Primescript 2 first strand cdna synthesis kit mix

All RNA samples were extracted with plant total RNA kit (OMEGA⁸) according to the manufacturer’s protocol. Samples were treated with the DNase digestion attached in the RNA kit to avoid genomic DNA contamination. The quality and quantity of the RNA samples were measured with NanoDrop ND1000 (Thermo Scientific). RNA samples with the absorbance ratios at both 260/280 and 260/230 nm being around 2.0 were selected. The integrity was assessed through 1.5% agarose gel electrophoresis to select the DNA-free RNA samples with well-defined bands. We added 1.2ug RNA for 30uL reverse-transcription system, which was equivalent to 400ng RNA for 10uL reverse-transcription system. The PrimeScript II first Strand cDNA Synthesis Kit MIX (Takara) was used according to the manufacturer’s instructions. The synthesized cDNA was diluted 15 times before used as the template for RT-qPCR.

Approximately 0.1 g (fresh weight) of pluripotent and non-pluripotent calli at 28 DAC were harvested respectively, and immediately frozen in liquid nitrogen for RNA isolation. Total RNAs were isolated using the RNeasy plant mini kit (Qiagen). Genomic DNA was eliminated by DNAse I (Invitrogen, Carlsbad, CA, USA) treatment, as recommended by the manufacturer. The purity of RNA samples was examined by use of the Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA was synthesized from 300 ng RNA using the PrimeScript II First Strand cDNA Synthesis Kit MIX (Takara Bio, Japan) with oligo (dT) primers in a final volume of 20 μL according to the manufacturer’s instructions.