An Internal Control for Evaluating Bisulfite Conversion in the Analysis of Short Stature Homeobox 2 Methylation in Lung Cancer

Document Type : Research Articles


1 Faculty of Biology, VNU University of Science, Hanoi, Vietnam.

2 Sorbonne Universités, UPMC Univ. Paris 06, École normale supérieure, PSL Research University, CNRS, INSERM, APHP, Laboratoire des Biomolécules (LBM), Paris, France.

3 175 Hospital, Ho Chi Minh City, Vietnam.

4 Department of Pathophysiology, Medical University, Ha Dong, Vietnam.


Objective: The methylation status is considered as powerful diagnostic, prognostic, and predictive biomarkers.
However, the limited DNA amount and conversion efficiency after bisulfite treatment are considerable hindrances in
quantitative methylation analysis. In this study, we designed an artificial internal control (IC) system that contained
the cytosine-free fragment (CFF) following CpG sequences of the SHOX2 promoter whose methylation status has
been described as a valuable biomarker of lung cancer. Its performance in quantifying DNA recovery and bisulfite
conversion efficiency as well as in detecting false-positive SHOX2 methylation was determined on samples from
lung cancer patients. Material and Methods: The IC system is composed of two pConIC and pUnIC plasmids that
both contain a cytosine-free (CF) sequence derived from the CFF and the CpG containing SHOX2 sequences. They
are identical in sequence, except that in the ConIC insert, all cytosines have been converted into thymines. Thus, the
ConIC can be used as calibrator of 100% bisulfite conversion efficiency, while the UnIC is the indicator in order to
evaluate the DNA recovery, bisulfite conversion efficiency of the SHOX2 promoter sequence by quantitative real time
PCR. Results: The copy number of the target sequences impacted on both DNA recovery rates and bisulfite conversion
efficiency. An amount of 0.005 ng pUnIC (106 copies) showed recovery rate of 18%, similar to that of pConIC, and a
bisulfite conversion efficiency of the SHOX2 reaching 98.7%. On the contrary, higher copy number of pUnIC showed
incomplete conversion (<85%) and over recovery (~42%). Using this calibrator/indicator couple, we were able to detect
false-positive SHOX2 methylation (3.77% instead of 0.03%) due to incomplete bisulfite conversion.Conclusion: Our
results proposed a customizable internal control using the ConIC/UnIC as calibrator/indicator to quantify simultaneously
and accurately the DNA recovery and bisulfite conversion efficiencies of individual sequence as well as whole genome
in methylation assays, thus promoting the validation of standardized clinical DNA methylation biomarker values to
progress toward clinical applications.


Main Subjects