Kapa sybr rapid quantitative pcr kit

Total RNA from the border zone of left ventricular myocardial tissues and CFs was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA was reverse transcribed with a First-Strand cDNA Synthesis Kit (Takara), and the cDNA samples were amplified with PCR (KAPA SYBR^® rapid quantitative PCR kit, KK4600) using specific primers. The primer sequences and annealing temperatures used in this study are listed in Supplemental Table S2 (see Supplemental Table S2).

RNA-Seq data from rose petals infected with B. cinerea were obtained from the National Center for Biotechnology Information (NCBI) database (accession number PRJNA414570). Clean sequencing reads were mapped to the rose reference genome, and the number of reads per kb per million reads (RPKM) were used to determine the gene expression levels. To confirm the RNA-Seq results, the transcript abundance of 6 RcWRKY genes was analyzed using qRT-PCR. To this end, cDNA was generated from rose petals inoculated with B. cinerea, using Takara Reverse Transcriptase M-MLV (Takara). Quantitative RT-PCR was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific), by using 1 μL of the first strand cDNA in the reaction with the KAPA SYBR rapid quantitative PCR kit (KAPA Biosystems). RhUbi was used as a housekeeping gene. The primers used for determining transcript abundance are listed in Additional file 3: Table S2.