Superscript 3 platinum sybr green one step qrt pcr kit

Plasma samples were mixed with Ribozol reagent (Thermo Fisher Scientific) to extract total RNAs. Total RNAs were used as template to perform reverse transcriptions using First Strand cDNA Synthesis Kit for RT-PCR (AMV, Sigma-Aldrich, USA). To detect the expression of LINC00311, qPCR reaction mixtures were prepared using Invitrogen SuperScript® III Platinum® SYBR® Green One-Step qRT-PCR Kit (Thermo Fisher Scientific) with 18S rRNA as endogenous control. All PCR reactions were performed 3 times and data were normalized using 2^-ΔΔCT method.

Gene expression in ΔcadA was measured by quantitative reverse transcription-PCR (qRT-PCR). The primers used for qRT-PCR are listed in Table 1. All primers were validated by performing a melt curve analysis with SYBR Green (Thermo Fisher Scientific Waltham, MA, USA) to ensure the amplification of a single specific product. In brief, total RNA was purified from mid-log phase TIGR4 and ΔcadA grown in THY (n = 3) using the RNeasy Midi kit and QIAcube (Qiagen, Valencia, CA, USA). Purified total RNA (7.5 ng/reaction) was transcribed into cDNA and qRT-PCR was performed using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA) as previously described [34 (link)]. Relative quantification of gene expression was determined by using the Stratagene Mx3005P qPCR system (Agilent, Santa Clara, CA, USA). Expression of target genes, speE, potD, cps4A, aguA, lys9, and nspC was normalized to the expression of gyrB and fold change determined by the comparative C_T method.

Total RNA from the cell lines and frozen tissues described above was extracted using TRIzol^® Reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The RIOK2 and NOB1 mRNA levels were detected with a one-step RT-qPCR reaction using a SuperScript^® III Platinum^® SYBR^® Green One-Step qRT-PCR Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The PCR primer sequences for RIOK2 and NOB1 were designed as follows: RIOK2, forward 5′-ACAACAGGCAAGATGGTCA-3′ and reverse 5′-GACGACAAGGCAATTAGATGAG-3′; NOB1, forward 5′-ACATACCAGTTGGAAGCAGAG-3′ and reverse 5′-CAGGTTCTCAGGCTCACAAG-3′. GAPDH was used as an internal control, and the primers were as follows: forward 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse 5′-GAAGATGGTGATGGGATTTC-3′. The RT-qPCR reaction conditions included reverse transcription at 50 °C for 3 min and then preheating at 95 °C for 5 min, which was followed by 45 cycles of denaturation at 95 °C for 15 sec, annealing at 60 °C for 30 sec, and extension at 72 °C for 30 sec. The RIOK2 and NOB1 mRNA levels were normalized to that of GAPDH. The experiment was performed in triplicate, and the results were analysed with the 2^−ΔΔCt method25 (link).