Labopass cdna synthesis kit

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the LaboPass cDNA synthesis kit (Cosmogenetech, Seoul, Korea). The quantitative real-time polymerase chain reaction (qPCR) was performed on a CFX96 Touch real-time PCR detection system (Bio-Rad, Hercules, CA, USA) using Labopass SYBR Green Q master mix (Cosmogenetech) according to the manufacturer’s protocol. The primers used were as follows: PVR, 5′-CTG GCT CCG AGT GCT TGC-3′ and 5′-GAG GTT CAC AGT CAG CA-3′. Glyceraldehyde 3-phosphate dehydrogenase was used as the internal control. Relative PVR transcript levels were determined using the 2^−ΔΔCt method, with all samples tested in triplicate and mean values used for further analyses.

Total mRNA was extracted from cells using TRIzol^TM Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. Reverse transcription was performed with 2 μg of pure RNA using Labopass cDNA synthesis kit (Cosmogenetech, Seoul, Korea). qRT-PCR was performed on a ViiATM 7 Real-time PCR system (Applied Biosystems, Waltham, MA, USA) using Luna universal qPCR master mix (New England Biolabs, Beverly, MA, USA). The relative quantities of target genes were calculated from triplicate samples after normalization to an internal control, 18S ribosomal RNA (18S rRNA). All oligonucleotide primers, listed in Table 1 below, were purchased from Macrogen company.

To estimate the effect of canadine on the gene expression of E3 ligases, the myotubes were pre-incubated with canadine prior to CT26 CM treatment, as described above. RNA purification from the cells and first-strand cDNA synthesis were performed according to the manufacturer’s instructions (Labopass™ cDNA synthesis kit, Cosmogenetech, Seoul, Korea). An RT-qPCR reaction was performed with the SYBR^® Green PCR Master Mix and conducted using the Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA, USA). All mRNA levels were normalized using glyceraldehyde 3-phosphate dehydrogenase mRNA as an internal control. The primers used for the amplifications are shown in Table 1.

Total RNA was extracted from bone marrow plasma cells using LaboPass Labozol reagent (Cosmo Genetech, Seoul, Korea). Complementary DNA (cDNA) was reverse transcribed from 1 μg of total RNA using LaboPass cDNA Synthesis kit (Cosmo Genetech) according to the manufacturer’s instructions. qPCR was performed using Labopass SYBR Green Q Master (Cosmo Genetech) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primers used were as follows: LGALS9, 5′-AGC TTC TCA GTG TGG ATC TTG-3′ and 5′-TCC TCA GGC GAT GGT AGT AT-3′. Each cDNA sample was tested in triplicate. The cycling conditions included initial template denaturation at 95°C for 3 min, followed by amplification for 40 cycles of 95°C for 10 s, 62°C for 20 s (for LGALS9) or 58°C for 10 s (for GAPDH), and 72°C (for LGALS9) or 68°C (for GAPDH) for 20 s. A final melting curve analysis was performed from 65 to 95°C at a rate of 0.05°C/s. The relative LGALS9 transcript level was determined using the 2^−ΔCt method.

Total RNA was extracted from mouse quadriceps muscle tissue and C2C12 cells using TRIzol reagent (Invitrogen™, Carlsbad, CA, USA). RNA purification and first-strand cDNA synthesis were performed following the manufacturer’s recommendation (Labopass™ cDNA synthesis kit, Cosmogenetech, Seoul, Republic of Korea). The RT-qPCR reaction was conducted with the SYBR^® Green PCR Master Mix and performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA, USA). All mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. The primers used for the amplifications are presented in Table S1.

Total RNA was isolated with the Qiagen RNeasy Plus Mini Kit according to the manufacturer’s protocol. Labo Pass™ cDNA synthesis Kit (Cosmogenetech) was used to synthesize cDNA from 1 μg total RNA per sample. The target cDNA was amplified using the following primers: iNOS forward: 5′-CCTGGTACGGGCATTGCT-3′,reverse: 5′-GCT CAT GCG GCC TCC TTT-3′, Arg-1 Forward: 5′-GGAATCTGCATGGGCAACCTGTGT-3′, reverse: 5’-AGGGTCTACGTCTCGCAAGCC A-3′, and GAPDH forward: 5′-TGGCAAAGTGGAGATTGTTGCC-3′, reverse: 5′-AAGATGGTGATGGGCTTCCCG-3′. The cDNA was used to evaluate the level of gene expression by qPCR using SYBR Green Master Mix (Bio-rad). The reaction mixture was incubated at 95 °C for 1 min, followed by 30 cycles of 95 °C for 10 s, 57 °C for 1 min and the relative iNOS and Arg-1 expression was calculated, respectively.

Cultures of 3T3-L1 preadipocytes were plated at a density of 2.5 × 10⁵ cells into a 60-mm dish and differentiated as mentioned above. Cells were lysed with TriZol reagent (Molecular Research Center, Cincinnati, OH, USA) at D5 to purify total RNA which was then used for cDNA synthesis (Labopass™ cDNA synthesis kit, Cosmogenetech, Seoul, Korea). Then, cDNA was used to estimate gene expression level during adipocyte differentiation by qPCR using SYBR Premix Ex TaqTM real time PCR Kit (Cosmogenetech) and Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA, USA). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level was used as an internal control to determine the relative mRNA level of adipogenic factors. Primers used for amplifications are listed in Table 1.