Quantstudio 5 real time pcr system

Manufactured by Thermo Fisher Scientific

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The QuantStudio 5 Real-Time PCR System is a thermal cycler designed for real-time polymerase chain reaction (PCR) analysis. It provides precise temperature control and optical detection capabilities to facilitate gene expression studies, genotyping, and other real-time PCR applications.

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1 382 protocols using quantstudio 5 real time pcr system

Quantitative Gene Expression Analysis

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Reverse transcription of 1 μg of total RNA was performed using the Superscript IV First-Strand cDNA Synthesis Kit (Life Technologies) using a mix of oligo dT and random hexamers following manufacturer’s instructions. Primers were designed using Primer Blast (Supplementary Table S2). Real-time PCR amplification was performed on a QuantStudio 5 Real-Time PCR System (Thermo Fisher) using the SYBR Green Master Mix (Roche). All PCRs were performed using a standardized protocol and data were analyzed with the QuantStudio 5 Real-Time PCR System (Thermo Fisher). For each sample, fold change (FC) was obtained by comparative quantification and normalization to expression of the GAPDHD, HPRT and PPIA housekeeping genes used as standard. Data are expressed as mean ± standard deviation (SD).

Laberthonnière C., Delourme M., Chevalier R., Dion C., Ganne B., Hirst D., Caron L., Perrin P., Adélaïde J., Chaffanet M., Xue S., Nguyen K., Reversade B., Déjardin J., Baudot A., Robin J.D, & Magdinier F. (2023). In skeletal muscle and neural crest cells, SMCHD1 regulates biological pathways relevant for Bosma syndrome and facioscapulohumeral dystrophy phenotype. Nucleic Acids Research, 51(14), 7269-7287.

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SARS-CoV-2 Extraction-Free RT-PCR Diagnosis

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All samples were initially screened at University College London Hospitals by real-time RT-PCR targeting the N gene and using the extraction-free SARS-CoV-2 diagnosis method [30 (link)]. In brief, 2 µL of sample in VTM was added to the RT-PCR mix comprised of 5 μL of 4X TaqMan Fast Virus 1-step Master Mix (Applied Biosystems, Waltham, MA, USA), SARS-CoV-2 N gene primers, RNase P primers and probes each at a final concentration of 250 nM and the reaction volume made up to 20 µL with nuclease-free water. Thermocycling was performed at 56 °C for 15 min for reverse transcription, followed by 95 °C for 20 s and then 45 cycles at 95 °C for 3 s, 60 °C for 30 s using an Applied Biosystems QuantStudio 5 real-time PCR system, and Cq values were determined using QuantStudio Design and Analysis Software (Thermo Fisher Scientific, Waltham, MA, USA).
Confirmatory real-time RT-PCR for presumptive positive samples was performed at UCL with the N1 gene (US CDC) protocol [31 ]. The TaqMan Fast Virus 1-Step MasterMix (Thermo Fisher Scientific) and QuantStudio 5 real-time PCR system were used. The positivity criterion was Cq < 40.

Cherkaoui D., Heaney J., Huang D., Byott M., Miller B.S., Nastouli E, & McKendry R.A. (2022). Clinical Validation of a Rapid Variant-Proof RT-RPA Assay for the Detection of SARS-CoV-2. Diagnostics, 12(5), 1263.

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Relative Quantification of Gene Expression

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All qPCR assays were previously published (vasa, gsdf, cyp19a1a, amh; Skaftnesmo et al., 2021 (link)). A qPCR reaction was prepared to contain 800 nM of each forward and reverse primer, 250 nM of the probe in a 6 µl reaction containing 1x concentration of the TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and 2 µl of a 1/20 diluted cDNA. The reaction was subjected to thermocycling in a QuantStudio 5 Real-Time PCR system (Thermo Fisher Scientific) with an initial hold at 50°C for 2 min followed by an initial denaturation step at 95°C for 2 min. Thermocycling was conducted for 40 cycles using a denaturation step at 95°C for 1 s followed by a combined annealing and extension step at 60°C for 30 s. Data was processed at Thermo Fisher cloud using the relative quantification app. QPCR was performed in duplicates in 384-well optical plates in a QuantStudio 5 Real-Time PCR system (ThermoFisher Scientific). No-template controls for each gene were run in all qPCR plates. The relative gene expression level was calculated using the 2−ΔΔCt method. All values were normalized to ef1a and calibrated to the average ΔCt of the controls of each sex.

F. L A., K. O S., E A., L K., R. B E., B N., P. G F., T. J H., R. W S, & A W. (2022). The Piwil1 N domain is required for germ cell survival in Atlantic salmon. Frontiers in Cell and Developmental Biology, 10, 977779.

