Primescript rt first strand cdna synthesis kit

The RNeasy Plant Mini kit (Qiagen, Hilden, Germany) was used to isolate citrus total RNA, and approximately 1 μg purified total RNA was used to synthesize cDNA by using the PrimeScript RT First Strand cDNA Synthesis Kit (TaKaRa, Otsu, Japan). The synthesized cDNA was diluted 1:10 as a template for real-time PCR. Primer Express software (PE Applied Biosystems, Foster City, CA, USA) was used to design real-time primers to avoid conserved regions. The GC content and length of the primers were 45–55% and about 21 bp, respectively. The PCR product sizes were 160–200 bp. These primers were tested to ensure amplification of single discrete band with no primer-dimers. Real-time PCR analysis was conducted using the ABI PRISM 7000 system (Applied Biosystems). A melting curve analysis was performed for each sample to verify the specificity of the reactions. Each reaction was performed with a 20 µL including 10.0 µL SYBR Green PCR Master Mix (TaKaRa, Otsu, Japan), 1.0 µL of cDNA template, 0.5 µL of sense and antisense primers (10 pmol L⁻¹), and 8 µL of ddH₂O with the following PCR parameters: 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The relative expression levels of the target genes were calculated using the 2^−ΔΔCt method by normalizing with citrus Actin according to a previously reported method [45 (link)]. Three biological repeats were assayed in this study.