Chamq universal sybr quantitative pcr qpcr master mix

MiRNAs were isolated using a miRcute miRNA isolation kit (Tiangen, Beijing, China). The cDNA was synthesized with a High Capacity RNA-to-cDNA Kit (Tiangen, Beijing, China). Real-time PCR was performed with SYBR PCR Mix (SYBR Green; Tiangen, Beijing, China) and primers for amplification of miR-1931 and miR-760-5p (Tiangen Biotech CO., LTD.) in a Light Cycler 480 PCR system (Roche, USA). U6 small nuclear RNA was used for normalization. For mRNA, total RNA was extracted using Trizol reagent and reverse transcribed with sequence-specific reverse transcription-PCR primers (HiScript III RT SuperMix, Vazyme, China). PCR was performed using a ChamQ Universal SYBR quantitative PCR (qPCR) Master Mix (Vazyme, China). Specific primers for mRNAs and the endogenous control (β-actin) were synthesized by Sangon Biotech (Shanghai, China). Gene expression was detected on the Light Cycler 480 PCR system. Each experiment was performed in duplicate in 96-well plates and repeated three times. Primer sequences were as follows: B4galnt 1, F: 5'-CTTCACCATCCTCGTAAGACAC-3', R: 5' -AGTGTCCGTAGTCGATCATAAC-3'; MMP-2, F: 5'—TTCCCCAGAGTCTACGTGCT-3', R: 5'-CGGCACTAAGACCTAACTGGA-3'; MMP-9, F: 5'-CATCCTTAACGCAGTTGCT-3', R: 5'-ATCCTGATCTTCATCGAGGTGA-3'; MMP-13, F: 5'-TTCAGCTCCAATAAATTGACA-3', R: 5'-GTAATATACTCAACGCTGGT-3'.

Total RNA was extracted from E. grandis using the CTAB (cetyltrimethylammonium bromide) method (85 (link)), and first-strand cDNA synthesis was initiated using HiScript III reverse transcriptase (catalog number R323-01; Vazyme, Nanjing, China). Quantitative real-time PCRs were performed using ChamQ universal SYBR quantitative PCR (qPCR) master mix (Vazyme, Nanjing, China) in a 96-well real-time PCR system (Bio-Rad). All of the reactions were performed with three technical replicates of three biological replicates. The EgUBI3 genes from E. grandis and the RiEF1a gene from R. irregularis were used as the internal controls for normalization. The relative expression levels of the genes were computed by the 2^−ΔΔCT method of relative quantification. A list of gene-specific primers used for qRT-PCR is given in Table S1 in the supplemental material.

We assessed the transcriptional profile of the SsCI72380 gene every 12 h over a period of 72 h under haploid and mating conditions as well as during the infection process (after inoculation of sugarcane plants of the smut susceptible variety “ROC22”) using quantitative real-time PCR (qRT-PCR). For the qRT-PCR, we used a ChamQ Universal SYBR quantitative PCR (qPCR) Master Mix (Vazyme, Q711), and the reaction was run on a Real-Time PCR System (CFX96, BioRad). Relative expression values were calculated with the 2^-ΔΔCt method using ACTIN as an internal control (Livak and Schmittgen, 2001 (link)). Three biological repeats each containing three technical replicas for each sample were performed. The primers used in this study are listed in Table 1.