Sybr premix ex taqtm perfect real time kit

The copy numbers of the subunit of the flagellar filament gene (fliC) (R. solanacearum), 16S rRNA gene (total bacteria), ITS I (F. solani), internal transcribed spacer (ITS) region (total fungi) and genes related to the production of the antagonistic substances lipopeptide (sfr (surfactin), fen (fengycin) and itu (iturin)) and polyketides (dfn (difficidin)) were quantified by quantitative PCR in all samples. Quantitative PCR (qPCR) assays were performed using the SYBR Premix Ex Taq^TM (Perfect Real Time) Kit (Takara Biotechnology Co., Dalian, China) with the ABI StepOne^TM Real-Time PCR System (Applied Biosystems, ‎Waltham, MA, USA). Each reaction was performed in a 20 µL volume. Detailed primer information and PCR steps are listed in Tables S1 and S2, respectively. Standard curves were developed by serially diluting the plasmids with known positive inserts to final concentrations of 10² to 10⁷ gene copies µL⁻¹. qPCR efficiencies ranged from 90% to 105%, and the R² values were greater than 0.99. Three independent technical replicates were used for each sample.

TRIzol Reagent (Invitrogen, United States) was used to isolate total RNA from flash-frozen myocardial tissues or H9c2 cells. cDNA was synthesized using a reverse transcription reagent kit (Takara Biotechnology, Japan) and then amplified in the Bio-rad CXF CONNECT Detector system using the SYBR^® Premix Ex TaqTM Perfect Real Time Kit (Takara Biotechnology, Japan). Relative expression was computed using the CT method. Table 1 lists the SYBR Green real-time PCR primers for mouse gene expression.

Total RNA was extracted from cultured cells using the Total RNA Kit I (Omega Bio-Tek, Doraville, GA, USA) according to the manufacturer's instructions. The cDNA was generated from 1 μg of total RNA using PrimeScript 1^st Strand cDNA Synthesis Kit (TaKaRa, Otsu, Japan) following the manufacturer's instructions. Quantitative real-time PCR was performed using the SYBR Premix Ex Taq^TM (Perfect Real Time) kit (TaKaRa, Otsu, Japan). The relative expression level of the target gene was calculated with the 2^−ΔΔCt method. The sequences of the primers used were provided in Supplementary Table 2.