Total RNA was isolated from the whole blood samples of the enrolled subject at the day of blood collection according to the manufacturer’s instructions (Mei5 Biotechnology, Co., Ltd). Then, the RNA samples were preprocessed using gDNA plus remover mix to remove genomic DNA contamination, and then, the RNA was reverse transcribed to cDNA using M5 RT Super plus Mix. HiPerSYBR Premix Estaq (Mei5 Biotechnology, Co., Ltd) was used to perform the real-time PCR on a Roche LightCycler 480 system, and the relative expression of the genes was calculated using the 2−ΔΔCt method after the normalization with the endogenous GAPDH mRNA expression. The primer sequences of the 6 DEMRs are listed in Table 1
Hiper sybr premix estaq
Manufactured by Mei5 Biotechnology
Sourced in United States, China
HiPer SYBR Premix EsTaq is a ready-to-use PCR master mix that contains all the necessary components for real-time PCR, including SYBR Green I dye, Taq DNA polymerase, dNTPs, and optimized buffer. It is designed for efficient and sensitive quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Reverse transcription, by Vazyme (2 mentions)
Prism v8, by GraphPad (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 system, by Roche (1 mentions)
6 well plate, by Corning (1 mentions)
Quantstudio 3 rt qpcr system, by Thermo Fisher Scientific (1 mentions)
Hiscript 2 q rt supermix, by Vazyme (1 mentions)
Cdna synthesis kit, by Transgene (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
All in one 5 rt mastermix kit, by Applied Biological Materials (1 mentions)
Smarter race 5 3 kit, by Takara Bio (1 mentions)
Rnaclean kit, by Aidlab (1 mentions)
Easyspin kit, by Aidlab (1 mentions)
M5 super qpcr rt kit with gdna remover, by Mei5 Biotechnology (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Superscript rt reagent kit, by Takara Bio (1 mentions)
Primer premier 6, by Premier Biosoft (1 mentions)
9 protocols using hiper sybr premix estaq
1
Quantification of Gene Expression from Whole Blood
2
Fluoride-Induced Cellular Responses: Investigating Apoptosis, Oxidative Stress, and Autophagy
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Based on the results of the CCK8 experiment, the experimental groups were set as the control (CK) and experimental (T1, T2, and T3, with NaF concentrations of 3.5, 4, and 4.5 mM, respectively). The cells were resuspended, and the density was adjusted to inoculate into 6-well plates (Corning, NY, USA). After 24 h, 350 µL of TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well, and the cells were gently lysed. Cells were collected into sterile, enzyme-free 1.5 mL centrifuge tubes. The total cellular RNA was extracted and stored at −80 °C. The RNA was reverse-transcribed to cDNA according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA), 2 × M5 HiPer SYBR Premix Es Taq (Mei5bio, Beijing, China) was used for qRT-PCR. Considering the studies indicating fluoride’s potential to induce cellular toxicity through oxidative stress, apoptosis, and autophagy, we selected specific genes for investigation. These included the apoptosis-related genes CASP3, CASP8, and P53; oxidative stress-related genes SOD1, SOD2, CAT, and GPX1; and autophagy-related genes LC3.
3
Quantitative RT-PCR Analysis of RNA
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Total RNA (500 ng) was subjected to reverse transcription with the HiScript II Q RT SuperMix (Vazyme, Nanjing, China). The generated mRNA was subsequently quantificated by using the HiPer SYBR Premix EsTaq (Mei5bio, Beijing, China) and the QuantStudio 3 RT-qPCR System (Applied BioSystems, Foster City, CA, USA). Primers used for real-time PCR are listed in Table S1 .
4
Quantitative Real-Time RT-PCR Analysis of Gene Expression
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Total RNA was extracted using a Yeast RNA kit R6870 (Omega, Norcross, GA, USA), while first-strand cDNA was synthesized by transcribing 1 mg of total RNA with a cDNA synthesis kit (TransGen, Beijing, China), according to the manufacturer’s instructions. Quantitative real-time reverse transcription-PCR (RT-PCR) was conducted with a CFX Connect Real-Time system (Bio-Rad, Hercules, CA, USA) using 2 × M5 HiPer SYBR Premix Es Taq (Mei5bio, Beijing, China) and 200 nM specific primer pairs (Table S2, Supplemental Material ), according to the manufacturers’ instructions. The reaction conditions were as follows: 95 °C for 30 s, followed by 35 cycles of 95 °C for 5 s, 60 °C for 30 s, and melt curve. The relative gene expression levels were calculated using the 2−ΔΔCt method and normalized to ACT1 mRNA levels.
