Pyromark vacuum prep workstation

Methylation status of IL6 gene promoter was quantitatively confirmed by pyrosequencing (PS) method. Briefly, bisulfite-modified DNA was amplified using forward primer (5′-AGGGATAATTTAGTTTAGAGTTTATTTGT-3′) and reverse primer (biotin-5′-CTCCCTCTCCCTATAAATCTTAATT-3′) through PCR, enabling the conversion of the PCR product to a single-stranded DNA template suitable for PS. All samples were heated to 95°C for 5 min and then amplified for 45 cycles of 95°C 45 s, 54°C 45 s, and 72°C 45 s, followed by a final extension at 72°C for 5 min. The quality and lack of contamination of the PCR products were checked on 2% agarose gels with ethidium bromide staining. After purification of PCR product using Sepharose beads on PyroMark Vacuum Prep Workstation (Qiagen), PS was performed according to the manufacturer’s specifications with sequencing primer (5′-ATAAGAAATTTTTGGGTGT-3′) using the PyroMark Q96MD System (Qiagen). A mean methylation index (MI) was calculated from the mean of the methylation percentage for all observed CpG sites. To set the controls for PS, we used universal methylated and unmethylated DNA that were consistently positive or negative with stable levels of methylation.

Alu methylation analysis was quantitatively performed on the bisulfite-treated DNA using pyrosequencing with the polymerase chain reaction (PCR) primers and conditions previously described (Bollati et al., 2007 (link)). In brief, the bisulfite-treated samples (50 ng) were amplified with a biotin-labeled primer via PCR, which enables the conversion of the PCR product to a single-stranded DNA template suitable for pyrosequencing. Confirmation of the quality of the PCR products and their freedom from contamination was established on 2% agarose gels with ethidium bromide staining. After purification of PCR products using Sepharose beads on PyroMark Vacuum Prep Workstation (Qiagen), pyrosequencing was carried out using the PyroMark Q96MD System (Qiagen) according to manufacturer’s instructions, including a single-strand binding protein. The percentage of methylatation was expressed for each DNA locus as % 5-mC divided by the sum of methylated and unmethylated cytosines. We tested each marker 3 times and used the average percentage in the statistical analysis. Three controls were included in every pyrosequencing run. One well was filled with water to ensure no contamination, and 2 wells were filled with CpGenome Universal methylated and unmethylated DNA (Chemicon, USA) to evaluate the repeatability of the assay.

The mutation analysis technique was described in a previous study.¹⁹ (link) Genomic DNA from 5 micrometers of formalin-fixed paraffin-embedded (FFPE) DLBCL sample was extracted with the QIAamp DNA FFPE Kit (QIAGEN GmbH, Germany) following the manufacturer specifications. The minimum necessary DNA concentration needed for the polymerase chain reaction (PCR) assay was 10 ng/mL.²⁰ (link) Isolated DNA was subjected to PCR amplification in a final volume of 25 mcL, containing 1 U of Platinum Taq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 10 pMol of primers flanking the codon 265 mutation region, 200 mM dNTPs, MgCl2 1.5 mM, 1× of the Taq DNA polymerase buffer, and 10–50 ng of genomic DNA. The primers were as follows: Forward 5′-AGACTGGGCTTGTCCCAC-3′ and Reverse bio-5′-AGATTTGGTGCAGGGGTTG-3′, generating a 175 bp amplicon. The reaction consisted of 45 °C of an initial denaturation step of 15 min at 95 °C, followed by 45 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s and a final extension step at 72 °C for 10 min. The PCR amplicons were confirmed after electrophoresis on agarose gel. Ten microliters of the PCR product were then sequenced using primer 5′-AGACTGGGCTTGTCCCAC-3′ and pyrosequencing was performed using the PyroMark Vacuum Prep Workstation and the PyroMark Q24 (Qiagen) according to the manufacturer's instructions.