Anion-exchange high-performance liquid chromatography (HPLC) analysis

We have developed a novel anion-exchange column for HPLC with electrostatic and hydrophobic properties. Both cytosine and thymine, corresponding to methylated and unmethylated cytosine after bisulfite modification, respectively, are captured by electrostatic interaction and then discriminated from each other by their hydrophobic interactions in our column [23]. As described previously, our column packing material was prepared using a three-step procedure [23]. Briefly, a reactor equipped with a stirrer was filled with 2000 mL of a 3% w/w aqueous solution of polyvinyl alcohol, and a mixture of 200 g tetraethylene glycol dimethacrylate, 100 g triethylene glycol dimethacrylate, 100 g glycidyl methacrylate and 1 g benzoyl peroxide was added to the solution. The contents were heated to 80 °C with stirring and polymerized at that temperature for 1 h in a nitrogen atmosphere. In this initial step, polymer particles with hydrophobic properties were formed.

Subsequently, a monomer solution of 100 g ethyl methacrylate trimethyl ammonium chloride dissolved in 100 mL ion-exchange water was added to the reactor, and the contents were polymerized at 80 °C for 2 h in a nitrogen atmosphere to prepare a polymerization composite. This composite was rinsed with ion-exchange water and then with acetone. In this second step, polymer chains with quaternary ammonium groups were introduced onto the surface of the polymer particles.

Finally, 10 g of polymer particles were dispersed in 100 mL ion-exchange water to prepare an unreacted slurry. Next, 10 mL N,N-dimethylamino propyl amine was added to the slurry with stirring, and the contents were allowed to react at 70 °C for 4 h. After completion of the reaction, the supernatant was removed using a centrifugal separator, Himac CR20G (Hitachi Koki Co., Ltd., Tokyo, Japan), and the remaining contents were rinsed with ion-exchange water. After rinsing, any additional supernatant was removed using a centrifugal separator. Rinsing with ion-exchange water was then performed an additional 4 times. In this final step, tertiary amino groups were introduced onto the surface of the polymer particles.

Bisulfite-converted genomic DNA in each sample and fully unmethylated and fully methylated control DNA (Qiagen) were amplified by PCR encompassing the candidate marker CpG sites using primers described in Additional file 1: Table S2. The PCR products were then subjected to HPLC analysis on an LC-20A system (Shimadzu Corporation, Kyoto, Japan) equipped with a stainless steel column (150 × 4.6 mm I.D.) filled with our original packing material. Eluent buffer A was 25 mmol/L MES-NaOH (pH 6.0), and Eluent buffer B was the same buffer containing 2 mol/L guanidine sulfate. The PCR products were separated on a gradient of 30–50% buffer B for 10 min at a flow rate of 1.0 mL/min. The separated PCR products were detected at 260 nm. HPLC analysis was performed at a column temperature of 70 °C.