Quantstudio 5 system

Manufactured by Thermo Fisher Scientific

Sourced in United States, China, Japan, Switzerland, France, Germany

The QuantStudio 5 system is a real-time PCR instrument designed for quantitative gene expression analysis. It features a 96-well block format and supports multiple fluorescent dye chemistries for diverse experimental requirements. The system provides precise temperature control and enhanced thermal uniformity to ensure reliable and reproducible results.

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QuantStudio 5 (776 citations) Applied Biosystems QuantStudio 5 Real-Time PCR System (24 citations) Quantstudio 5 Flex Real-Time PCR System (23 citations) QuantStudio 5 RT-PCR system (22 citations) QuantStudio™ 5 Dx Real-Time PCR System (7 citations) QuantStudio 3 and 5 systems (5 citations) Applied Biosystems QuantStudio 5 system (5 citations) QuantStudio 5 Real-Time PCR System for Human Identification (5 citations) QuantStudio 5 Real Time Detection System (5 citations) QuantStudio 5 real-time RT-PCR systems (3 citations)

207 protocols using quantstudio 5 system

RNA Isolation and qPCR Analysis of K7M2 Cells

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The K7M2 cell line was homogenized in 1 mL of TRIzol-reagent by vortexing, and its RNA contents were isolated using Direct-zol™ RNA MicroPrep (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions. The amount and purity of RNA were determined using a Jenway™ Genova Nano Micro-volume Spectrophotometer (Fisher Scientific, Loughborough, UK). Samples were then treated with 1U DNase I enzyme to eliminate any contaminating genomic DNA. cDNA was synthesized from 500 ng of RNA using an Applied Biosystems™ High-Capacity Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA).
PCR was performed using a QuantStudio™ 5 system (Life Technologies Magyarország Ltd., Budapest, Hungary) in a 96-well block, using Gapdh as a reference gene, with a reaction volume of 10 µL, containing 1× SensiFAST™ Probe Lo-ROX mix (Meridiane Bioscience, Memphis, TN, USA), 400 nM of probe primer mix (forward and reverse), and 20 ng cDNA. FAM-conjugated TaqMan™ Gene Expression Assays (Thermo Scientific, Waltham, MA, USA) were used to amplify the target loci—Gapdh: Mm99999915_g1, Trpa1: Mm01227437_m1, and Trpv1: Mm01246302_m1. The K7M2 PCR products were electrophoresed on a 2% agarose gel containing 0.01% ethidium bromide at 70 V for 40 min and visualized using a Molecular Imager BioRad Gel Doc XR+ (BioRad Laboratories, Hercules, CA, USA) with Image Lab 6.0.1 build 34 software.

Hudhud L., Rozmer K., Kecskés A., Pohóczky K., Bencze N., Buzás K., Szőke É, & Helyes Z. (2024). Transient Receptor Potential Ankyrin 1 Ion Channel Is Expressed in Osteosarcoma and Its Activation Reduces Viability. International Journal of Molecular Sciences, 25(7), 3760.

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Quantifying Gene Expression in Cell Lines

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Total RNA was prepared from the exponentially growing cells after treatments using the Qiazol reagent and miRNAeasy Mini Kit following the manufacturer’s manuals (Qiagen). First strand cDNA was synthesized on the Veriti Thermal Cycler (Applied Biosystems) from 1 μg of total RNA using the SuperScript IV First-Strand Synthesis System (Invitrogen). Quantitative PCR was performed on a QUANT STUDIO 5 system (Life Technologies) using SYBR Green Mix (Applied Biosystems). Primers validated by the vendor were used (Bio-Rad POLQ: qHsaCID0018136; PARP1: qHsaCED0045162; LIG1: qHsaCID0008449). GADPH primer sequences were forward: 5’-CAGCCTCCAGATCATCAGCA-3’ and reverse: 5’-TGTGGTCATGAGTCCTTCCA-3’). Comparative Ct method was used to calculate the relative expression of each gene to the expression of the housekeeping gene GADPH.

