Takara genomic dna extraction kit

Genomic DNA extraction was performed using the TaKaRa Genomic DNA Extraction Kit (TaKaRa Co., Dalian, China). Genomic DNA (2 ug per sample) was bisulfite modified with the Epitect Bisulfite Kit Protocol (Qiagen), and the modified DNA was amplified using the following primers: BSQ1 forward, 5′-gaaggatttcggttaatttgggg-3′, and reverse, 5′-caaactcgccaaataactacctacg-3′; and BSQ3 forward, 5′-ggttgattatttgaggttaggtgtt-3′, and reverse, 5′-aaaacaattttcaaccaaccatc-3′. The modified DNA was amplified by PCR using 0.2 µM of each primer, 2 units of Hot Start Taq DNA polymerase, and 0.2 mM of each dNTP per reaction. Cycling programs were 95°C for 10 minutes, and then 40 cycles of 95°C for 30 seconds, 54°C for 30 seconds, and 72°C for 30 seconds, followed by a 5-minute incubation at 72°C. The PCR products were examined by gel electrophoresis in 1.5% agarose to confirm that a single band had been obtained and were then sequenced by Invitrogen. Methylation-specific PCR (MS-PCR) was carried out on bisulfate-treated DNA. The primers used were Un-methylated KLF4 forward, 5′-ggttgattatttgaggttaggtgttt-3′, and reverse, 5′-cccaaataacaaaaattacaaacat-3′; and Methylated KLF4 forward, 5′- gttgattatttgaggttaggtgttc-3′, and reverse, 5′-cgaataacgaaaattacaaacgta-3′. Umbilical cord blood DNA was used as a negative control, and it was methylated in vitro by using the Sss1 (CpG) methylase (New England Biolabs).

mRNA expressions of AQP4 and AQP9 in the discrete cortex, and mRNA expressions of intercellular cell adhesion molecule-1 (ICAM-1) and nuclear factor-κB (NF-κB) in the hippocampus were determined by qPCR. Total RNA was extracted from the discrete cortex or hippocampus (n=6 rats per group at each time point) using a Takara Genomic DNA Extraction Kit (TaKaRa Bio, Inc., Shiga, Japan) at 6, 24, and 72 h after reperfusion. Extracted total RNA was then reverse transcribed to generate cDNA. The reverse transcription reaction was then amplified using SYBR Green qPCR system (TaKaRa Bio Inc.). The fold change in relative mRNA expression was determined using the 2^−ΔΔC_t method and β-actin as an internal control [19 (link)]. All the forward and reverse primers used in the study were shown in Table 1.