Transstart tiptop green qpcr supermix

mRNA level was measured with quantitative polymerase chain reaction (qPCR). Nucleospin RNA II isolation kit (Macherey-Nagel, Duren, Germany) was used to extract total RNAs from frozen tissues according to the manufacturer’s protocol. A PrimeScript^TM RT reagent kit (TransGen Biotech, China) was used to perform RNA reverse transcription into cDNA, and then qPCR was performed using the TransStart TipTop Green qPCR SuperMix (TransGen Biotech, China) according to the manufacturer’s instructions. The primer sequence for human VEGF were 5′-CTACCTCCACCATGCCAAGT-3′ (F) and 5′-GCAGTAGCTGCGCTGATAGA-3′ (R). The thermal conditions for the qPCR assay in the Applied Biosystems StepOne^TM Real-Time PCR thermal cycler (SN-271004043, Thermo Fisher Scientific, United States) were as follows—cycle 1: 95°C for 10 min, cycle 2 (×40): 95°C for 10 s and 58°C for 45 s. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and relative quantities were calculated using the 2^–ΔΔCt method. Triplicate independent experiments were performed.

The levels of gene-specific mRNA were assessed using RT-qPCR as per our previous study [54 (link)]. In brief, total RNA was carefully extracted from frozen mycelia using a FastRNA Pro Red Kit (MPbio, Irvine, CA, USA) as per the manufacturer’s instructions. Total RNA (500 ng) was reverse-transcribed and cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA eraser (TaKaRa, Japan), according to the manufacturer’s instructions. qPCR was performed using an ABI StepOne thermocycler (Applied Biosystems, Foster City, CA, USA) and the TransStart TipTop Green qPCR SuperMix (TransGen, Shanghai, China) with 200 nM of forward and reverse primers (Additional file 1: Table S1). Thermal cycling was conducted under the following conditions: an initial denaturation step at 95 °C, followed by 40 amplification cycles of 5 s at 94 °C and 60 s at 64 °C. For transcription analysis, an SYBR green assay with reference to the sar1 gene was performed [57 (link)]. Melt curves were obtained after each RT-qPCR run to confirm the specificity of amplification and the absence of primer dimers.

The levels of gene-specific mRNA were assessed using RT-qPCR, according to our previous study, with some modification [59 (link)]. In brief, the total RNA of 50 mg fresh weight cells was extracted using a FastRNA Pro Red Kit (MPbio, Irvine, CA, USA), according to the manufacturer’s instructions. Synthesis of cDNA from total RNA was performed using the PrimeScript RT Reagent Kit with gDNA eraser (TaKaRa, Japan) as per the manufacturer’s instructions. For RT-qPCR, the TransStart TipTop Green qPCR SuperMix (TransGen, Shanghai, China) was used with 200 nM of forward and reverse primers (see Additional file 1: Table S1). Gene transcription was analyzed using SYBR green assays. Transcription levels of target genes were normalized to that of the sar1 gene [60 (link)]. Thermocycling was performed in an ABI StepOne Plus thermocycler (Applied Biosystems, Foster City, CA, USA).

For RT-qPCR analysis, L4 stage worms were washed with dH₂O for at least three times to remove the bacteria. dH₂O was removed as much as possible, and 1 mL RNAiso plus (Takara, Tokyo, Japan, Code 9108) was added and then quickly frozen in liquid nitrogen. RNA extraction was carried out, as previously described [45 (link)]. Concentration and purity of RNA samples were determined with a NanoDrop spectrophotometer and gel electrophoresis, respectively. Samples were stored at −80 °C for future use. Reverse transcription was performed with 1 μg RNA per sample using PrimeScript^® RT reagent Kit with gDNA Eraser (Takara, Tokyo, Japan, Code RR047A). Diluted cDNA and custom-designed primers were mixed with SYBR Green (TransStart TipTop Green qPCR SuperMix, TransGen Biotech, Beijing, China, Code AQ141), and samples were analysed using a ABI 7900HT analyzer (Applied Biosystems, Foster City, CA, USA). RT-qPCR data presented were from at least three independent biological replicates, and all values were normalized to tubulin as an internal control.

For analysis of gene expression, total RNA was isolated using Trizol according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg of total RNA with the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa Bio Engineering Co., Ltd., Dalian, China). The relative mRNA expression from the transgenic plants was analyzed by quantitative reverse transcription PCR (qRT-PCR) using the TransStart TipTop Green qPCR SuperMix (Transgen, Yongtaizhuang North Road, Haidian District, Beijing, China), according to the manufacturer’s instructions. β-actin was used as an internal control. Amplification of targeted genes was performed using the LightCycler^® 480 II system (Roche, Penzberg, Germany). The analysis of relative mRNA expression data was performed using the 2^−ΔΔCt method [55 (link)]. qRT-PCR primers were designed to avoid conserved regions and to amplify 150 to 300 bp products (Table S2). qRT-PCR was performed with three independent biological replicates.

Total RNAs were extracted and isolated through Trizol reagent (Invitrogen) as guided by the manufacturer’s instructions. For reverse transcription, Prime Script TMRT-reagent kit (TransGen Biotech, China) was used while Trans Start Tip Top Green qPCR Super Mix (TransGen Biotech, China) was used for qPCR according to the instructions given by the manufacturer. The primers sequences were as follows. MALAT1: 5′-GCA​TTA​ATT​GAC​AGC​TGA​CCC​A-3′ (F), 5′-GCT​TGC​TCC​TCA​GTC​CTA​GCT​T-3′ (R); FUT4: 5′-ACAACTGTTCCCGATTCACG-3′(F), 5′-TGCCCTCCTCACCTTTCTC-3′(R); STAT3: 5′-CTT​TGA​GAC​CGA​GGT​GTA​TCA​CC-3′ (F), 5′-GGTCAGCATGTTGTACCACAGG-3′(R); GAPDH: 5′-AGC​CCA​TCA​CCA​TCT​TCC​AG-3′ (F), 5′-ACC​CAT​CAC​AAA​CAT​GGG​GG-3′. GAPDH was used to normalize the data and the relative measurements were determined using the 2^−ΔΔCt method. All the experiments were performed independently in triplicate.