Liu Q., Palomero L., Moore J., Guix I., Espin R., Aytés A., Mao J.H., Paulovich A.G., Whiteaker J.R., Ivey R.G., Iliakis G., Luo D., Chalmers A.J., Murnane J., Pujana M.A, & Barcellos-Hoff M.H. (2021). Loss of TGFβ signaling increases alternative end-joining DNA repair that sensitizes to genotoxic therapies across cancer types. Science translational medicine, 13(580), eabc4465.

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Quantitative Real-Time PCR Analysis

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Total RNA was extracted from cells with the NucleoSpin RNA II Extraction Kit (Macherey-Nagel) following the manufacturer’s recommendations. Total RNA was converted into cDNA using SuperScript IV (Life Technologies) and oligo-dT primers. Real-time QPCR reactions were performed with 2X Fast SYBR Green PCR Master Mix (Life Technologies) and 50–400 nM of each primer. Fluorescence was detected using the QuantStudio 5 System (Life Technologies) under a standard temperature protocol. Primer pairs were either designed with Primer-BLAST or retrieved from PrimerBank and supplied by Exxtend (online supplemental table 4). geNorm (https://genorm.cmgg.be/) was used to determine the normalisation factor (using gene expression of TATA-box binding protein (TBP), hydroxymethylbilane synthase (HMBS) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) and calculate normalisation factors (E−ΔC) for each sample. The final relative expression values were determined based on the Pfaffl40 (link) method.

Romanelli Tavares V.L., Guimarães-Ramos S.L., Zhou Y., Masotti C., Ezquina S., Moreira D.D., Buermans H., Freitas R.S., Den Dunnen J.T., Twigg S.R, & Passos-Bueno M.R. (2021). New locus underlying auriculocondylar syndrome (ARCND): 430 kb duplication involving TWIST1 regulatory elements. Journal of Medical Genetics, 59(9), 895-905.

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RNA Isolation and qPCR Analysis

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Total RNA was isolated from sorted cells using the RNAeasy Mini kit (Qiagen). The isolated RNA was transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Probes were purchased from Life Technologies and the assays were performed using the TaqMan Fast Advanced Master mix (Thermo Fisher Scientific) on 384-well reaction plates (Life Technologies). The quantitative PCR (qPCR) was performed on the Quant Studio 5 system (Life Technologies). In all of the experiments, GAPDH (Mm99999915_g1 and Hs00230829_m1, Thermo Fisher Scientific) was used as a reference gene to calibrate gene expression.

Afzali A.M., Nirschl L., Sie C., Pfaller M., Ulianov O., Hassler T., Federle C., Petrozziello E., Kalluri S.R., Chen H.H., Tyystjärvi S., Muschaweckh A., Lammens K., Delbridge C., Büttner A., Steiger K., Seyhan G., Ottersen O.P., Öllinger R., Rad R., Jarosch S., Straub A., Mühlbauer A., Grassmann S., Hemmer B., Böttcher J.P., Wagner I., Kreutzfeldt M., Merkler D., Pardàs I.B., Schmidt Supprian M., Buchholz V.R., Heink S., Busch D.H., Klein L, & Korn T. (2024). B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4. Nature, 627(8003), 407-415.

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Quantitative RT-PCR Analysis of Gene Expression

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Total RNA of tissue or cells was extracted using TRIzol Reagent (Invitrogen), and measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA was synthesized using HiScript Q RT SuperMix (Vazyme, Nanjing, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) with the Applied Biosystems^TM QuantStudio^TM 5 system. The cycling parameters were as follows: hold stage, 95 °C for 3 min; PCR stage, 40 cycles of 95 °C for 10 s and 60 °C for 30 s; melt curve stage, 95 °C for 15 s and 60 °C for 1 min, which was concluded by the melting curve analysis process. Triplicate PCR reactions were performed for each sample. The murine housekeeping gene GAPDH was used to normalize the gene expression using a comparative method (2^−ΔΔCq) [10 (link)]. The primers were designed by Primer Premier 5 software (Premier Biosoft, San Francisco, CA, USA). All primer sequences are listed in Additional file 1: Table S1.

