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

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

Methylation profiles of miR34 gene family in Vietnamese patients suffering from breast and lung cancers.

The three genes encoding small non­coding microRNA (miR)34a, MIR34b and MIR34c act as tumor­suppressor genes. Their aberrant expressions regulated by DNA methylation have been frequently found in various types of cancer. In the present study, the DNA promoter methylation profiles of the MIR34 gene family were analyzed using the methylation specific polymerase chain reaction in order to clarify their association with breast and lung cancer, non­cancerous or normal adjacent tissues. The methylation frequency of MIR34a was significantly higher in breast cancer (49.37%) compared with normal adjacent tissues (30.38%). The methylation frequency of MIR34b/c was 59.49 and 62.03% in breast cancer and normal adjacent tissues, respectively. MIR34a methylation showed a significant concordance with that of MIR34b/c only in breast cancer tissue. MIR34a methylation was significantly associated with cancer and the invasive ductal carcinoma type of breast cancer (P=0.015 and P=0.02, respectively). Methylation frequency of MIR34a and MIR34b/c was 48.42 and 56.84% in lung cancer, and 47.22 and 51.39% in pulmonary diseases, respectively. No significant association was observed between the methylation status of MIR34a and MIR34b/c, and the clinicopathological features of lung cancer or with those of non­cancerous pulmonary diseases. Promoter methylation of MIR34a and MIR34b/c occurs frequently and concomitantly in breast and lung cancer, as well as in pulmonary diseases tissues, but not in breast normal tissues adjacent to tumor. These results of the present study emphasize the involvement of MIR34 methylation in human diseases, including cancer. Furthermore, MIR34a methylation may be a promising marker for a subtype of breast cancer.