Amplitaq gold 360 buffer kit

Primers (Invitrogen, USA) were designed to specifically amplify ASTL (NCBI gene accession number NM_001002036) among the 134 zinc metalloproteases in the human genome. N-terminus primers: Fw-5′-GCGCCCCTGGCCTCCAGCTGCGCA-3′ and Rv-5′- CACGACACCACTACCACCCATGGG-3′; C-terminus primers: Fw-5′-GGCTGCAGCCCAAGTGGCCCCAGG-3′ and Rv-5′-AGCAACACCGGGGGCACCTGCTCC-3′; catalytic domain primers: Fw-5′-GAGGTCCCCTT CCTGCTCTCCAGC-3′ and Rv-5′-GGCATGGGACCC TCTCCCACGGGG-3′ yielded amplimers of 237, 309, 579 base pair respectively. For PCR, AmpliTaq gold 360 buffer kit was utilized (# 4398853, Applied Biosystems, USA).

Pyrosequencing was used to determine DNA methylation level at individual cytosines (CpGs) in DNA extracted from paracarcinoma and ccRCC carcinoma samples. EZ DNA Methylation Kit (Zymo Research, D5001) were used to perform bisulfite conversion with five hundred nanograms of DNA. Converted DNA was amplified using the AmpliTaq Gold 360 buffer Kit (Applied Biosystems, 4398853) and then the promoter methylation of LRG1 gene was detected by PCR with primers mapping to the homologous promoter CpG1 and CpG2 regions of LRG1. PCR primers are shown in Table 1. Pyrosequencing was performed using PyroMark Q24Gold reagents (Qiagen, 970802), and data were analyzed by PYROMARK Q24 1.0.10 software (Qiagen). Background nonconversion levels were <3%.