Quantitative PCR using the bacteria-specific 16S rRNA gene was performed using the method described elsewhere [27 (link)]. The 16S rRNA gene primers (338F: 5′-ACTCCTRCGGGAGGCAGCAG-3′, 806R: 5′-GGACTACCVGGGTATCTAAT-3′) were used to amplify the 16S rRNA gene in the extracted DNA. A 10 μL reaction mixture contained 5 μL of 2X ChamQ Vazyme SYBR qPCR master mix, 0.4 μL of each primer (0.4 μΜ), 1 μL of DNA template, and 3.2 μL nuclease-free molecular-grade water. A CFX ConnectTM Real-Time System (Bio-Rad CFX Connect Real-Time System) was used to perform qPCR. The following qPCR regime was used: denaturation at 94 °C for 3 min, 40 cycles at 94 °C for 15 s, annealing at 50 °C for 30 s, and elongation and acquisition of fluorescence data at 72 °C for 30 s. A standard curve was constructed using known amounts of target template generated by PCR amplification of the target gene from genomic DNA of E. coli, as previously described (see Section 2.3.1 ).
Cfx connect real time system
Manufactured by Bio-Rad
Sourced in United States, China, Japan, Germany, Singapore, United Kingdom, Switzerland, Canada, Spain, Italy, Hong Kong, France
The CFX Connect Real-Time System is a qPCR (quantitative Polymerase Chain Reaction) instrument manufactured by Bio-Rad. It is designed to perform real-time PCR experiments, enabling the quantification of DNA or RNA targets in a sample.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (244 mentions)
Trizol, by Thermo Fisher Scientific (168 mentions)
Iscript cdna synthesis kit, by Bio-Rad (136 mentions)
Rneasy mini kit, by Qiagen (134 mentions)
Primescript rt reagent kit, by Takara Bio (88 mentions)
Itaq universal sybr green supermix, by Bio-Rad (85 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (74 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (67 mentions)
Iq sybr green supermix, by Bio-Rad (62 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (62 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (42 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (33 mentions)
Trizol reagent, by Takara Bio (31 mentions)
Sybr premix ex taq, by Takara Bio (28 mentions)
Cfx manager 3, by Bio-Rad (28 mentions)
Cfx manager software, by Bio-Rad (27 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (27 mentions)
M mlv reverse transcriptase, by Promega (26 mentions)
Rneasy kit, by Qiagen (25 mentions)
Sybr green, by Bio-Rad (25 mentions)
1 737 protocols using cfx connect real time system
1
Quantitative PCR for 16S rRNA Gene Analysis
2
Quantifying Fungal Biomass and Gene Expression
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For mycelial qRT-PCR, total RNA extraction was performed using the RNeasy® Plant Mini Kit (Qiagen, Germany), and cDNA synthesis was completed using the PrimeScript RT Reagent Kit (RR036A, Takara, Dalian, China) according to the manufacturer's instructions. The qRT-PCR was performed using SYBR Premix Ex Taq II (RR820A, Takara, Dalian, China) and the CFX Connect Real-Time System (Bio-Rad, Hercules, USA).
For biomass assay, four-day-old T. guizhouense mycelia growing at 37 °C under SSF were collected and mixed evenly, and the total DNA was extracted with the EZNATM Soil DNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA). The number of T. guizhouense copies was determined with SYBR Premix Ex Taq II (RR820A, Takara, Dalian, China), and the amplification of the fragment using the extracted DNA as a model was performed on a CFX Connect Real-Time System (Bio-Rad, Hercules, USA).
For biomass assay, four-day-old T. guizhouense mycelia growing at 37 °C under SSF were collected and mixed evenly, and the total DNA was extracted with the EZNATM Soil DNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA). The number of T. guizhouense copies was determined with SYBR Premix Ex Taq II (RR820A, Takara, Dalian, China), and the amplification of the fragment using the extracted DNA as a model was performed on a CFX Connect Real-Time System (Bio-Rad, Hercules, USA).
