This methylation study indicated that cyclin A1 is a potential tumor marker …

• Abstract
• Background
• Methods
• Results
• Discussion
• Conclusion
• References
• Figures
• Tables

Figure 1 Schematic representation of inverse correlation between promoter methylation and expression of CCNA1 in CC cell lines. (A) Diagram of methylated and non-methylated sequences after bisulfite modification covering the area of both primers (underlined) in the promoter region of CCNA1. M, DNA size marker. Top panel, 10-bp ladder; bottom four panels, 100-bp ladder. Neg, negative. (B) Duplex MSP analysis of cell lines. Upper and lower arrows indicate non-methylated and methylated amplicons, respectively. MSP, methylation-specific PCR. (C-E) RT-PCR of the CCNA1 gene after 28, 30 and 35 cycles, respectively. (F) RT-PCR of the GAPDH gene as an internal control.

Figure 2 Intra- and inter-assay variation of the duplex MSP. (A) Duplex MSP of a mixture of CCNA1 complete and non-methylated CC cell lines, SiHa and HeLa(S), respectively. M, DNA size marker; Neg, negative; 0, 25, 50, 75, 100 Met (%) represent the proportion of SiHa DNA in the mixture, varied from 0 to 100%, respectively. The upper and lower bands are non-methylated and methylated bands, respectively, indicated by labeled arrows. (B) Graphical comparison between measured CCNA1 methylation, percentage intensity of methylation amplicon (x-axis), and actual methylation, the proportion of SiHa DNA (y-axis). The bar height indicates the mean and error bars, T, represent standard deviation (SD) across experiments.

Figure 3 CCNA1 methylation and expression in microdissected cervical tissues. (A) Duplex MSP and CCNA1 PCR; E and CNT are epithelium and connective tissue cells from normal cervix; N and T are adjacent histological normal and cancer cervical epithelium from CC, respectively. Arrows indicate non-methylated, methylated, CCNA1 cDNA and GAPDH cDNA, respectively. (B) Bisulfite sequencing at the CCNA1 promoter, with circles denoting the methylation status of each selected clone. Black and white circles are methylated CG dinucleotides, and non-methylated CpG dinucleotides and TG dinucleotides, respectively.

Figure 4 Schematic representation of methylation-specific PCR in CC. (A) PCR analysis of CC: M, DNA size marker; Neg, water; N and T, matched normal cervices and tumors, respectively. (B) Bar graph demonstrating the frequency of DNA methylation. Numbers on the y-axis are the percentage of positive methylation cases. Sample types are on the x-axis. WBC, normal cervix, Low-grade SIL, High-grade SIL, microinvasive cancer and squamous cell CCs number are 43, 25, 13, 11, 5, and 30, respectively. The methylation frequencies of each tissue type are represented by the height of each rectangular bar.