Rt pcr machine

To evaluate the mRNA levels of TRAP, cathepsin K, matrix metalloproteinase-9 (MMP-9) and carbonic anhydrase II, the RAW 264.7 cells were stimulated with RANKL, treated with melittin and cultured for the indicated periods of time (0, 3, 5, 7, 9 and 11 days). Total RNA was extracted using TRIzol reagent (Gibco-BRL). cDNA synthesis was performed using the GeNet Bio kit (GeNet Bio, Cheonan, Korea). Quantitative PCR amplification was performed using a RT-PCR machine (Bio-Rad, Hercules, CA, USA) and SYBR-Green Supermix (Toyobo, Tokyo, Japan). We used the following PCR protocol: 95°C for 3 min; 40 cycles (15 sec, 95°C/1 min, 58–62°C); and 72°C/45 sec; and 60°C to 95°C per cycle for melting curve analysis. The oligonucleotide primers used in this analysis are listed in Table I.

The mRNA expression of IL-1β, IL-6 and TNF-ɑ were measured by quantitative RT-PCR, and β-actin was used as an internal control for mRNAs. IPEC-J2 cells were cultured on a six-well plate at a density of 5 × 10⁶ cells per well for 48 h, and then divided into five groups as follows: (a) control group (con), where cells were kept in DMEM without FBS; (b) model group (mod), where cells were cultured in DMEM without FBS containing 5 μg/ml LPS; (c) low-dose berberine-treated group (low); (d) middle-dose berberine-treated group (mid); and (e) high-dose berberine-treated group (hig). Total RNA was isolated from the IPEC-J2 cells with Trizol reagent according to the manufacturer’s instructions and reverse transcribed into cDNA using the PrimerScript RT reagent kit. PCR amplification was performed with a SYBR Green PCR kit in a Bio-Rad RT-PCR machine in triplicate independently. The following primer sequences were used: IL-1β F: 5′-CGTGCAATGATGACTTTGTCTGT-3′, R: 5′-AGAGCCTTCAGCATGTGTGG-3′; IL-6 F: 5′-GCTGCAGTCACAGAACGAGT-3′, R: 5′-GGACAGGTTTCTGACCAGAGG-3′; TNF-α F: 5′-TTGAGCATCAACCCTCTGGC-3′, R: 5′-GGCATACCCACTCTGCCATT-3′. PCR parameters were: 3 min 95°C for one cycle, 5s 95°C, annealing temperature 30s for 40 cycles. Reaction specificity was controlled by post-amplification melting curve analyses. The expression fold-changes were analysed by the 2^−ΔΔCt relative quantitative method.

Total RNA was isolated using an RNeasy kit (Qiagen, 74004) including an optional DNase digestion step. Typically 500 ng RNA was used as the starting template to create cDNA using a high capacity cDNA reverse transcription kit (Applied Biosystems, 4368814) and heating samples to 25°C for 10 min, 37°C for 2 h and 85°C for 5 min. RT-qPCR was undertaken in 96 well plates using 4.5μl 1:10 cDNA and 10.5 μl of a master mix containing (Sigma-Aldrich, S4438) or SYBR Green PCR Master Mix (Applied Biosystems, 4309155). Primer sequences are listed in Supplementary Table 1. Plates were run on a BioRad RT PCR machine typically using the following programme: 95°C 2 min, 40x (95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec), 95°C for 30 sec.

Total RNA was extracted from Ishikawa cells using TRIzol reagent (Ambion, Austin, TX, USA). A total of 1 μg of RNA was used to synthesize complementary DNA using superscript IV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and oligo-dT primer. Using one-tenth of the volume of the complementary DNA, gene expression was quantitatively analyzed using an RT-PCR machine (Bio-Rad, Hercules, CA, USA). Amplification was performed using AccuPower PreMix (Bioneer, Daejeon, Korea) and programmed with 40–45 cycles as follows: denaturation at 95°C for 10 minutes, annealing at 58°C–60°C for 30 seconds, and extension at 72°C for 30 seconds. The PCR products were subjected to electrophoresis using a 2% agarose gel and visualized with G-box software (Syngene, Cambridge, UK). The gene expression levels of the genes of interest were normalized to that of β-actin using ImageJ software.