5
Quantifying Antibiotic Resistance Genes
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Residual genomic DNA was completely removed by wipe Mix, and cDNA was synthesized via reverse transcription (Vazyme Biotech Co., Ltd, Nanjing, China). qPCR was performed on the CFX96 real-time PCR detection system (Bio-Rad Laboratories Co., Ltd, Hercules, CA, USA) using HiPer SYBR Premix EsTaq (Mei5 Biotechnology, Co., Ltd, Beijing, China). The mecA primer set was designed using primer Basic Local Alignment Search Tool (BLAST) for the region spanning 36–335 bp, and the housekeeping gene gryB was used as an internal standard for normalization (Table S1 in the supplemental material). The thermal cycling conditions were initial denaturation at 95°C for 2 min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The experiment was repeated three times. Fold changes in mecA expression were illustrated using GraphPad Prism v8.0 (GraphPad Software Inc., San Diego, CA, USA). The mecA gene was amplified using the synthesized cDNA as template via conventional PCR. Products were sequenced using Sanger sequencing and the sequences were comparatively analyzed.
6
Quantifying Fungal and Plant Gene Expression
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Total RNA was extracted from the F. oxysporum mycelia and the plants using TRIzol reagent (Invitrogen) according the manufacturer's instructions. Reverse transcription was performed using the All‐In‐One 5× RT MasterMix Kit (Abm). HiPer SYBR Premix EsTaq (Mei5bio, 2× M5) was used for qPCR to analyse the expression pattern. The relative expression of each gene was calculated using the 2−∆∆Ct method as previously described. All experiments were repeated three times. The primers used in RT‐qPCR are listed in Table S3 .
7
Comprehensive RNA Extraction and Analysis
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Total RNAs used for RACE and RNA‐seq were extracted using Trizol reagent (Thermo Fisher Scientific) and purified using the RNAclean Kit (Aidlab Biotechnologies). Total RNAs used for RT‐PCR were extracted using EASYspin Kit (Aidlab Biotechnologies), following manufacturer's instructions. First‐strand cDNA was synthesized using M5 Super qPCR RT Kit with gDNA remover (Mei5bio). The 5′‐ and 3′‐ RACE were conducted with SMARTer RACE 5′/3′ kit (TaKaRa) following manufacturer's instructions. Real‐time PCR was performed using a CFX96 real‐time system (Bio‐Rad) and Hiper SYBR Premix Es Taq (Mei5bio). Three replicates were performed and rice ACTIN1 was used as the internal control (Livak and Schmittgen, 2001 (link)). Primers used for qRT‐PCR are listed in Table S2 .
8
Quantifying Antibiotic Resistance Genes
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Residual genomic DNA was completely removed by wipe Mix, and cDNA was synthesized via reverse transcription (Vazyme Biotech Co., Ltd, Nanjing, China). qPCR was performed on the CFX96 real-time PCR detection system (Bio-Rad Laboratories Co., Ltd, Hercules, CA, USA) using HiPer SYBR Premix EsTaq (Mei5 Biotechnology, Co., Ltd, Beijing, China). The mecA primer set was designed using primer Basic Local Alignment Search Tool (BLAST) for the region spanning 36–335 bp, and the housekeeping gene gryB was used as an internal standard for normalization (Table S1 in the supplemental material). The thermal cycling conditions were initial denaturation at 95°C for 2 min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The experiment was repeated three times. Fold changes in mecA expression were illustrated using GraphPad Prism v8.0 (GraphPad Software Inc., San Diego, CA, USA). The mecA gene was amplified using the synthesized cDNA as template via conventional PCR. Products were sequenced using Sanger sequencing and the sequences were comparatively analyzed.
9
Extraction and Quantification of Total RNA
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Total RNA was extracted using Trizol reagent (15596026, Invitrogen, Carlsbad, United States) following manufacturer’s protocols. Concentration and purity of RNA were checked with a NanoDrop 2000C (Thermo Scientific, Waltham, MA, United States). Samples had an optical density ratio at 260/280 nm > 1.9 and <2.2. Reverse transcription of the total RNA was conducted using SuperScript™ RT reagent kit (RR047A, TaKaRa, Japan). All primers used were designed with Primer Premier 6 (Premier Biosoft, United States) based on GenBank data. Primer sequences are listed in Table 1 . The housekeeping gene GAPDH was used as an internal control. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the 2 × M5 HiPer SYBR Premix EsTaq (MF787-01, Mei5bio, China).
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