Sybr green qpcr mix with rox

Reverse transcription was completed using the SPARKscript II RT Plus Kit (Sparkjade, Jinan, China), after total RNA was extracted using Trizol reagent (Sparkjade, China). Using 2 × SYBR Green qPCR Mix (With ROX) (Sparkjade, China), real-time PCR was performed on a LightCycler 480 real-time PCR machine (Roche, Basel, Switzerland). The exact primers (Azenta, Suzhou, China) were utilized to amplify the genes for Collagen I (Coli), alkaline phosphatase (Alp), runt-related transcription factor 2 (Runx2), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh). Cycle threshold (Ct) values were utilized to compare relative mRNA expression, while Gapdh was used as an internal control. The experimental data were computed utilizing the 2^−ΔΔCt approach [46 (link)]. Each experiment was repeated three times.

An aliquot (200 μL) of overnight pure cultures of both S. aureus strains was individually added to 20 mL TSB broth, and incubated at 37 °C with shaking at 220 rpm until log or stationary growth phases. The total RNA of all S. aureus strains was extracted with SPARKeasy Improved Bacteria RNA kit (AC0402; SparkJade, Jinan, China). Three biological replicates were prepared for each strain. RNA was demonstrated to be intact by 1% agarose gel electrophoresis; its concentration was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
The total RNA of the strains was reverse-transcribed to generate cDNA with a SPARK script II RT Plus Kit (AG0304; SparkJade) for real-time quantitative PCR. Amplification of samples was performed using the 2 × SYBR Green qPCR Mix (With ROX; AH0104; SparkJade) in an Eppendorf Mastercycler ep realplex 4 system (Eppendorf, Mannheim, Germany). The samples were tested in triplicate in three independent experiments using the following conditions: 94 °C for 3 min followed by 40 cycles of amplification at 94 °C for 15 s, 55 °C for 15 s, and 72 °C for 25 s. The relative transcriptional levels of the chosen genes were calculated using the 2^ΔΔCT threshold cycle (C_T) method [13 (link)]. The expression of rpoB was determined as normalization control. The genes used in this study are listed in Table 1.

Total RNA from rat liver samples was extracted by TRIzol reagent (Ambion, Austin, United States). 1 μg of total RNA in each sample was reverse-transcribed into cDNA using SPARKscript Ⅱ RT Plus Kit (With gDNA Eraser) (Sparkjade, Qingdao, China). The mRNA expression of the target gene was quantified by 2 ×SYBR Green qPCR Mix (With ROX) (Sparkjade, Qingdao, China). The expression of rat β-actin was used as the internal reference. Relative gene expression was detected in triplicate using the ABI StepOne Plus system (Applied Biosystems, CA, United States). The primer sequences used in the present study are listed in Supplementary Table S3.

Reverse transcription was conducted using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, R323-01) following the manufacturer’s instructions. Real-time PCR was performed using 2 × SYBR Green qPCR Mix (with ROX) (SparkJade, AH0104). Data are shown as the fold change = 2^-ΔΔCt mean ± s.d. The primers are listed in Table S1.