Over the last two decades, profiling DNA (cytosine) methylation in the human genome is used as a tool to detect, monitor and treat various disorders, as well as to investigate the effects of ageing or lifestyle factors.
To translate DNA methylation variation to DNA sequence changes, researchers have been broadly relying on DNA conversion protocols, namely via bisulfite conversion. During this process, non-methylated cytosines are converted into uracils, while methylated cytosines remain intact. Despite the promise of this approach, it is known that bisulfite conversion may lead to DNA fragmentation and loss, consequently, obstructing downstream analysis. On top, if bisulfite conversion is not complete, it can cause overestimation of DNA methylation levels, hence misinterpretation of the outcome(s).
To assess these variables and provide the community with a solution, we developed qBiCo – a novel, patent-pending quality-control (QC) method to address the qua(nt/l)ity of bisulfite-converted DNA. qBiCo is a 5-plex, TaqMan probe-based, real-time qPCR assay, which simultaneously detects both single- and multi-copy, converted and non-converted, genomic fragments, to give estimates of the sample’s converted DNA concentration, conversion, fragmentation and potential PCR inhibition.
The method has been optimized and fully validated in our laboratory (TRL-4), showcasing that qBiCo is reliable, robust and sensitive down to pg level. Aiming to scale-up and further establish the usability of qBiCo by other laboratories, we have also successfully transferred and validated it in six different qPCR platforms from BioRad, Qiagen and Thermo Fisher Scientific.
Simultaneously, we evaluated the performance of several commercial, broadlyused, bisulfite conversion kits using 5 different input DNA amounts, demonstrating large variability in their performance. Besides bisulfite conversion, our method is applicable to also enzymatic-based conversion protocols.
Our current work includes the conduction of an inter-laboratory collaborative study to achieve testing at the end-user environment (TRL-5). Overall, using this quality control approach we offer the community with a tool to standardize the popular DNA conversion process, in order to prevent inconsistent data and false outcomes, regardless of the downstream analysis.
Speaker: Faidra Karkala, Erasmus MC