capable to detect the different global DNA methylation levels in both cell types. IF staining and 5mC ELISA were used to quantify the global DNA methylation in the cells These routine methods served as reference in order to verify the results from the Raman measurements.

Objectives

Methods

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Discussion

Conclusion