Primescript rtase 1st strand cdna synthesis kit

Total RNA was extracted using the E.Z.N.A. Plant RNA Kit (Omega Bio-tek, Norcross, GA, USA). Equal quantities of RNA from three biological replicates at each stage were pooled to construct a complementary cDNA library. RNA concentration and quality was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and RNA integrity was assessed using the RNA Nano 6000 Assay Kit and the Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA). Equal quantities of RNA from three biological replicates at each stage were pooled to construct each library. First-strand cDNA was synthesized using the PrimeScriptTM RTase 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China). cDNA library construction was performed as described below and sequencing was performed at Biomarker Technologies Co, LTD (Beijing, China) using an Illumina HiSeq2500 platform (Illumina, San Diego, CA, USA).

Total grapevine RNA was extracted from powdery mildew-inoculated grapevine leaves, and total Arabidopsis RNA was extracted from leaves infected with powdery mildew, PstDC3000 or B. cinerea, using the E.Z.N.A.^® Plant RNA Kit (Omega Bio-tek, Norcross, GA, USA). First-strand cDNA was synthesized using the PrimeScript^TM RTase 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China) and diluted 6-fold with sterile water. Quantitative real-time PCR analysis was carried out using SYBR Green (TaKaRa Biotechnology) in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster, CA, USA), and cycling parameters were: 95 °C for 30 s, and 42 cycles of 95 °C for 5 s, and 60 °C for 30 s. The grape ACTIN1 gene (GenBank Acc. No. AY680701) or Arabidopsis ACTIN1 gene (GenBank Acc. No. AT3G18780) was used as an internal control. The sequences of gene-specific primers used for the qRT-PCR reactions are provided in Table 1. Each reaction was performed in triplicate for each of the three biologically replicated sets of cDNA samples. Relative expression levels were analyzed with the IQ5 software using the normalized expression method.