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Quantification of Gene Expression in Plant Roots

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Six days after the start of bacterial treatment, total RNA was extracted from roots using TRIzol™ Reagent (Sigma, Neustadt, Germany) according to the manufacturer’s instructions. DNA was destroyed with DNaseI (Syntol, Moscow, Russia), and the first-strand cDNA was synthesized with M−MLV reverse transcriptase (Fermentas, Waltham, MA, USA) and oligo(dT)15 used as a primer. Further, 2 μL of diluted cDNA was used for qPCR. The primers were designed with PrimerQuest™ Tool based on the cDNA sequences (Table 1).
Quantitative PCR was performed with a set of predefined reagents EvaGreenI (Synthol, Russia) and QuantStudio™ 5 Real-Time PCR System by Thermo Fisher Scientific (Singapore). Ten-fold cDNA dilution series were used to confirm the efficiency of each pair of primers and reliability of the fold changes. Hv α-tubulin (GenBank Accession No. AK250165) was used as an internal reference for the real-time qPCR analysis. Gene expression was quantified with QuantStudio™ 5 Real-Time PCR System by Thermo Fisher Scientific (Singapore). Each experimental treatment was assayed in three biological replicates.

Arkhipova T., Sharipova G., Akhiyarova G., Kuzmina L., Galin I., Martynenko E., Seldimirova O., Nuzhnaya T., Feoktistova A., Timergalin M, & Kudoyarova G. (2022). The Effects of Rhizosphere Inoculation with Pseudomonas mandelii on Formation of Apoplast Barriers, HvPIP2 Aquaporins and Hydraulic Conductance of Barley. Microorganisms, 10(5), 935.

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RT-qPCR Analysis of Gene Expression

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TRIzol^™ Solution (Thermo Fisher Scientific^®) was used for extracting Total RNA from cells and tissues according to the manufacturer’s instructions. Total RNA was quantified by NanoDrop^™ ND-1000 (Thermo Fisher Scientific^®) or Quibit^™ 4 (Thermo Fisher Scientific^®). 50 ng or RNA was reverse transcribed using the cDNA synthesis and gDNA removal QuantiTect^® Reverse Transcription Kit (Thermo Fisher Scientific^®). Real time PCR was performed using the iTaq^™ Universal SYBR^® Green Supermix (Bio-Rad Laboratories, Inc.), 10 ng of cDNA and on a QuantStudio^™ 5 Real-Time PCR System or a 7500 fast real time PCR system (both from Thermo Fisher Scientific^®). Relative gene expression was calculated using the comparative Ct method, normalized to GAPDH. Primers are listed in Supplementary Table 3. Primers were obtained from Integrated DNA Technologies, Inc.

Feichtenschlager V., Zheng Y.J., Ho W., Chen L., Callanan C., Chen C., Lee A., Ortiz J., Rappersberger K, & Ortiz-Urda S. (2023). Deconstructing the role of MALAT1 in MAPK-signaling in melanoma: insights from antisense oligonucleotide treatment. Oncotarget, 14, 543-560.

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Chromatin Immunoprecipitation of HA-Tagged Proteins

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Samples were cross-linked in 3% formaldehyde solution in PBS, and cross-linking was quenched with 0.2 M glycine. Nucleus enrichment was performed as described (Fiil et al. 2008 (link)). Samples were sonicated in lysis buffer (50 mM Tris-HCL at pH 8, 10 mM EDTA, 1% SDS) and further processed as described (Stracke et al. 2010 (link); Binkert et al. 2014 (link)). The chromatin was immunoprecipitated with anti-HA antibody (ChIP-grade; Abcam, ab9110) overnight at 4°C, after which cross-linking was reversed for 2 h at 85°C. DNA was purified using QIAquick PCR purification kit (Qiagen) before analysis with a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific) and the following primer sets: Pro_{FT_-100}-Fw (5′-AGAGGGTTCATGCCTATGATA C-3′), Pro_{FT_-100}-Rv (5′-CTTTGATCTTGAACAAACAGGTG-3′) (Bu et al. 2014 (link)), Pro_{FT_-1185}-Fw (5′-TTATCCTGGTCGTGCAAATG-3′), and Pro_{FT_-1185}-Rv (5′-CAAGCGGCCATATTATGGAA-3′) (Song et al. 2012 (link)). qPCR data were analyzed according to the percentage of input method (Haring et al. 2007 (link)). Each reaction was performed in technical triplicates; data shown are representative of three independent biological repetitions.

Arongaus A.B., Chen S., Pireyre M., Glöckner N., Galvão V.C., Albert A., Winkler J.B., Fankhauser C., Harter K, & Ulm R. (2018). Arabidopsis RUP2 represses UVR8-mediated flowering in noninductive photoperiods. Genes & Development, 32(19-20), 1332-1343.

