Tissue genomic dna extraction kit

By methylation-specific polymerase chain reaction (methylation-specific polymerase chain reaction, MSP) method, collection of esophageal squamous cell carcinoma patients who resection of carcinoma tissue and paired normal tissue adjacent to carcinoma, using tissue genomic DNA extraction kit (QIAGEN GmbH) to extract DNA, the DNA methylation kit (ZYMO RESEARCH) for DNA denaturation and bisulfite conversion. The transformed DNA was used as a template for PCR amplification. PRDM5 methylation-specific primers and non-methylation-specific primers (Servicebio) were designed. The methylation-specific primers were upstream 5-TTGTTTCGGGTTTCGCGTTC-3 and downstream 5′-ATTCCTACTACGAAAACGCG-3′. PRDM5 geneno-methylation-specific primers were upstream 5′-TAGTTTTGTTTTGGGTTTTGT-3′ and downstream 5′-CCATTCCTACTACAAAAA CACA-3′. PCR reaction system: DNA template 1 L, 5 × PCR buffer 10 L, upstream and downstream primers 0.5 L each, with ribozyme free water added up to 20 L. PCR reaction conditions: pre-denaturation at 95 °C for 120 s, denaturation at 95 °C for 60s, annealing at 55 °C for 60s, elongation at 72 °C for 60s, 40 cycles. PCR products were analyzed by the gelatinization imaging system. For example, the target band amplified by gene methylation-specific primers indicated positive methylation of the PRDM5 gene, and the gene methylation rate was calculated.