Quant qrt pcr kit

RNA isolation and qRT-PCR analysis were performed as described previously with some modifications [8 (link)]. Briefly, Xoo strains were grown in M210 liquid medium at 28 °C till OD₆₀₀ of 0.8, and harvested by centrifugation at 12,000 g for analysis of gene expression. For T3SS-related gene assays, the harvested bacterial cells were sub-cultured in XOM2 medium [43 (link)] overnight at 28 °C and collected again. Total RNA was extracted with RNAprep pure Cell/Bacteria Kit (Tiangen, Beijing, China) and treated with DNase and cDNA was synthesized from total RNA using the FastQuant RT Super Mix (Tiangen, Beijing, China). RT-qPCR was performed using Quant qRT-PCR kit (Tiangen, Beijing, China) in Applied Biosystem’s 7500 (Applied Biosystems, Foster City, CA, USA) with gene specific primers, and gyrB was used as a reference gene (Additional file 2: Table S1). The relative expression ratio was calculated using 2^–∆∆Ct method [44 (link)]. These experiments were performed in three biological replicates and triplicate PCR.

Total RNA was reverse transcribed using M-MLV reverse transcriptase (Invitrogen). Semi-quantitative PCR was performed using gene-specific primers, with ß-actin as a loading control (S1 Table). The intensity of PCR products was analyzed using the Lane 1D software (Sagecreation). Quantitative PCR was performed using Quant qRT-PCR Kit (Tiangen) with gene-specific primers (S1 Table). RNA sequencing was performed on Illumina HiSeq 2000, using 12 hpf mRNA libraries constructed by TruSeq RNA Library Preparation Kit. The data were aligned and analyzed as described [65 (link)].

Reverse transcription quantitative PCR (qRT-PCR) was performed as previously report (Liu et al., 2019 (link)) to determine the transcriptional levels of DEGs. All qPCR reactions were performed using Quant qRT-PCR kit [Tiangen Biotech(Beijing) Co., Ltd., Beijing, China]. The primers used for qRT-PCR were listed in Supplementary Table S1. The standard curve of qRT-PCR was determined with six-serial dilutions of first-chain cDNA template. The comparative threshold (2^–ΔΔCt) method was used to assess the relative expression levels. For every primer pair, a standard curve was performed to analyze the amplification efficiency and to identify potential primer dimers.