Sybr green qpcr master mix

Bladder cancer and normal bladder cell lines were used to extracted total RNA using cell total RNA isolation kit (Foregene, China) according to manufacturer’s protocol. cDNA was synthesized using UeIris II RT-PCR System for First-Strand cDNA Synthesis (US Everbright, China). qRT-PCR was performed using SYBR Green qPCR Master Mix (US Everbright, China) on CFX Connect System (Bio-Rad, United States). Gene expression levels were normalized to the “housekeeping” gene GAPDH. The primers were designed and synthesized by Sangon Biotech (Shanghai, China) and detailed primer sequences were listed below: ACER2: (forward primer: 5′- CCT​TTG​GGT​TCT​GAT​GTG​TGC​TTT​G-3′; reverse primer: 5′-GGA​CAC​TGA​CCA​CCA​CCT​TGA​AC-3′); GAPDH: (forward primer: 5′- CAA​GGC​TGT​GGG​CAA​GGT​CAT​C-3′; reverse primer: 5′- GTG​TCG​CTG​TTG​AAG​TCA​GAG​GAG-3′).

In this study, five randomly selected candidate genes for DEGs were used for RT-PCR analysis. The specific primers for these five genes and actin gene (an internal control) were designed by Primer 5.0 (Supplementary Table 2). The qRT-PCR reaction (10 μL) was formulated using the 2 X SYBR Green qPCR Master Mix (US Everbright^®Inc., Suzhou, China). All qRT-PCRs were carried out on a CFX96 Touch^TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States). Three biological replicates (from three independent RNA samples) were used for qRT-PCRs. For each biological replicate, three technical replicates were also conducted. The average threshold cycle (Ct) from three biological replicates was employed to calculate the gene expression fold change by the 2^–ΔΔCT method (Kong et al., 2019a (link),b (link)).

RNA extraction, cDNA library construction, and library sequencing on Illumina HiSeq2500 platform of 12 samples were strictly implemented by Biomarker Technologies (Beijing, China) in accordance with standard procedures (Kong et al. 2020 ). Raw data were filtered by fastp (Chen et al. 2018 (link)) and mapped to the Nipponbare genome (MSU v7.0) using hisat2 (Kim et al. 2015 (link)) with default parameters. The mapped reads were counted by featureCounts (Liao et al. 2014 (link)) and differentially expressed genes (DEGs) in QTLs were identified by DEseq2 with |log₂^{fold change}|≥ 1 and a False Discovery Rate (FDR) < 0.01 (Kong et al. 2020 ). The heatmap of DEGs was also conducted by TBtools (Chen et al. 2020 (link)).
Finally, three randomly selected candidate DEGs in QTLs were verified by RT-PCR according to the previously described method (Kong et al. 2019 ). All primers of RT-PCR were designed by Primer 5.0 software (Additional file 4: Table S3). The qRT-PCR reaction (10 μL) was formulated using the 2 X SYBR Green qPCR Master Mix (US Everbright®Inc., Suzhou, China). All qRT-PCRs were carried out on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The gene expression fold change was calculated by the 2^−ΔΔCT method from three biological replicates.

Rice salt-responsive (OsGolS1) or cold-responsive GT8 genes (OsGAUT21, OsGATL2, and OsGATL5) from microarray analyses were verified by using qRT-PCR. Primers of these four rice GT8 genes were designed by Primer 5.0 (Table S1). The qRT-PCR reaction (10 μL) was formulated using the 2 X SYBR Green qPCR Master Mix (US Everbright^® Inc., Suzhou, China). All qRT-PCRs were carried out on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The actin gene was employed as an internal control [37 (link)]. Three biological replicates (from three independent RNA samples) were used for qRT-PCR. For each biological replicate, three technical replicates were also used. The average threshold cycle (Ct) from three biological replicates was employed to calculate the gene expression fold change by the 2^−ΔΔCT method [37 (link),49 (link)].

All RNAs were extracted by the TRIzol Ragent (Invitrogen, Beijing, China) and reverse transcriptions of all RNAs were performed using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Shanghai, China), according to their instruction manuals. The qRT-PCR reaction (10 μL) was formulated using the 2X SYBR Green qPCR Master Mix (US Everbright^®Inc., Suzhou, China). Primers of rice 19 STP genes were designed by Primer 5.0 and displayed in Table S1. All qRT-PCRs were carried out on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The actin gene was used as an internal control [32 (link)]. Three biological replicates (from three independent RNA samples) of each treated point were used for qRT-PCR. For each biological replicate, three technical replicates were used. The average threshold cycle (Ct) from three biological replicates was used to calculate the gene expression fold change by the 2^−ΔΔCT method [4 (link),32 (link)].