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Pelargonidin Modulates Antioxidant Pathways

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Cells (3×10⁵ cells per 100-mm dish) were incubated with pelargonidin (10, 30 and 50 µM), and 0.1% DMSO (control) in MEM containing 1% FBS. RNA was extracted from each group by GeneJET RNA Purification Kits (Thermo Fisher Scientific, Rockford, IL). Then, 1 µg of RNA was used for reverse transcription by Taqman Reverse Transcription Reagents (Thermo Fisher Scientific, Rockford, IL). Gene expression was measured on the transcription level by quantitative real-time PCR on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, Rockford, IL) and quantified by the ∆∆Ct method. The data from triplicate experiments were subjected to statistical analysis. The primers are listed as follows: Nrf2: 5’-GGCTCAGCACCTTGTATCTT-3’ and 5’-CACATTGCCATCTCTGGTTTG-3’; NQO1: 5’-GAGAAGAGCCCTGATTGTACTG-3’ and 5’-ACCTCCCATCCTCTCTTCTT-3’; GAPDH: 5’-AACAGCAACTCCCACTCTTC-3’ and 5’ -CCTGTTGCTGTAGCCGTATT-3’; and HO-1: 5’-CTCCCTGTGTTTCCTTTCTCTC-3’ and 5’-GCTGCTGGTTTCAAAGTTCAG-3’.

Li S., Li W., Wang C., Wu R., Yin R., Kuo H.C., Wang L, & Kong A.N. (2019). Pelargonidin reduces the TPA induced transformation of mouse epidermal cells –potential involvement of Nrf2 promoter demethylation. Chemico-biological interactions, 309, 108701.

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Quantification of KAR2 Gene Expression

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The sample taken during the bioreactor cultivation was centrifuged (14,000 rpm, 5 min) and the cell pellet was stored at −80°C. Total RNA was isolated from the cell pellets according to the protocol provided by Invitrogen (http://tools.thermofisher.com/content/sfs/manuals/easyselect_man.pdf). The cDNA was prepared using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, USA). qPCR was performed in QuantStudio™ 5 Real-Time PCR System (ThermoFisher Scientific, Waltham, USA), using the 5 × HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX) (Solis BioDyne, Tartu, Estonia). Relative expression of KAR2 was monitored (primers q34 and q35), using ACT1 as a reference (primers q24 and q25) and no-template as well as no-RT controls. The nucleotide sequences of the primers are provided in Supplementary Table 1. The fold-change of KAR2 expression was evaluated using the ΔΔc_t method, relating all expression data to the expression of KAR2 in the non-producing control strain in the sample taken 3 h after induction.

Raschmanová H., Zamora I., Borčinová M., Meier P., Weninger A., Mächler D., Glieder A., Melzoch K., Knejzlík Z, & Kovar K. (2019). Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris. Frontiers in Microbiology, 10, 335.

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Real-Time qPCR for Gene Expression Analysis

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RNA was extracted using the RNeasy RNA isolation kit (Qiagen, cat#74104, Hilden, Germany), DNAse treatment was performed following the manufacturer’s instructions. For cDNA synthesis, 500 ng of template total RNA were reverse transcribed using the high-capacity cDNA reverse transcription kit (Thermofisher scientific, Waltham, MA, USA, cat#4368814). Real-time qPCR amplification was performed using PowerUp SYBR^® Green Master Mix (Applied Biosystems, Waltham, MA, USA, cat#A25742) and specific PCR primers in a QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA). Oligonucleotides used are provided in Table S1. Relative quantification of each target, normalized to an endogenous control (GAPDH or TBP), was performed using the comparative Ct method (Applied Biosystems, Waltham, MA, USA). Error bars indicate SD of three technical replicates.

Bakaric A., Cironi L., Praz V., Sanalkumar R., Broye L.C., Favre-Bulle K., Letovanec I., Digklia A., Renella R., Stamenkovic I., Ott C.J., Nakamura T., Antonescu C.R., Rivera M.N, & Riggi N. (2024). CIC-DUX4 Chromatin Profiling Reveals New Epigenetic Dependencies and Actionable Therapeutic Targets in CIC-Rearranged Sarcomas. Cancers, 16(2), 457.

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RNA Extraction and qRT-PCR Analysis

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The total RNA was extracted from cell samples using TRIzol (SIGMA), and 1 µg of total RNA was reversed transcribed to cDNA using the 5 × PrimeScript RT Master Mix (Takara) according to the manufacturer’s instructions. The synthesized cDNAs were then subjected to quantitative real-time PCR analysis using the 2 × ChamQ Universal SYBR qPCR Master Mix (Vazyme) according to the manufacturer’s instruction. Real-time PCR for each sample was performed in a 20 µl reactions system using the QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific). The primer sequences used for PCR amplification can be found in supplementary table 5.

Yang T., Chi Y., Wang X., Xu C., Chen X., Liu Y., Huang S., Zhu X., Zhang H., Zhuo H, & Wu D. (2024). PRL-mediated STAT5B/ARRB2 pathway promotes the progression of prostate cancer through the activation of MAPK signaling. Cell Death & Disease, 15(2), 128.

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