Maxime pcr premix kit

Mouse liver RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was reverse transcribed with the Maxime PCR premix Kit (iNtRON Biotechnology, Seongnam, Korea). Quantitative RT-PCR using SYBR Green 2× master mix kit (m.biotech., Inc., Seoul, Korea) was run in hexaplicate on a CFX96 Touch real-Time PCR Detection System (Bio-Rad, Hercrules, CA, USA). The 18s was used for the relative quantization of the target genes based on the comparative ∆∆ threshold cycle (Ct) method, as previously described [26 (link)]. Primer sequences are shown in Table 2.

Bacteria colony PCR amplification was performed using a Maxime PCR PreMix kit (iNtRON Biotechnology, Korea). The PCR mixture contained 1.0 μl of each of the 27F and 1492R primers [28 (link)], 1.0 μl diluted bacterial isolates, and 22.0 μl distilled water. Thermal cycling consisted of 95°C for 10 min, and 35 cycles of 95°C for 40 sec, 55°C for 40 sec, 72°C for 60 sec, and then incubation at 72°C for 5 min. The PCR products were electrophoresed through a 1% agarose gel and purified using Expin^TM PCR Purification Kits (GeneAll Biotechnology, Korea) following the manufacturer’s instructions. Purified PCR products were sequenced using an ABI3700 automated DNA sequencer at Macrogen (Seoul, Korea). The sequences were aligned using MAFFT [29 (link)] and edited manually with MEGA v.5.0 [30 (link)]. Phylogenetic trees were constructed using the neighbor-joining method with 1000 bootstrap replications. Reference sequences were retrieved from the EzBioCloud database [31 (link)]. Based on species identification, the number of species were compared between each part using Kruskal-Wallis rank sum test and Dunn’s test as a post hoc test adjusted by the false discovery rate of Benjamini and Hochberg [32 ]. Sequences generated from the study were deposited in Genbank under accession numbers KY992547-KY992562.

Extraction of total cellular RNA was performed using an RNeasy mini kit (Cat. no. 74106, Qiagen, CA). Reverse transcription of total RNA (1 µg) was performed using SuperScript II reverse transcriptase (Cat. no. 18064, Invitrogen) and an oligo (dT) primer (Cat. no. 18418012, Invitrogen). One-tenth of the cDNA from the first-strand reaction was amplified using a Maxime PCR premix kit (Cat. no. 25167, iNtRON Biotechnology). The PCR primers used for detection of mRNA levels are shown in Supplementary Table S2.

Total RNA was extracted from MCF-7 and TAMR-MCF-7 cells using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol. 1 μg total RNA was reverse-transcribed using Maxime RT-PreMix Kit (Intron biotechnology, Gyeonggi-do, Korea) to synthesize cDNA, and the obtained cDNA was amplified by PCR using a Maxime PCR PreMix Kit (Intron biotechnology, Gyeonggi-do, Korea) and electrophoresed on 2% agarose gel. Detailed primer sequences used in the experiments are provided as Supplementary Information (Supplementary Table S1). Quantitative polymerase chain reaction was carried out using SYBR green (biorad, San Francisco, CA) incorporation with gene-specific primers. The forward primer for P2X7 was: 5′- TATGAGACGAACAAAGTCACTCG-3′ and the reverse one was: 5′- GCAAAGCAAACGTAGGAAAAGAT-3′. The primers for 18 S were: forward: 5′- CCATCCAATCGGTAGTAGCG-3′; reverse: 5′- GTAACCCGTTGAACCCCATT-3′. Relative gene expression was calculated by ΔΔCt analysis relative to 18 S.