All samples were investigated by Seegene NTM PCR test (Seegene, Soul, South Korea). Overall, 5 µl of an aliquot of the supernatant was mixed with 15 µl of master mix, which contains 10 µl×2 Anyplex PCR master mix, 2 µl 10×MTB/NTM oligonucleotide mix, and 3 µl 8- methoxypsoralen. CFX96 Touch RT-PCR Detection System (Bio-Rad Laboratories Inc., USA) was used for amplification and identification of NTM, which detects the 16 S rRNA gene. For quality control, the kit contains in-house extraction controls, positive and negative amplification, and internal control in the master mix, and we processed them in each run. The interpretation of data was done automatically by CFX Maestro software that showed the results to threshold and cutoff values. Positive and negative controls were used for quality control in each run.
Cfx96 touch rt pcr detection system
Manufactured by Bio-Rad
Sourced in United States
The CFX96 Touch RT-PCR Detection System is a real-time PCR instrument designed for gene expression analysis, genotyping, and other molecular biology applications. The system provides accurate and reliable quantification of nucleic acids with a wide dynamic range. It features a 96-well format and precise temperature control for consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (3 mentions)
Quantitect sybr green kit, by Qiagen (3 mentions)
Quantitect reverse transcription kit, by Qiagen (3 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Cfx manager software, by Bio-Rad (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Transstart green qpcr supermix, by Transgene (2 mentions)
Hardshell low profile thin wall 96 well skirted pcr plates, by Bio-Rad (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Cfx manager 3, by Bio-Rad (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
40 protocols using cfx96 touch rt pcr detection system
1
Rapid Detection of Nontuberculous Mycobacteria
2
Zinc Modulates Gene Expression
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Cells were seeded into 60 mm petri dishes (Primaria, Corning, US) and after a monolayer was formed, cell medium was replaced by SMCM supplemented with 40 μM or 120 μM Zn2+ solutions. After incubation for 24 h, total RNA was isolated by RNeasy Mini Kit (Qiagen, US). The concentration and purity of total RNA were determined by spectrophotometer (Nanodrop 2000, US) at 230 nm, 260 nm and 280 nm. The range of A260/A280 was 2.00–2.05 and the range of A260/A230 was 2.20–2.29. Then 500 ng total RNA was inversely transcripted into cDNA by RT2 First Strand Kit (Qiagen, US), according to the manufacturer’s protocol. RT-PCR was performed in a CFX96 Touch RT-PCR Detection System (Bio-Rad, US) using microarray. cDNA was mixed with RT2 SYBR Green Master Mix (Qiagen, US) and RNase-free water. In the 96-well PCR array plate, 25 μl of the mixture was added to each well. After 10 min incubation at 95 °C, cDNA was amplified according to the following parameters: denaturing for 15 s at 95 °C, then annealing for 1 min at 60 °C. 40 cycles were performed. After the amplification, Ct values were collected and analyzed by Bio-Rad CFX Manager 3.1 (Bio-Rad, US). The cutoff Ct value was 35. The ΔΔCt method was used to calculate the fold change.
3
Quantitative Analysis of Gene Expression
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Total RNA was extracted from 15 embryos, using the Dynabeads mRNA DIRECT kit (Invitrogen), according to the manufacturer’s instructions. First-strand cDNA was synthesized by reverse transcription of mRNA using the Oligo(dT) 12-18 primer and SuperScript TM III reverse transcriptase (Invitrogen). RT-qPCR was performed using SYBR Green, a fluorophore that binds double-stranded DNA, in a final reaction volume of 20 µL using the CFX96 touch RT-PCR detection system (Bio-Rad, Hercules, CA, USA). Gene expression was quantified by the 2−ΔΔCt method, with normalization to the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PCR primers used to amplify each gene are listed in Supplemental Table S1 .
4
Identifying Centrosome Clustering Genes
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To identify genes associated with centrosome clustering, we used a custom‐designed plate with mitotic spindle‐associated genes18 selected based on our previously published study (Figure S1 ).17 The RNeasy plus mini kit (Qiagen® Cat# 74104) was used to extract total RNA from Beas‐2B and NSCLC cell lines according to the manufacturer's instructions. The iScript cDNA synthesis kit (Bio‐Rad Cat# 1708841) was used for reverse transcription, using 500 ng RNA and SSO advanced Universal SYBR® Green Supermix (Cat# 172‐5271). The CFX96 Touch RT‐PCR Detection System (Bio‐Rad®) was used with the following cycle: 95°C for 10 min (1 cycle), 95°C for 15 s, 60°C for 1 min, 95°C for 15 s for 40 cycles followed by 95°C for 15 s, and 60°C for 1 min. PCR product specificity was confirmed by melt curve analysis. Data analysis was performed using CFX Manager Software (Bio‐Rad®). Data were normalized that of actin and GAPDH from the same sample, and fold changes in gene expression were calculated using the ΔΔCt method.
