Sybr green premix pro taq hs qpcr kit ag11701

RT-qPCR was used as the reference to test the accuracy of the ratiometric fluorescent biosensor. A total of 3.75 μL of RNAs were reverse transcribed by Mir-X™ miRNA First-Strand Synthesis (Takara, Kusatsu, Japan) on T100TM Thermal Cycler (Bio-Rad, Shanghai, China) to synthesize cDNAs. RT-qPCR was carried out by SYBR^® Green Premix Pro Taq HS qPCR Kit AG11701 (Accurate Biology, Shanghai, China) on a CFX96TM Real-Time System (Bio-Rad, China). The calibration line was drawn based on the real-time fluorescence curves obtained at different concentrations of miR-92a-3p.

Isolation of total RNA from cells entailed the use of Trizol reagent, followed by reverse transcription of 1 μg of total RNA into first-strand cDNA utilizing a reverse transcriptase kit (Thermo Fisher, America). To quantify mRNA levels, qPCR was executed employing the SYBR® Green Premix Pro Taq HS qPCR Kit AG11701 (Accurate Biotechnology, China) on a Real-time fluorescence quantitative PCR system (Bio-Rad, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was enlisted as the internal control, with the 2-∆∆Ct method serving as the calculation methodology. Comprehensive insight into primer sequences employed for qRT-PCR, along with those pivotal for RIP, was collated within Table Supplementary 2 for qRT-PCR and Table Supplementary 3 for RIP.

Three 24 h treatments including submergence (the whole plant was submerged with a water level of 1~2 cm above the shoot tip), waterlogging (water level above the soil for about 1 cm) and a control (CK) were set. After digging out from the soil, the seedlings were immediately washed with tap water. Plants were divided into three parts: ‘leaves’ (twigs and needles), stems, and roots. All materials were stored at −80 °C. RNA extraction and reverse transcription were performed within one month. The mRNA concentration was unified at 100 ng/µL before reverse reaction. cDNA was stored at 4 °C for one month before qPCR. The qPCR experiment was performed using a SYBR^® Green Premix Pro Taq HS qPCR Kit AG11701 produced by Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China, and the reaction was carried out with a StepOnePlus instrument, Applied Biosystem, America. Three biological replicates were conducted in the experiments. Three technical replicates were set for each treatment/organ/gene. The actin gene was exploited as the reference gene [48 (link)], and the qPCR primers are shown in Table S1.