Sybr premix ex tap 2

Amplification of the cDNA sequences employed the expression of aminomethyl transferase- AMT, nitrate transporter- NRT, Nitrate reductases- NR, Glutamate synthase- GS, Glutamine synthetase- GOGAT, N fixation-nifH, endo-glucanase- β-1, 4-GA, and chitinase- CHI, genes in the root tissues of sugarcane during plant-microbes interaction at tillering phase were analyzed in greenhouse condition after treatment with strains (AF1 and EF1) in GT42 sugarcane varieties with control plants. The primer sequences used in this study are presented in Supplementary Table 2. The qRT-PCR reaction mixture consisted of SYBR Premix Ex Tap™ II (TaKaRa, Japan), 10 μL of SYBR Premix, 1 μL of each primer (10 μM), 2 μL of RNA template (10 × diluted cDNA), and 6 μL of ddH₂O in a total volume of 20 μL with five repeats in Real-Time PCR Detection System (Bio-Rad, United States). Amplification was started with a denaturation step of 94°C for 2 min, followed by 40 cycles of 94°C for 30 s, 60°C for 20 s, and 72°C for 30 s, and the last extension at 72°C for 2 min. To standardize qRT-PCR data, the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was utilized as the reference gene, and the relative expression of all genes was quantified using the 2^–ΔΔCt technique (Livak and Schmittgen, 2001 (link)).

The expression of nifH, CAT, PAL, SOD, CHI and GLU genes in leaf tissues of sugarcane during plant-microorganism interaction was analyzed under a greenhouse condition after inoculation of selected strains (CY5 and CA1) in GT11 and GXB9 sugarcane varieties. Both sugarcane varieties were provided by Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China. The relative expression of all genes was measured by calculating the difference in the expression level of the inoculated sample and the control at 60 and 120 days with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene.
Real-time quantitative PCR (RT-qPCR) was conducted with SYBR Premix Ex Tap™II (TaKaRa, Japan) and the reaction was completed in RT-PCR System (Bio-Rad, USA). The total reaction volume was 20 μL, the composition of the reaction mixtures and conditions were followed by Niu et al. [98 ]. The list of primers used is presented in Table 5. The specificity of the reaction was confirmed by melting curve analysis at the end of the amplification and 2^−△△Ct method was used for quantification of relative gene expression [107 (link)]. Each RT-qPCR experiment was conducted in triplicate.

To validate the results of pyrosequencing analysis, we determined the expression levels of 10 DEGs by qRT-PCR. Total RNAs from each sample were extracted using TRIzol reagent (Invitrogen) and qRT-PCR was performed using SYBR® Premix Ex Tap™ II (TaKaRa) according to the manufacturer’s instructions. qRT-PCR cycles were carried out on a Step One Real-Time PCR system (ABI) as follows: 30 s at 95 °C for denaturation, followed by 40 cycles of 5 s at 95 °C for denaturation, 30 s at 60 °C for annealing. Fluorescence data was collected at 60 °C. A melting curve was performed from 60 °C to 95 °C (held for 1 s per 0.1 °C increase) to examine the specificity of the amplified product. Primers used in qRT-PCR for validation of differentially expressed genes are shown in Additional file 1: Table S1, an actin gene from woodland strawberry was selected as the reference gene. Expression quantification and data analysis were performed in accordance with Bustin et al. [23 (link)].