Bai X., Li M., Wang X., Chang H., Ni Y., Li C., He K., Wang H., Yang Y., Tian T., Hou M., Ji M, & Xu Z. (2020). Therapeutic potential of fucoidan in the reduction of hepatic pathology in murine schistosomiasis japonica. Parasites & Vectors, 13, 451.

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Quantification of ASFV viremia in animals

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Viremia in experimentally inoculated animals was quantified at different days post-infection using quantitative PCR (qPCR). Genomic DNA was extracted from 200 μL of EDTA anti-coagulated blood using a Zymo Quick-DNA miniprep DNA extraction kit (Ref: D3025, Zymo research, Irvine, CA, USA). For the detection of ASFV genome copies in tissues, approximately 0.01 g to 0.025 g of splenic, submandibular, and gastrohepatic lymph tissue was weighed and DNA extracted using the Qiagen DNeasy Blood and Tissue Kits (Cat# 69506, Qiagen, Hilden, Germany). qPCR was performed using QuantStudio^TM 5 system (Applied Biosystems, Waltham, MA, United States). Each reaction was conducted in duplicates in a 10 μL reaction mixture containing 2.23 μL nuclease-free water, 5 μL EXPRESS qPCR Supermix (Invitrogen, Waltham, MA, USA), 0.3 μL of forward primer (10 μM), 0.3 μL of reverse primer (10 μM), 0.15 μL of TaqMan^® probe (10 μM), 0.02 μL of ROX reference dye, and 2 μL template DNA. The plasmid standard dilutions, primers, and qPCR conditions are described by Abkallo et al. [19 (link)]. Data, in eds file format, were exported and analysed on a QuantStudio™ design and analysis software (Applied Biosystems, United States). Results were analysed on GraphPad Prism (version 6) for Windows (GraphPad Software, San Diego, CA, USA).

Abkallo H.M., Hemmink J.D., Oduor B., Khazalwa E.M., Svitek N., Assad-Garcia N., Khayumbi J., Fuchs W., Vashee S, & Steinaa L. (2022). Co-Deletion of A238L and EP402R Genes from a Genotype IX African Swine Fever Virus Results in Partial Attenuation and Protection in Swine. Viruses, 14(9), 2024.

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Mitochondrial DNA Extraction and Quantification

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mtDNA extraction and measurement were made using the adopted and modified protocol from Bronner et al. [31 (link)]. The cells were seeded at the concentration of 2 × 10⁶ cells per well onto 10 cm culture plates, followed by priming and exposure to epoxomicin or MG-132 in serum-free media. Cells were washed once with DPBS (Gibco^®, Thermo Fisher Scientific) and lysed using 1% Nonidet^® P 40 Substitute (NP-40) (Cat.no. M158, VWR Life Science). The mtDNA was isolated in microtubes using a commercial kit (Macherey-Nagel, Düren, Germany). The amplification and measurement of mitochondrial cytochrome b (mt-CYB) and house-keeping gene GAPDH (Table 1) were made using Quantstudio^TM 5 System and SYBR^® Green chemistry (both from Applied Biosystems). The thermo-cycling program consisted of 40 cycles at 95 °C for 30 s and at 60 °C for 60 s, with an initial cycle at 95 °C for 10 min. The melting curve analysis was performed at 72 °C for 30 s and at 95 °C for 60 s, as well as at 60 °C for 30 s and at 95 °C for 30 s. Finally, mtDNA was quantified and analyzed using the 2^ΔΔCt method.

Sridevi Gurubaran I., Hytti M., Kaarniranta K, & Kauppinen A. (2022). Epoxomicin, a Selective Proteasome Inhibitor, Activates AIM2 Inflammasome in Human Retinal Pigment Epithelium Cells. Antioxidants, 11(7), 1288.