3
Quantitative PCR Assay for Gene Expression
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Quantitative PCR assay was performed according to the protocols described by Tang et al.4 (link) Briefly, total RNA was extracted by TRizol reagent (KeyGen Biotech, Nanjing, China) according to the manufacturer’s instruction. cDNA was then synthesized with PrimeScript™ RT Master Mix (Perfect Real Time) obtained from TAKARA Co., Ltd. (Tokyo, Japan). Quantitative RT-PCR was conducted by SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) on CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA, USA). The specific primers were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and are shown in Supplementary Table 1
4
Quantifying Liver Gene Expression in Mice
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SYBR green quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect mRNA levels of PCSK9, LDLR, ABCA1, ABCG1, and SR-B1. The Trizol method was used to extract total RNA from mouse liver tissue. RNA yield and purity was confirmed by measuring the ratio of the absorbance at 260 and 280 nm. cDNA was synthesized using the SuperScript III First-Strand Synthesis System. The qRT-PCR reaction, containing target genes and SYBR Green PCR master mix, was performed on a Bio-Rad CFX connect real-time system (Bio-Rad, USA). The qRT-PCR containing target genes and SYBR Green PCR master mix was carried out on a Bio-Rad CFX connect real-time system (Bio Rad, USA) at 95 °C for 3 min, cycled at 95 °C for 10 s, 56 °C for 30 s and 72 °C for 30 s for 42 cycles. Melt curves were performed from 56.0 °C to 95.0 °C with intervals at 0.5 °C for 5 seconds. Relative RNA levels were determined by analyzing the changes in SYBR Green fluorescence by the 2−ΔΔCT method according to the manufacturer’s instructions. GAPDH was amplified in parallel and the results were used for normalization. The PCR product was confirmed by gel electrophoresis on a 2% agarose gel stained with ethidium bromide. Purity of amplified PCR products was determined by melting point analysis using ICycler software. All experiments were performed in triplicate.
5
Quantitative RT-PCR Analysis of Gene Expression
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Overnight bacterial cultures were diluted 1:100 into fresh LB and grown to an OD600 of 1 in LB. Three hundred microliters of the bacterial cells was collected by centrifugation at 10,000 × g for 1 min, followed by RNA isolated with a bacterial total RNA kit (Zomanbio, Beijing, China). Qualities of the RNA samples were determined by measuring OD260/OD280 and OD260/OD230. Samples with both ratios of ≥2.0 were used for following experiments. A 1.0-μg amount of RNA was used to synthesize cDNA with reverse transcriptase and random primers (TaKaRa, Dalian, China). The same amount of cDNA was mixed with the SYBR II green supermix (Bio-Rad, Beijing, China) and specific primers (Table S2). Real-time PCR was performed with the CFX Connect real-time system (Bio-Rad, USA). Since (p)ppGpp has been shown to inhibit the expression of rRNA and ribosomal protein genes (52 (link)), we used previously reported housekeeping gene PA1805 as the internal control for normalization (53 (link)). Primers were designed with a software program (Primer Premier 5; Bio-Rad, USA), and the sequences ae listed in Table S2. Melting curve analyses were performed with the CFX Connect real-time system (Bio-Rad, USA) to verify the specific amplification of target regions. Each sample was tested in triplicate.
6
Quantification of miRNA and mRNA Levels
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Total RNA was extracted as previously de ned. For mature miRNAs, All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia) was used to generate 10 µL cDNA. Then using miR-speci c primers and universal adaptor PCR primers (Genecopoeia), qRT-PCR was performed with All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia) on CFX Connect Real-Time System (Bio-Rad). For mRNA, the reverse transcription was carried out using HiScript II Q RT SuperMix (Vazyme), and qRT-PCR was performed with ChamQ™ SYBR Color qPCR Master Mix (Vazyme) on CFX Connect Real-Time System (Bio-Rad) (21) . Speci c primers for qRT-PCR of mRNA were listed in Supplementary Data. The U6 small nuclear RNA or GAPDH was used as an endogenous control. Each experiment was repeated at least three times and the results were analyzed using 2 -△△CT method.
