Itaq universal sybr green supermix kit

The RT-qPCR reactions were performed using a Bio-Rad CFX96 Real-Time PCR Detection System using iTaq™ Universal SYBR® Green Supermix Kits (Bio-Rad Laboratories Inc., Hercules, CA, USA) to detect the mRNA expression level associated with each gene. The chicken β-actin gene was used as an internal control. The analysis was carried out using the 2^−ΔΔCt method, as described previously (Livak and Schmittgen, 2001 (link)).

Ginsenoside panaxatriol was obtained from Must Bio-Technology (Chengdu, China). PTX was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) (11995-040), F-12 nutrient mixture (Ham) and fetal bovine serum (FBS) were bought from Life Technologies (Grand Island, NY, USA). MammoCul medium (human) and supplements were purchased from STEMCELL Technologies (Vancouver, BC, Canada). CellTiter-Glo luminescent cell viability assay kits were purchased from Promega Corporation (Madison, WI, USA). iScript gDNA Clear cDNA Synthesis Kits and iTaq Universal SYBR Green Supermix Kits were purchased from Bio-Rad Laboratories (Hercules, CA, USA). p-IRAK1 S376, IRAK1, p-P65 S536, P65, p-ERK1/2, ERK1/2, BAX, BCL-2 and MCL-1 antibodies were supplied by Cell Signaling Technology (Danvers, MA, USA). Beta-actin antibody was purchased from Sigma–Aldrich (St. Louis, MO, USA).

Retrotranscription (RT) was performed with 1 µg of total RNA using the iScript cDNA synthesis kit (Bio Rad, Hercules, CA, USA, #1708891), according to the manufacturer’s instructions. Each RT reaction was completed in duplicate. Real-Time RT-PCR analysis was performed using Biorad C1000 Touch or Biorad CFX Connect thermocyclers and CFX Real-Time PCR detection systems, according to the manufacturer’s instructions. PCR efficiency was measured for each primer pair on cDNA standards. PCR reactions were performed in 96-well plate using the iTaq™ Universal SYBR^® Green Supermix kit (Biorad #1725124), with 5 pmol of each primer and 1 µL of ten-fold diluted RT reaction in a final volume of 10 µL. Reactions were run using the manufacturer’s recommended cycling parameters (95 °C for 3 min, 40 cycles of 95 °C for 15 s and 60 °C for 30 s). No-template controls were included for each primer pair and each PCR reaction was completed in duplicate. Melting curves were analyzed to verify the specificity of each amplification reaction. The primers used for the qPCR reactions are indicated in Table S3. The amplification of EF1α was used as a control for normalization. For each gene, differences between samples were calculated using the ΔΔCt method.

The total RNA from flushed tibial shafts or the middle 20 mm of small intestines were isolated by utilizing Trizol reagent (Invitrogen, Carlsbad, CA, USA). The first-strand cDNA was synthetized using the Super-Script^TM First-Strand Synthesis Kit (Takara Bio USA Inc, Mountain View, CA, USA). RT-qPCR was performed using the iTaq™ Universal SYBR^® Green Supermix kit (BIO‐RAD Laboratories Inc., Hercules, CA, USA). The relative target gene expression was normalized to β-actin. The data were calculated using the method previously described^{53 (link)}. The primers for the genes of interest are listed in Supplemental Table 1.