7500 system sds software

Adherent cells (2.0 × 10⁶/mL, 2 mL) cultured in 6-well plates were incubated with or without AS (10 μg/mL) for 2 h. Subsequently, cells were stimulated with LPS (100 ng/mL) for 4 h. Total RNA was extracted from the harvested cells using a Trizol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA with a PrimeScript RT reagent Kit (TOYOBO, Japan). The primers for SR-A, MARCO, SR-BI and β-actin (Table 1) were added to the PCR tubes. Reactions were set up for qPCR per manufacturer’s instructions containing 2 μL of cDNA, 10 μL of SYBR green mix (TOYOBO, Osaka, Japan), 1 μL of specific forward primer (10 μM stock), 1 μL of specific reverse primer (10 μM stock), and 6 μL of distilled water. Amplification reactions were performed in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) for 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 40 s. C_T values were determined using the 7500 System SDS Software (v.1.2.3; Applied Biosystems, Foster City, CA, USA). Expression ratios were calculated according to the 2^−△△CT method described [31 (link)]. Each sample was analyzed in quadruplicate.

Total RNA was isolated from cells using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Two μg of isolated RNA were reverse transcribed to cDNA with RevertAid Reverse Transcriptase, RiboLock RNase Inhibitor, and Oligo(dT)₁₈ anchored primer (all Thermo Scientific, USA). qRT-PCR was performed using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, USA) on a sequence detection system 7500 (Applied Biosystems, CA, USA) and analyzed by 7500 System SDS Software (version 1.3.1). The sequence of primers used in this study and PCR cycling conditions are listed in S2 Table. Expression of the TATA-box binding protein (TBP) was used as an endogenous control for standardization. Ct values were determined for the internal control (TBP) and the test genes at the same threshold level in the exponential phase of the PCR curves. Relative quantification (comparative Ct (ddCt) method) was used to compare the expression level of the tested genes with the internal control and was represented in relative units. Dissociation curve analysis was performed after every run to check the specificity of the reaction. Three reactions (each in triplicate) were run for each gene, and the standard error of mean was calculated.

An established diagnostic RT-PCR protocol [41 (link)] was adapted for GE quantification using PDV26 and PDV27. GEs were calculated by 7500 System SDS Software (Applied Biosystems, Foster City, USA) based on the standard curve of a cDNA plasmid. A different RT-PCR was used to determine the presence of the BamHI marker mutation in DWV genomes. A 764 nt fragment flanking the novel BamHI site was amplified using PDV16 and PDV28. Half of the PCR product was incubated with 20U BamHI for 30 min at 37°C and subjected to agarose gel electrophoresis. The appearance of two bands (488 and 276 nt) after BamHI digestion proved the presence of recombinant DWV.

Reactions were performed using the TaqMan™ System (Applied Biosystems). The gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as the endogenous control for the reaction. The expression of tumor necrosis factor alpha (TNF-α, Mm00443258_m1), interleukin 1 beta (IL-1β, Mm00434228_m1), interleukin 6 (IL-6, Mm00446190_m1), interleukin 10 (IL-10, Mm01288386_m1), Cx3cl1 (Mm00436454_m1), Ccl20 (Mm01268754_m1), Cxcl1 (Mm.PT.5842076891), and LIF (Mm.PT.58.13926050) was quantified in the hypothalamus. Hypothalami were extracted 1 day after the introduction of the HFD in anti-LIF or IgG-treated mice. For the determination of relative transcript expression, real-time PCR reactions were performed in duplicate as follows: 3.0 μl TaqMan Universal PCR Master Mix 2X, 0.25 μl of the primers and probe solution, 2.75 μl water, and 4.0 μl cDNA. The values of relative gene expression were obtained by analyzing the results using 7500 System SDS software (Applied Biosystems).