Abi prism 7700 dna sequence detection system

RNAs were isolated from each sample group using a plant TRIzol reagent (Invitrogen, Shanghai, China). RNA quantity and quality were analyzed using a NanoDrop ND-430 spectrophotometer (Agilent, Santa Clara, CA, USA). About 1.0 μg RNAs of each sample were reverse transcribed to cDNA library using ReverAid First Strand cDNA Synthesis Kit (Thermo Scientific, Shanghai, China). qRT-PCR assay (three independent repeats) was applied using a SYBR Premix Ex Taq Kit (TaKaRa, Dalian, China) on an ABI PRISM 7700 DNA Sequence Detection System (Applied Biosystems, Shanghai, China). All primers were listed in Table S1.

Total RNAs were extracted using TRIzol reagent and reverse-transcribed, as described [29 (link),41 (link)]. Quantitative real-time RT-PCR analysis (qRT-PCR) was performed and normalized with the internal control β-actin (ACTB) using commercial ready-to-use primers/probe mixes (Assays on Demand Products, Applied Biosystems): CD147 (assay Hs 00936295_m1), MCT4 (assay Hs00358829_m1), ACTB (assay Hs 9999903_m1) and ACE2 (assays Hs 01085331_m1 and Hs 01085333_m1) [29 (link),41 (link)]. The ABI PRISM 7700 DNA Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used [29 (link),41 (link)].

To validate the TMT data, eight DEPs were randomly selected for analysis of their corresponding gene transcript levels via qRT-PCR. Total RNA was extracted from grapevine rootstock leaves using a TRIzol kit according to the manufacturer’s protocol (Promega, Beijing, China). Residual contaminating DNA was removed by RNase-free DNase I (TaKaRa, Dalian, China). QRT-PCR was performed using a SYBR Premix Ex Taq Kit (TaKaRa, Dalian, China) and an ABI PRISM 7700 DNA Sequence Detection System (Applied Biosystems, Shanghai, China). The sequences of the primers used were designed using Primer Premier 5 software (Premier Biosoft International, Palo Alto, CA, United States). The actin gene (AB073011) was used as an internal standard to calculate relative fold differences based on comparative cycle threshold (2^–ΔΔCt) values. Details of the primer sequences used in this study are presented in Supplementary Table 1. The qRT-PCR procedure was as follows: 1 μL of a 1/10 dilution of cDNA in H₂O was added to 5 μL of 2 × SYBR^® Green buffer together with each primer at 0.1 μM, and H₂O was added until the final volume reached 10 μL. The reactions were run in accordance with the following procedure: 50°C for 2 min; 95°C for 10 min; and then 40 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s. PCR was performed in 96-well optical reaction plates.

Total RNA was extracted using a TRIzol Kit according to the manufacturer’s protocol (Promega, Beijing, China). Residual DNA contamination was removed by RNase-free DNase I (TaKaRa, Dalian, China). QRT-PCR was performed using the SYBR Premix Ex Taq Kit (TaKaRa, Dalian, China) and an ABI PRISM 7700 DNA Sequence Detection System (Applied Biosystems, Shanghai, China). The primer sequences were designed using Primer Premier 5 software (Premier Biosoft International, Palo Alto, CA, USA) (Additional file 1: Table S1). Actin gene was used as an internal standard to calculate relative fold-differences based on comparative cycle threshold (2^−ΔΔCt) values. The qRT-PCR procedure was as follows: 1 μL of a 1/10 dilution of cDNA in H₂O was added to 5 μL of 2× SYBR® Green buffer, with 0.1 μM of each primer and H₂O to a final volume of 10 μL. The reactions were run as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s in 96-well optical reaction plates.