5
Saliva-based Genotyping of Taste Receptors
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Saliva was collected using the Oragene·DNA (OG-575) collection kit for Assisted Collection (DNA Genotek). Participants were fasted for a minimum of 30 min before the saliva sample was provided. Genetic material from saliva was extracted by ethanol precipitation according to the manufacturer’s protocol (DNA Genotek). Real-time polymerase chain reaction (RT-PCR) was used to identify the genotypes of the participants with respect to the CD36 (rs1761667; Assay ID C___8314999_10), TAS1R2 (rs35874116; Assay ID C_____55646_20), and TAS2R38 SNPs (rs713598; Assay ID C___8876467_10). Taqman fluorescent oligonucleotide probes were obtained for each SNP (Thermo Fisher Scientific, Waltham, MA, USA, Cat. #4351379). A BioRad CFX96 Touch™ RT-PCR Detection System in tandem with the Bio-Rad CFX Manager Software was used to detect the fluorescent signals and to produce a graphical representation which allowed for allelic discrimination.
6
Gene Expression Analysis in Liver Tissue
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Livers (~100 mg) were homogenized in TRIzol reagent to extract total RNA as per the manufacturer's instructions (Life Technologies). Total RNA (~1 µg) was reverse transcribed into cDNA using an iScript cDNA kit (Bio‐Rad). RT‐PCR reactions were performed using appropriately diluted cDNA in triplicate with (β‐actin) serving as an internal control, and gene expression levels were quantified using a CFX96 Touch RT‐PCR detection system (Bio‐Rad) according to the manufacturer's recommendation. Relative quantification of amplified DNA was performed using the delta‐delta Ct method (Choi et al., 2010 ; Mahmood et al., 2016 ). Primers used for gene expression analysis are presented in Table S1 (https://figshare.com/articles/figure/_/12821813 ).
7
Quantitative Analysis of Cell Signaling Genes
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The mRNAs of CXCR4, CXCL12, VEGF, VEGFR, TIMP1, MMP9, and MMP2 were taken from cells and tissues using the miRNeasy Mini Kit (Qiagen NV, Venlo, the Netherlands) under the manufacturer’s instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to obtain cDNA template using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) along with the CFX96 Touch RT-PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The primer sequences are shown in Table 1 . Relative expression quantity of target genes was normalized to that of the internal control group (β-actin) and was calculated using the 2−ΔΔCT method. The experimental procedures were repeated for at least three times.
8
Multimodal Analysis of Assembled DNA Duplexes
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The morphologies of assembled DNA duplexes were examined with AFM (Nanoscope IIIa, USA). The gel electrophoresis was imaged on an Alliance Ld2 (Uvitec, Cambridge, U.K.). Fluorescence measurements were carried out on a Hitachi F-4500 fluorescence spectrofluorometer (Tokyo, Japan). Confocal fluorescence imaging measurements were performed on a confocal laser scanning fluorescence microscope (CLSM, FV1200, Olympus, Japan). Flow cytometric analysis was performed on a BD FACSAria (Becton, Dickinson and Company, USA). The qRT-PCR was performed on a CFX96 touch RT-PCR detection system (Bio-Rad, USA).
9
Quantifying Boric Acid-Induced Laccase Expression
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To monitor the expression of laccase‐like family genes in response to boric acid excess, Citrus leaves and Arabidopsis shoots and roots were treated with boric acid. Real‐time (RT) PCRs were set up with 2× SYBR Green qPCR Master Mix (Takara Bio). Amplification levels were detected with a CFX96 Touch RT‐PCR Detection System (Bio‐Rad). Quantification was performed using three independent biological replicates. β‐actin and tubulin were set as internal controls for Citrus and GAPDH and UBQ5 (Joseph et al., 2018 (link)) for Arabidopsis. To monitor the relative abundance of miR397 in Arabidopsis treated with different levels of boric acid, stem‐loop RT‐PCR was conducted with a TaqMan® MicroRNA Assay Kit (Takara Bio) as previously described (Huang et al., 2016 (link)). All the primers employed for RT‐PCR are listed in Table S5 . Relative levels were expressed as 2 ‐ Δ Δ C t excel 2016 (Microsoft, Redmond, WA, USA).
10
Quantitative RT-PCR Protocol for HeLa Cells
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Total RNA was isolated from HeLa cells using TRIzol reagent (MRC, TR118-200). Complementary DNA (cDNA) was synthesized from the reverse transcription of 900 ng of total RNA using HiScript® II Q RT SuperMix for qPCR (Vazyme, R222-01) according to the product instruction. ChamQ SYBR qPCR Master Mix (Vazyme, Q311-02/03) was used for RT-PCR. Samples were detected by a CFX96 Touch™ RT-PCR Detection System (Bio-Rad, USA). All primers used for RT-PCR were listed in Additional file 1 : Table S1.
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