Reverse transcription system a3500 kit

Total RNA of heart tissue was extracted by Trizol method using the RNA isolation kit (Solarbio, Shanghai, China) and the purity and density of RNA determined from the OD260/OD280 ratio using a UV-spectrophotometer. RNA samples were preserved at −80°C. For qRT-PCR, primers for miR-21, B cell lymphoma 2 (Bcl-2), BCL-2-associated X protein (Bax) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using the Primer5.0 software using the RNA sequences from the Genbank (Table 2) and synthesized by Shanghai Gene Pharma Co., Ltd. The reverse transcription of total RNA (30 μg) was conducted according to Reverse Transcription System A3500 kit (Promega, USA) followed by PCR on the ABI 7500 (Applied Biosystem, CA, USA). The PCR conditions were: 95°C for 3 min followed by 40 cycles of 95°C for 30s, 55°C for 30s, 72°C for 1 min, and finally, 72°C for 5 min. For quantitation, PCR recipe included pre-mix Ex-Taq or SYBR Green Mix 12.5μl, Forward Primer 1μl, Reverse Primer 1μl, DNA template 4μl and ddH₂O up to 25μl. GAPDH was used as internal reference. The melting curve was used to evaluate the credibility of PCR results. Ct value (point at which the amplification curve reaches its maximal) and 2^−ΔΔCt were calculated to determine the relative expression of target genes [46 (link)].

RAW 264.7 macrophages were plated in 12-well plate (5 × 10⁵/well). Cells pretreated with the test samples, positive control (parthenolide, 10 μM), or solvent vehicle (0.1% DMSO in culture medium) for 1 h were incubated with 100 ng/mL LPS for additional 4 h. Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription of RNA was performed with the Reverse Transcription System A3500 kit (Promega, Madison, WI) according to the manufacturer's protocol. Relative quantification of gene expression was performed with SYBR® Green Real-Time PCR Master Mix (TOYOBO, Osaka, Japan) and conducted with the Eppendorf Mastercycler ep realplex (Hauppauge, NY). The following primers were used: TNF-α (5′-TTCTCATTCCTGCTTGTGG-3′; 5′-ACTTGGTGGTTTGCTACG-3′), IL-6 (5′-CTTCTTGGGACTGAT G-3′; 5′-CTGGCTTTGTCTTTCT-3′), and IL-1β (5′-GATCCACACTCTCCAGCTGCA-3′; 5′-CAACCAACAAGTGATATTCTCCATG-3′). The primers for the mouse housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5′-CCTTCCGTGTTCCTACC-3′ and 5′-CAACCTGGTCCTCAGTGT A-3′ [9 (link)].

Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription of RNA was performed with the Reverse Transcription System A3500 kit (Promega, Madison, WI, USA) according to the manufacturer′s protocol. The complementary DNA (cDNA) was subsequently subjected to Real-Time PCR to quantify the transcripts of TNF-α and IL-6 using SYBR^® Green Real-time PCR Master Mix (TOYOBO, Osaka, Japan). The following primers were used: TNF-α (5′-TTC TCATTC CTG CTT GTG G-3′; 5′-ACT TGG TGG TTT GCT ACG-3′), IL-6(5′-GAG GAT ACC ACT CCC AAC AGA CC-3′; 5′-AAG TGC ATC ATCGTT GTT CAT ACA-3′). The primers for the mouse housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5′-CCT TCC GTG TTC CTA CCC-3′; 5′-CAA CCT GGT CCTCAG TGT AG-3′). PCR was performed according to the following conditions: 94 °C for 3 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 45 s, extension at 72 °C for 30 s, followed by a final extension at 72 °C for 10 min. The quantified fold changes in messenger RNA (mRNA) in each sample were normalized to GAPDH expression and calculated using the 2exp (−ΔΔCt) method.