Tb green premix ex taq 2 kit

Total RNA was extracted using Trizol reagent (Invitrogen, Cat. 15596026, Waltham, MA, USA) and digested with RNase-free DNase I (TaKaRa, Dalian, China), and 2 μg of total RNA was used for the first-strand cDNA synthesis (TaKaRaPrimeScript™ II 1st strand cDNA synthesis kit, Code No. 6210A) based on the manufacturer’s protocol. QRT-PCR was performed with ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Waltham, MA, USA) using the TB green™ premix Ex Taq™Ⅱ kit (Takara, Dalian, China). The total volume of 20 μL reaction mixture consisted of 10 μL 2 × TB Green Premix Ex Taq Ⅱ (ROX plus), 0.8 μL gene-specific forward and reverse primers (10 μM each), 1 μL cDNA template, and 7.4 μL RNase-free water. Reaction conditions were 95 °C for 5 min followed by 40 cycles of 10 s at 95 °C, 30 s at 60 °C. Planarian elongation factor 2 (Djef2) was utilized as the reference gene in all of the experiments [32 (link)]. Expression ratios were determined with the 2^−ΔΔCT method, which was described by Livak and Schmittgen [33 (link)]. The primers used in this work were listed in Supplementary Excel File S1.

To verify the accuracy and reliability of differentially expressed RNAs (DE RNAs) screened by RNA-seq, six DE mRNAs, six DE lncRNAs, and six DE miRNAs were randomly selected for RT-qPCR. We used the PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Dalian, China) to convert RNAs to cDNAs. The random hexamers were used for DE mRNAs and DE lncRNAs, and stem-loop RT primers were used for DE miRNAs. RT-qPCR was performed using the TB Green^® Premix Ex Taq™Ⅱ Kit (Takara, Dalian, China) according to the manufacturer’s instructions. GAPDH for mRNAs and lncRNAs and U6 for miRNAs were used as endogenous controls. All primers used in RT-qPCR are designed using Primer Premier 5 software and exhibited in Supplementary Data S1. RT-qPCR was carried out in the LightCycler480 system (Roche, Swiss Confederation) with three replicates for each sample. The data were expressed as values relative to the E10 group. The relative expression levels of DE mRNAs, DE lncRNAs, and DE miRNAs were calculated using the 2^−ΔΔCT method, and one-way ANOVA in SPSS 20.0 was used to analyze the data. Differences were considered significant at p < 0.05.
All tools, databases, and related information used in this study are shown in Supplementary Data S2.

After total RNA quality control, total RNA was reverse-transcribed into cDNA by using an All-In-One 5× RT MasterMix kit (ABM, Richmond, BC, Canada), and experiments were carried out according to the kit instructions. The RT-qPCR primers of GAPDH, FHIT, and PIAS1 genes were designed using Primer Premier 5.0 software, and the primers were synthesized by Shengong Bioengineering Co., Ltd. (Shanghai, China) The detailed information of RT-qPCR primers is given in Table S4.
Then, RT-qPCR was performed using a TB Green Premix Ex TaqⅡkit (Takara, Shanghai, China); the reaction system of RT-qPCR is shown in Table S5 and the amplified conditions are shown in Table S3.
Finally, the 2^−∆∆Ct method was used to calculate relative gene expression, using the following formula:

In this study, an Applied Biosystems QuantStudio 3 real-time fluorescence quantitative PCR system was used to reverse transcribe the extracted total RNA into cDNA according to the instructions of a PrimeScript™ RT Master Mix Kit (RR036A, TAKARA). Then, based on the cDNA sequences of the goat PDGFRA, WNT5A, BMPR2, and BMPR1A genes published by NCBI, specific primers were designed with Primer 5.0 (Table 3) and synthesized by Shanghai Bioengineering Co., Ltd. Finally, a TB Green “Premix Ex Taq” II Kit (RR820A, TAKARA) was used for qRT–PCR. Six samples were tested for each month, and three technical replicates were performed.

TRIzol Reagent (Cat#15596018, Invitrogen, United States) was used to isolate the total RNA of samples according to the manufacturer’s protocol. The RevertAid First Strand cDNA Synthesis Kit (Cat#RR036A, TAKARA, Japan) was used to synthesize cDNA from 500 ng of total RNA. Real-time quantitative PCR (qRT-PCR) was carried out by using TB Green Premix Ex Taq II Kit (Cat#RR820A, TAKARA, Japan) on BioRad CFX96 system. Expression levels of the target genes were normalized to a housekeeping gene GAPDH. Gene expression values are stated as 2^–ΔΔt. The sequences of the primers are listed in Supplementary Data 1. All results were performed by Student’s t-test or Linear regression using GraphPad Prism 8. Statistical analysis was expressed as mean with SEs, and differences were considered significant when a value of p < 0.05.

To detect the transcript levels of the target genes, total RNA was extracted from the brain and colon tissues of each group of mice. After the RNA concentration is determined, it is transcribed to cDNA using the PrimeScript™ RT kit (Takara, RR047A). RT-qPCR was performed using the TB Green Premix Ex Taq II Kit (Takara, RR820A). The program was 95 °C (30 s), followed by 40 cycles of 95 °C (5 s) and then 60 °C (34 s). Primers are listed in Supplementary Material Table S1. Analyzed using the 2^−ΔΔCt method [22 (link)].

Total RNA was extracted from OSCC tissue and ANT using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The cDNA was generated in the T100 gradient PCR machine (Bio-rad, USA) using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). RT-qPCR was performed using TB Green Premix Ex Taq II Kit (Takara, Japan) on an AriaMx Real-time quantitative PCR machine (Agilent, USA). The PCR reaction conditions were 95℃ for 30 s, followed by 40 cycles at 95℃ for 5 s and 60℃ for 30 s. Each reaction was performed in triplicate. The expressions of the angiogenic genes including ANG, bFGF, EGF, HGF, HB-EGF, VEGF, PDGF-BB, Leptin, and MMPs including MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1, TIMP-2, and TIMP-4 were analyzed by RT-qPCR. GAPDH gene was used as the internal control. The fold change relative to the control group was measured by the 2^−∆∆Ct method. Primer sequences used in this study are listed in Table S2.