Sybr green pro taq hs premixed qpcr kit

Used Trizol kit (TIANGEN BIOTECH CO., LTD.) to extract total RNA from tubers of pigmented potatoes in different growth periods. Use Evo M-MLV reverse transcription premixed kit (including gDNA removal reagent for qPCR) Ver 2 (Accurate Biology), used SYBR ^® Green Pro Taq HS premixed qPCR kit (including ROX) (Accurate Biology) conducts real-time fluorescent quantitative PCR reaction. StGAPDH was the reference gene. The cDNA obtained by reverse transcription was diluted 10 times and used as a template. The primers of all genes (Table 1) were designed with SnapGene 4.3.6 software and synthesized by Sangon Biotech (Shanghai) Co., Ltd. All primers were diluted 10 times before being used. Reaction procedure: 95 °C 2 min, 95 °C 15 sec, 60 °C 30 sec, 40 cycles, Melt Curve Stage, 2^{- ΔΔ Ct} method (Livak and Schmittgen, 2001 (link)).

According to the instructions, the total RNA of tissue samples was extracted by using MolPure^® Cell/Tissue Total RNA Kit (YEASEN CAT#19221ES50). The cDNA of samples was synthesized from 2 µg RNA via Evo M‐MLV reverse transcription master mix (Accurate Biology, CAT#AG11706). The qRT-PCR was performed using a SYBR Green Pro Taq HS premixed qPCR kit (Accurate Biology, CAT# AG11701). The ITGAL primer sequence is as follows: forward 5′-CTG​CTT​TGC​CAG​CCT​CTC​TGT-3′ and reverse 5′-GCT​CAC​AGG​TAT​CTG​GCT​ATG​G-3′. GAPDH: forward 5′-CGG​AGT​CAA​CGG​ATT​TGG​TCG​T-3′ and reverse 5′-TCT​CAG​CCT​TGA​CGG​TGC​CA-3′. The 2−ΔΔCT calculation method was used to determine the relative target gene level.

Total RNA was isolated from cultured cells using TRIzol reagent (Accurate Biology). cDNA was synthesized with Evo M-MLV reverse transcription master mix (Accurate Biology) from 500 ng of RNA from each sample. A SYBR@ Green Pro Taq HS premixed qPCR kit (Accurate Biology) was used for qPCR. Data are shown as ‘2-ΔΔCT’. The primers are listed in Supplementary Table 1.

Total RNAs were isolated by using TRIzol Reagent (CW0580S, CWBIO) according to the manufacturer’s instructions. Then, cDNAs were synthesized with the Reverse Transcriptase kit (E047-01B, Novoprotein) as we previously reported (12 (link)). After that, qRT-PCR analysis was conducted by using the SYBR Green Pro Taq HS premixed qPCR kit (AG11701-S, Accurate Biotechnology) and the ABI PRISM 7900 (Applied Biosystems). In details, an initial denaturation step of 95°C for 30s, followed by 40 cycles of 95°C for 5s and 60°C for 30s. The results were calculated by using 2^-ΔΔCt method. The primer sequences were shown in Supplementary Table S1. GAPDH was used as internal reference.

Total RNA was extracted from the cell lines using AG RNAex Pro RNA Kit (AG21101, Accurate Biotechnology, Changsha, Hunan, China). Subsequently, cDNA was synthesized and reverse transcribed using the Evo M-MLV RT Kit with gDNA Clean for RT-qPCR (AG11705, Accurate Biotechnology, Changsha, Hunan, China). Real-time polymerase chain reaction (RT-PCR) was performed with the SYBR Green Pro Taq HS premixed qPCR kit (AG11701, Accurate Biotechnology, Changsha, Hunan, China), and expression levels were calculated using the 2^{^(−ΔΔCt)} method. mRNA expression was normalized to the expression level of β-actin mRNA. All primers were provided by Accurate Biotechnology Co., Ltd. (Changsha, Hunan, China), and the detailed primer sequences are shown in Supplementary Table S1. All data are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Quantitative RT-PCR was employed to analyze the expression levels of nitrate reductase (NR), nitrite reductase (NiR), asparagine synthetase (AS), asparaginase (ASPG), glutamine synthase (GS), glutamate synthase (GOGAT), cell wall apoplastic invertase (CWINV1), and vacuolar invertase (VI2) genes in the leaves, stem, and roots of poplar. Total RNA was extracted by using RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (TIANGEN, Beijing, China). The purity and concentration of the extracted RNA were checked by the Microvolume Spectrophotometer (Colibri LB 915, Bad Wildbad, Germany) and then verified through agarose gel electrophoresis. Reverse transcription kit (PrimeScriptTM RT reagent Kit with gDNA Eraser, Takara, Japan) was used to synthesize the first strand of cDNA. Gene-specific primers (Supplement Table S1) were designed by Primer3 (http://primer3.ut.ee/, accessed on 21 February 2022). Quantification of gene amplification was performed on the real-time fluorescent quantitative PCR instrument (Applied Biosystems StepOne^TM, Beijing, China) with the fluorescent agent AG SYBR Green Pro Taq HS premixed qPCR kit (Accurate Biology, Changsha, China).