Rna purification kit

5 mL blood was obtained from each participant, and serum was prepared via centrifugation at 2000 × g for 10 min. Total RNA was obtained by using the RNA purification kit (Epicenter, Chicago, IL, USA). 2 μg of RNA from each person was reversely transcribed by using the cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The following primers were used for real-time PCR and synthesized by TaKaRa (Dalian, China), miR-124 (forward primer: 5′-CTAGTCTAGAGTCGCTGTTATCTCATTG. TCTG-3′ and reverse primer: 5′-CGCGGATCCTCT GCTTCTGTCACAGAATC-3′), miR-135 (forward primer: 5′-AGCATAATACAGCAGGCACAGAC-3′ and reverse primer: 5′-AAAGGTTGTTCTCCACTCTCTCAC-3′), and U6 snRNA (5′-CTCGCTTCGGCAGCACA-3′) as an internal control. The qRT-PCR reaction was performed using the following condition: 1 cycle of 95°C for 40 s, followed by 45 cycles of 95°C for 10 s, 60°C for 20 s, and 1 cycle of 60°C for 50 s, and maintained at 4°C. Relative gene levels were normalized to the level of U6 snRNA and calculated by using the 2^−ΔΔCT method.

Total nucleic acids were extracted as previously described (Angel, 2012 (link)). We followed bacterial RNA, which has been proven to be a better indicator of active community variations than DNA (Blazewicz et al., 2013 (link)). The obtained RNA was purified using an RNA purification kit (Epicenter, Madison, WI, USA) according the manufacturer's instructions. The RNA was reverse-transcribed to cDNA using ImProm-II™ Reverse Transcriptase (Promega, Madison, WI, USA) in the presence of Recombinant RNasin Ribonuclease Inhibitor (Promega) following the manufacturer's protocol. The resulting cDNA was purified by PCR purification kit (Bioneer, Daejeon, Republic of Korea) and quantified spectrophotometrically with Nanodrop (Thermo Scientific).