Ez dna methylation kittm

Bisulfite treatment was performed using the EZ DNA methylation kitTM (Zymo Research). Specific genomic regions were amplified by nested PCR (primers are listed in Table D in S1 File) and the purified product was sequenced with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).

DNA was isolated from 10 tissue sections of 10 μm thickness by proteinase K digestion and a tissue DNA extraction 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, 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).

Bisulfite treatment was performed using 250 ng of genomic DNA with the EZ DNA Methylation KitTM (Zymo Research, Orange, CA, USA) as described elsewhere [16 (link)]. The bisulfite conversion protocol described for the cervical scrapes was adapted to the biopsies with an additional pretreatment incubation for 10 min at 95 °C, and denaturation was performed at 95 °C for 20 min.
After bisulfite treatment, methylation of the promoter regions of the cellular genes CADM1-m18, MAL-m1, and hsa-miR-124-2 was tested using the prototype PreCursor-M kit (Self-Screen BV, Amsterdam, The Netherlands). This is a quantitative multiplex methylation-specific PCR (qMSP), based on the TaqMan technology. Samples were run in single, separate reactions on an ABI 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. In addition, a methylation-independent β-actin was included in the kit as a sample reference to determine the total amount of converted human DNA present in the reaction. To assure sample quality, samples with Cq-values for β-actin >32 were considered of poor DNA quality (invalid) and excluded from the analysis.

Genomic DNA (2μg) was modified by bisulfite treatment (EZ DNA methylation Kit^TM, Zymo Research) and amplified by FastStart^™ DNA polymerase (Roche) using primers listed in S1 File. Amplified products were cloned and single colonies were analyzed for CpG methylation by direct sequencing (ABI 3130XL). For pyrosequencing, PCR products were analyzed using PyroMak Q24 (QIAGEN).