Hemical assay on the same four prostate cancer samples. As expected, methylation scores were identical to that derived via spectrometry (Fig. 7b and Additional fileFig. 5 Performance of gene-specific assay for 100, 50, 25, 5, and 0 methylated DNA samples. a Gel electrophoresis images of RPA amplifications and photo of HRP/TMB reactions. Top: I-CBP112MedChemExpress I-CBP112 post-MBD enrichment. Bottom: input controls i.e., before MBD enrichment. b Corresponding absorbance measurements. Blue: post-MBD enrichment. Red: input control. c Calibration plot of HRP/TMB response to methylation changes. Error bars represent SD, n =Wee et al. Clinical Epigenetics (2015) 7:Page 6 ofFig. 6 GSTP1 methylation in samples. Detecting GSTP1 methylation in cell lines (a ), cells before and after 5-Aza treatment (d ) and in prostate cancer patients (g ). a, d, g Gel electrophoresis images of RPA amplifications and photos of HRP/TMB reactions. b, e, h Corresponding absorbance measurements. Blue: post-MBD enrichment. Red: input control. c, f, i Normalized HRP/TMB response (score) to methylation changes. Error bars PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28250575 represent SD, n =Fig. 7 Electrochemical detection of GSTP1 methylation in urine DNA. a Conceptual schematic of the electrochemical detection of HRP oxidized TMB (TMBox). Photo of disposable SPCE is shown. On the electrode surface, TMBox is reduced back to TMBred. The resulting current is proportional to the amount of TMBox. b Current measurements of the four patient samples. Blue: post-MBD enrichment. Red: input control. c Comparison of methylation scores for patient samples determined electrochemically (blue) and by absorbance (red). Error bars represent SD, n =Wee et al. Clinical Epigenetics (2015) 7:Page 7 of1: Figure S6). This not only demonstrated the feasibility of an electrochemical MBD-based assay for detecting DNA methylation in urine (and possibly other bodily fluids), it also demonstrated that our methylation assay was readout agnostic and may be adapted to other readout methods for added convenience as a diagnostic assay. To the best of your knowledge, this is also the first electrochemical assay for methylation detection via MBD.Conclusions In conclusion, we have developed simple and rapid approaches to detect total genomic DNA methylation and at specific gene sequences. The assays were first developed on a colorimetric readout for naked-eye evaluation and subsequently onto electrochemical approach for convenient diagnostic and research applications. We also described a novel MBD protocol with improved stringency by exploiting the competitive binding of nonhuman carrier DNA to sequester excess MBD. The high sensitivity, ease and low sample requirements of our approach may be useful for routine diagnostics and a wide range of applications unlike traditional methods. Availability of supporting data The data sets supporting the results of this article are included within the article and its additional files. MethodsDNA preparationTo demonstrate the feasibility of the assay, a 119-bp DNA fragment model system over the GSPTP1 gene was PCR amplified using standard procedures using the Kapa2G Robust PCR kit (KapaBiosystems, USA) with forward and reverse primers (AACCCCCTTATCCCTCC GTCGTGTGGCTTTTAC and AAACAGGTTCCTCCG AAGATTTCACACAACACT, respectively (Integrated DNA Technologies, Australia). Amplicons were then purified using QIAquick Gel extraction kit (Qiagen, Australia). To generate methylated sequences, amplicons were treated with SssI methyltransferase (New England Biola.
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