Plant rna isolation kit

Total RNA was extracted from pear peel, strawberry receptacle, pear calli, and tomato fruit samples by the Plant RNA Isolation Kit (Foregene, Chengdu, China), and first-strand RNA was synthesized using a Primer Reverse Transcription Master Mix Kit (Takara, Tokyo, Japan). Primers for RT-qPCR are listed in Supplemental Table S1. PyTubulin, PyActin, Fv26S, FvActin, SlTubulin, and SlActin expression levels were used as normalized genes, and relative genes expression was determined by the 2^−ΔΔCT method (Pirrello et al. 2006 (link)). Three biological replicates were used for all analyses.

The response of DcCHIs to injection of the inducer was analyzed based on transcriptome data of D. cambodiana in previous reports (Supplementary Table 1; Zhu et al., 2016 (link)). Tissue expression, stress response expression, and overexpression analysis of DcCHIs were determined by quantitative real-time PCR (qRT-PCR) analysis. Total RNA from different samples were extracted using plant RNA Isolation Kit (FOREGENE, Chengdu, China) according to the manufacturer’s instructions. cDNA was synthesized from the total RNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Dalian, China). The qRT-PCR analysis was carried out in triplicate using the Mx3005P Real-Time PCR System (Stratagene, La Jolla, CA, United States). qRT-PCR conditions were set as follows: 5 min at 95°C for initial denaturation, followed by 40 cycles of denaturation for 10 s at 94°C, annealing for 30 s at 60°C, and extension for 30 s at 72°C. The actin genes in D. cambodiana (Zhu et al., 2016 (link)) and tobacco (Liu et al., 2012 (link)) were used as the internal controls to normalize qRT-PCR data. Primers used in this study were listed in Supplementary Table 2.

Total RNA from diseased peels of calcium-deficient apples and calcium-sufficient apple peels at 0, 14, and 21 DAS as well as transiently infected apple peel samples was extracted using the Plant RNA Isolation Kit (Foregene, Chengdu, China) and first strand ribonucleic acid was synthesized using a Primer Reverse Transcription Master Mix Kit (Takara, Tokyo, Japan). The RT-qPCR primers are shown in Table S1. The expression levels of MdTUB (TUB, accession number GO562615) and MdUBQ (UBQ, accession number MDU74358) were used as normalization genes, and the expression of relative genes was determined with the 2^−ΔΔCT method. Three biological replicates were used for all analyses.