Cdna kits

Analyzed RNA expression was using qPCR assay as described previously [18 (link)]. Briefly, RAW 264.7 cells (1.5 × 10⁵ cells/well) were cultured in 6-well plates. 0, 10, 20, 40, and 80 μM EK100 were treated before LPS treatment. TRIzol reagent with SuperScript™ II Reverse Transcriptase and RNaseOUT™ Recombinant RNase Inhibitor extracted RNA. The RNA which was being collected was transformed into cDNA by using cDNA kits (Roche, Mannheim, Germany). The following PCR primer sequences were used: IL-6_ forward (F): 5′-CCGGAGAGGAGACTTCACAG-3′, and IL-6_reverse (R): 5′-TCCACGATTTCCCAGAGAAC-3′ (Sequence ID: NM_013693.3); TNF-α_F: 5′-TCAGCCTCTTCTCATTCCTG-3′, and TNF-α_R: 5′-TGAAGAGAACCTGGGAGTAG-3′ (Sequence ID: NM_013693.3); GAPDH_F: 5′-GGCCTTCCGTGTTCCTACC-3′, GAPDH_R: 5′-TGCCTGCTTCACCACCTTC-3′ (Sequence ID: BC023196.2). The thermal cycler parameters were followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. StepOne Plus Real-Time PCR Systems (Applied Biosystems, Carlsbad, CA, USA) were applied to PCR reactions, which also operated using SYBR green working solution. The following steps about thermal cycler parameters were used 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, and 60 °C for 30 s.

Total RNA was extracted from the whole blood of 30 healthy participants, 30 MAP patients, and 30 SAP patients according to standard procedures. Total RNA was reverse-transcribed into cDNA kits (Roche, Penzberg, Germany) using random primers and Transcriptor First Strand cDNA Synthesis according to the manufacturer's instructions. 6 differentially expressed circRNAs were measured by qPCR using a ViiA 7 Real-time PCR System (Applied Biosystems). The reaction conditions were as follows: 95°C for 10 minutes and 40 cycles of 95°C for 10 seconds, 60°C for 60 seconds. RNA levels were normalized to human β-actin. All qPCR reactions were performed in triplicate. Different primers were designed for circRNAs, rather than the more commonly used convergent primers (Figure 2). Table 2 lists all the primers.

We apply TRIzol reagent (Invitrogen) in concordance with supplier's protocol to isolate total RNA from cells. Subsequently, the first strand of transcripts was used to synthesize cDNA kits (Roche, Mannheim, Germany). After that, the reverse transcription was achieved. qRT-PCR was realized with SYBR Green I Master (Rox) on the LightCycler® 480 System (Rox). The U6 or GAPDH was identified as a standard about the relative gene. Their expression quantity was figured out applying the 2^−ΔΔCt method.