7
Quantification of miRNA and mRNA Expression
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Total RNA was extracted as previously defined. For mature miRNAs, All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia) was used to generate 10 μL cDNA. Then using miR-specific primers and universal adaptor PCR primers (Genecopoeia), qRT-PCR was performed with All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia) on CFX Connect Real-Time System (Bio-Rad). For mRNA, the reverse transcription was carried out using HiScript II Q RT SuperMix (Vazyme), and qRT-PCR was performed with ChamQ™ SYBR Color qPCR Master Mix (Vazyme) on CFX Connect Real-Time System (Bio-Rad) [21 (link)]. Specific primers for qRT-PCR of mRNA were listed in Supplementary Data . The U6 small nuclear RNA or GAPDH was used as an endogenous control. Each experiment was repeated at least three times and the results were analyzed using 2-△△CT method.
8
Real-time Fluorescent RT-CPA for Virus Detection
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Real-time fluorescent reverse transcription-CPA (RT-CPA) was carried out to detect the efficiency and specificity of the primers using a Bio-Rad CFX Connect Real-Time System (America). The total RNA extracted from the healthy leaves of White Burley was used as a negative control. A real-time fluorescent RT-CPA reaction was performed in a total volume of 20 µl containing 8 U of Bst DNA polymerase, 10 U of AMV reverse transcription polymerase, 0.3X ThermoPol buffer, 12.5 mM betaine, 2 mM MgSO4, 0.1 mM dNTP, 2.5 µM SYTO™ 16 Green Fluorescent Nucleic Acid Stain (Thermo Fisher), 1 µM BPCPF, 0.6 µM BPDR, 0.6 µM BPMBR, 0.2 µM BPBF, 0.2 µM BPBR, and 50 ng RNA template. Reactions were carried out with a Bio-Rad CFX Connect Real-Time System (America) at isothermal temperature (60 °C) for 90 min. FAM was chosen as the selector fluorescent light channel with an excitation wavelength of 495 nm and an emission wavelength of 517 nm.
Specificity tests were performed by applying the real-time fluorescent RT-CPA assay to the total RNA extracted from the leaves of White Burley infected by BPMV, SBMV, ToRSV, ArMV, and TRSV.
Specificity tests were performed by applying the real-time fluorescent RT-CPA assay to the total RNA extracted from the leaves of White Burley infected by BPMV, SBMV, ToRSV, ArMV, and TRSV.
9
Quantitative PCR Analysis of RNA Expression
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RT was performed as above. For qPCR analysis results in Supplementary Figs. 2A and 3C , 2 µL of first strand cDNA was taken for qPCR analysis in Promega GoTaq® qPCR Master Mix (Ref: A6001). Reactions were run at 95 °C for 3 min, followed by 50 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 20 s with melt curve analysis in the end in a BioRad CFX ConnectTM Real-Time System. For all N1 TaqMan qPCR analysis results, RT was performed as above in RT section by taking 5 µL of sample in 25 µL RT reaction. For qPCR analysis, 25 µL of PCR1 top up reaction mix with 1.5 µL of CDC-N1 primer/probe set (IDT 10006713/sub part 10006600) was added to above 25 µL RT reaction mix. Reactions were run at 95 °C for 3 min, followed by 45 cycles of 95 °C for 15 s and 55 °C for 45 s in a BioRad CFX ConnectTM Real-Time System.
10
Thermal Shift Assay for SOD1 Protein
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Thermal shift assays were performed on the Bio-Rad CFX ConnectTM Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA) using a Protein Thermal ShiftTM Dye Kit (Thermo Fisher Scientific, USA). SOD1 proteins (30 μM) in 20 mM Tris (pH 8.0) buffer containing 100 mM NaCl were used in the thermal shift assays. The temperature was measured from 4 °C to 95 °C at intervals of 1 °C to calculate the Tm value of proteins. Data were collected as three individual endpoint readings for each 1 min cycle. The predefined experimental method of the CFX instrument was chosen to enable software calculation of the first derivative values for the denaturation curve raw data points. The melting temperature of a protein (Tm, the temperature at which there is 50% denaturation) was determined by the temperature at the maximum velocity of the fluorescence curve, which is the temperature at the local minimum point of first derivatives [-d(fluorescence)/dT].
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