Tb green premix ex taq 2 mix

Total RNA was extracted from S. mutans using MasterPure Complete DNA and RNA Purification Kit (Epicentre, USA). Genomic DNA was then eliminated and double-stranded cDNA synthesized using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan). Quantitative real-time PCR (qRT-PCR) was then performed using TB Green Premix Ex Taq II mix (Takara, Japan) on an Applied Biosystems QuantStudio 6 unit (Thermofisher, USA). Validated primers used in qRT-PCR are listed in S5 Table. Data were collected using QuantStudio Real-Time PCR Software v1.3, normalized to reference gene 16S rRNA levels, and analyzed using the 2-^ΔΔCT method. Each experiment was performed in triplicate.

Total RNA was isolated from different samples using TRIzol reagent (Invitrogen, Carlsbad CA, United States) and then treated with RNase-free DNase I (TaKaRa, Dalian, China). The first-strand cDNA was synthesized using 2.0 μg of total RNA per 20 μl reaction and an oligo (dT) primer. Tenfold diluted cDNA, a set of gene-specific primers (Supplementary Table S1), and TB Green^® Premix Ex Taq^™ II mix (TaKaRa, Dalian, China) were mixed for qRT-PCR to determine the transcriptional levels of the maize genes on the CFX96^™ real-time PCR detection system (Bio-Rad, Hercules, CA, United States). The expression level of GAPDH mRNA was determined and used as an internal control. The relative expression level of each gene was calculated using the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)). Differences between the treatments were then analyzed using Student’s t-tests. All experiments were carried out at least three times.

Col-0, atg5-1, atg7-2 was grown on 1/2 MS medium plates for 7 d, harvested and total RNA was extracted, to analyze the gene expression of FREE1 by qRT-PCR. The autophagy related genes are measured by the use of the 7-d-old Col-0. The seeds were cultivated on 1/2 MS + Fe medium plates for 7 d and then transferred to 1/2 MS − Fe liquid medium, the samples were harvested respectively after 0 d, 1 d, 2 d and 3 d treatment as the previous designed time points. Total RNA could be extracted with the Hi-Pure Plant RNA Mini Kit (Magen, R4151). The first-strand cDNA was synthesized from 1 µg total RNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, RR047Q). Quantitative real-time PCR was performed on the CFX96 Touch real time PCR detection system (BioRad) using TB Green^® Premix ExTaq™ II mix (TaKaRa, RR820Q). Relative abundance of target transcripts was determined by the 2^−ΔΔCT method, using the levels of Arabidopsis Ubiquitin10 transcript as the internal control. The specific primers used in this study were listed in Supplemental Table S1.