FAQ

Introduction

DNA methylation is essential for normal gene expression, X inactivation, genomic imprinting, chromatin modification, and silencing of endogenous retroviruses. Gene-specific DNA methylation analysis, a most common methodology to detect gene methylation, is difficult to find untargeted loci changes and unable to provide whole picture of DNA methylation changes in genome. The measurement of global methylation can lead to identify new disease biomarkers, which would not be accomplished by gene specific methylation techniques.

Several methods have been developed to measure global methylation, but numerous drawbacks exist. In general, those methods are time consuming, costly, imprecise, and less reliable. For example, recently developed bisulfate-conversion-based high throughput methods such as next generation sequencing and microarray share the similar shortcomings in addition to possible bisulfate conversion errors.  In broader areas of basic research and clinical applications, there is an urgent demand for the advancement in technologies for measurement of global methylation to overcome those disadvantages. 

Here we report a novel approach, which directly measures the percentage of global methylation in DNA samples. NanoMeth, a unique kit with qPCR based technology, has been developed to measure global methylation of genomic DNA which conquers the technical limitations stated above.

Check qPCR efficiency if it is first run in your system (optional)

If the efficiency of the PCR is between 90–110%, you do not need do efficiency correction when calculate methylation percentage. The PCR efficiency can be evaluated by performing a dilution series experiment using the 100X PCR control (included). We recommend a 4 to 10 fold dilution series.
To adjust PCR efficiency to 90–110% you can:
Change primer concentration
Adjust extension timeand/or temperature
Adjust annealing time and/or temperature
Several parameters can affect the efficiency of the PCR (ranked from most to least frequent):
·         Your gDNA samples may contain PCR inhibitors
·         Inaccurate sample and reagent pipetting
·         The standard curve may not have been properly analyzed