8%) as stage III/IV. Thirty-three of the patients presented with lymph node metastases. This study was approved by the Research Ethics Committee of Shihezi University School of Medicine, P. R. China. Written informed consent was obtained from all of the patients. All specimens were handled and made anonymous according to the ethical and legal standards. DNA isolation and bisulfate conversion DNA was isolated
from 10 tissue sections of 10 μm thickness by proteinase K digestion and a tissue DNA extraction check details kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s protocol. As an internal control, all purified genomic DNA samples were successfully tested by polymerase chain reaction (PCR) with human β-actin primers For: 5′-CAGACACCATGGTGCACCTGAC-3′ and Rev: 5′-CCAATAGGCAGAGAGAGTCAGTG-3′, indicating that the suitable quality and quantity of DNA can be used to detect the profile of miR-34a methylation. Genomic DNA was stored at −20°C until use as a template for each PCR reaction. The genomic DNA was treated with bisulfite through an EZ DNA Methylation KitTM according to the manufacturer’s instructions (Zymo Research, Orange, ASP2215 supplier CA, USA) (Catalog No. D5001). This treatment combines bisulfate conversion and DNA clean-up. The converted DNA was measured by an ND-1000 spectrophotometer (NanoDrop Technologies,
Inc., Wilmington, DE, USA). Quantitative analysis of DNA methylation The sequence of the CpG island was identified by the UCSC genome browser (http://genome.ucsc.edu/).
Given that the genomic region upstream the p53 binding site in the miR-34a gene revealed a prominent CpG island, we selected Lck the area with proximal promoter activity in previous experiments [22]. The analyzed region and the CpG sites of the miR-34a promoter are shown in Figure 1. We designed primer sets for the methylation analysis of the miR-34a promoter region by EpiDesigner software (http://epidesigner.com; Table 1). For each reverse primer, an additional T7 promoter tag was added for in vivo transcription, and a 10-mer tag was added to the forward primer to adjust for differences in melting temperature. The DNA methylation of miR-34a was quantitatively analyzed by the MassARRAY platform (SEQUENOM) as previously described [25]. The 5 μl PCR mixture contained 10 ng of bisulfite-treated DNA, 25 mM dNTP, 0.2 U of Hot Start TaqDNA polymerase (Sequenom, Selleckchem LY333531 Sequenom Inc., San Diego, CA, USA), and a 1 μM mixture of forward and reverse primers. The cycles included pre-heating at 94°C for 4 min, followed by incubation for 45 cycles of 94°C for 20 s, 62°C for 30 s, and 72°C for 60 s and then by incubation at 72°C for 3 min. Two microliters of a shrimp alkaline phosphatase (SAP) mix containing 1.7 μl of H2O and 0.3 μl (1.7 U) of SAP (Sequenom) was added to digest redundant dNTPs with the following program: 37°C for 20 min, 85°C for 5 min, and 4°C thereafter.