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Quantification of Angiogenic Factors in mdx Mice

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TaqMan Gene Expression Assays (Thermofisher, Waltham, MA, USA) were used to assay Tek (Tie2) (Mm004432343_m1), Angpt1 (Mm00456503_m1), Angpt2 (Mm00545822_m1) [17 (link)], ActB (Mm02619580_g1), AP3D1 (Mm00475961_m1), and GusB (Mm01197698_m1). qPCR products were generated using the QuantStudio^TM 5 system with TaqMan Fast Advanced Master Mix (Applied Biosystems, Waltham, MA, USA). Quantification was performed using Design & Analysis Software v2.4.3 (Thermofisher, Waltham, MA, USA). Expression of Tek, Angpt1, and Angpt2 was normalized to the geometric mean of control genes (ActB, AP3D1, and GusB) validated for use in mdx mice [18 (link)].

Lin Y., McClennan A, & Hoffman L. (2023). Characterization of the Ang/Tie2 Signaling Pathway in the Diaphragm Muscle of DMD Mice. Biomedicines, 11(8), 2265.

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Gene Expression Analysis by qPCR

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According to the gene sequences of TvAP33 and TvActin (GenBank accession no. KF747377.1), primers were designed according to the gene sequences of TvAP33 and TvActin (GenBank accession no. KF747377.1), respectively, and synthesized for detection by real-time PCR (qPCR) (Table 1). The amplification efficiency and specificity of the primers were tested by qPCR with gradient-diluted complementary DNA (cDNA) as a template. The qPCR (RT-PCR) was run on a QuantStudio ^TM 5 system (Applied Biosystems, Thermo Fisher Scientific) with SYBR qPCR Master Mix (Vazyme, Nanjing, China). As described in the manufacturer’s instructions, the reaction mixture (20 μl) contained 2 × ChamQ Universal SYBR qPCR Master Mix (10 μl), forward primer (10 μM, 0.4 μl), reverse primer (10 μM, 0.4 μl), cDNA (2 μl) and ddH₂O (7.2 μl). The amplification protocol consisted of a predenaturation step at 95 °C for 30 s; 40 cycles of a circular reaction at 95 °C 10 s and 60 °C for 30 s; and a melting curve at 95 °C for 15 s, 60 °C for 30 s and 95 °C for 15 s. The messenger RNA (mRNA) expression levels of the target genes, such as TvAP33, were normalized to those of the TvActin primers. Relative expression levels were calculated by the 2^−△△Ct method.

Zhang Z., Deng Y., Sheng W., Song X., Li Y., Li F., Pan Y., Tian X., Yang Z., Wang S., Wang M, & Mei X. (2023). The interaction between adhesion protein 33 (TvAP33) and BNIP3 mediates the adhesion and pathogenicity of Trichomonas vaginalis to host cells. Parasites & Vectors, 16, 210.

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Liver RNA Extraction and qRT-PCR Analysis

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First, total RNA was extracted from 20 mg liver samples of each rat (five rats per group) using TRIzol reagent (Accurate Biotechnology Co., Ltd., Changsha, China) following the manufacturer’s protocol. First-strand cDNA was generated using 1 µg of total RNA with an SYBR Green I reagent kit (Accurate Biotechnology Co., Ltd., Changsha, China). The real-time PCR reaction was performed using a QuantStudioTM 5 system (Applied Biosystems, Stony Creek, VA, USA) and the QuantStudioTM design and analysis software (v.1.5.1). All primers were designed according to the Primer 5.0 website and purchased from Sangon Biotech (Shanghai, China), which were listed in Table S2. The target mRNA was normalized to GAPDH; GAPDH served as an endogenous control. The changes in the relative expression were determined using the 2^−ΔΔCt method.

Liu B., Zhang S., Sun L., Huang L., Zhang R., Liu Z, & An L. (2023). Unravelling the Link between Psychological Distress and Liver Disease: Insights from an Anxiety-like Rat Model and Metabolomics Analysis. International Journal of Molecular Sciences, 24(17), 13356.

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