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

Abstract

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