Quantstudio 12k flex

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Japan, Switzerland, Finland

The QuantStudio 12K Flex is a real-time PCR system designed for high-throughput gene expression and genotyping analysis. It features 12 independently controllable thermal blocks for flexible sample processing, and supports a wide range of sample types and reaction volumes. The system is capable of performing up to 12 separate experiments simultaneously.

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QuantStudio 12K Flex system (147 citations) QuantStudio 12K Flex instrument (21 citations) QuantStudio 12K (19 citations) QuantStudio 12K Flex real-time PCR instrument (10 citations) QuantStudio 12k Flex Software v.1.2.2 (10 citations) QuantStudio™ 12K Flex Real-Time PCR (9 citations) ABI QuantStudio 12K Flex Real-Time PCR System (7 citations) QuantStudio 12K Flex machine (7 citations) QuantStudio 12K Flex RT-PCR system (6 citations) QuantStudio 12K Flex AccuFill system (4 citations)

237 protocols using quantstudio 12k flex

Quantifying Gene Expression Changes in Leukemia Cells

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RNA was isolated from shSCR-BV173 and shSTAT5-BV173 cells, untreated or treated with Doxycycline at 2.5 μg/ml, using the RNeasy Plus Mini Kit (Qiagen, Limburg, The Netherlands) and reverse-transcribed (2 μg) with the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA, USA). Quantitative PCR was performed with the QuantStudio 12k Flex (Life Technologies) instrument and QuantStudio 12k Flex software, using the following primers: MYC FW 5’-GTCACACCCTTCTCCCTTCG-3’; MYC RV 5’-ATGTCTCCTCCCAGCAGCTC-3’; TRIB3 FW 5’-TGCGTGATCTCCAAGCTGTGT-3’; TRIB3 RV 5’-GCTTGTCCCACAGGGAATCA-3’; ITGα5 FW 5’-GGCTTCAACTTAGACGCGGA-3’; ITGα5 RV 5’-GGCTGGCTGGTATTAGCCTT-3’; ITGβ1 FW 5’-CCGCGCGGAAAAGATGAATTT-3’; ITGβ1 RV 5’-CCACAATTTGGCCCTGCTTG-3’; PIM1 FW 5’-ACACGGACTTCGATGGGACC-3’; PIM1 RV 5’-GATGGTCTCAGGGCCAAGCA-3’; IDH2 FW 5’-GCCGGCACTTTCAAAATGG-3’T; IDH2 RV 5’-TCCTTGACACCACTGCCATC-3’; UBC FW 5’-GTCGCAGTTCTTGTTTGTGGATC-3’; UBC RV 5’-GTCTTACCAGTCAGAGTCTTCACGAAG-3’; GAPDH FW 5’-CCCATCACCATCTTCCAGGAG-3’; GADPH RV 5’-CTTCTCCATGGTGGTGAAGACG-3’.

Minieri V., De Dominici M., Porazzi P., Mariani S.A., Spinelli O., Rambaldi A., Peterson L.F., Porcu P., Nevalainen M, & Calabretta B. (2018). Targeting STAT5 or STAT5-regulated pathways suppresses leukemogenesis of Ph+ acute lymphoblastic leukemia. Cancer research, 78(20), 5793-5807.

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Quantitative PCR for IL-17A Expression

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cDNA was synthesized from total RNA obtained in 2.7 using Superscript IV VILO master mix (Invitrogen, Waltham, MA, USA), according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed as described previously (Joseph et al., 2012 (link)) using the primer pairs described below, Power SYBR green PCR Master mix (Applied Biosystems, Waltham, MA, USA), and a QuantStudio 12K Flex (Applied Biosystems) equipped with QuantStudio 12K Flex software (Applied Biosystems). Next, relative gene expression was calculated using cycle threshold (Ct) values: ΔCt (Ct of IL-17A – Ct of GAPDH) (Nobles et al., 2010 (link); Robinson et al., 2020 (link); Silver et al., 2006 (link)). The following forward and reverse primers, respectively, were used (all from Eurofins Scientific, Luxembourg): GAPDH, 5′-AGCTTGTCATCAACGGGAAG-3′ and 5′-TTTGATGTTAGTGGGGTCTCG-3’; IL-17A, 5′-TGTGAAGGTCAACCTCAAAGTCT-3′ and 5′-GAGGGATATCTATCAGGGTCTTCAT-3’.

Tokano M., Matsushita S., Takagi R., Yamamoto T, & Kawano M. (2022). Extracellular adenosine induces hypersecretion of IL-17A by T-helper 17 cells through the adenosine A2a receptor. Brain, Behavior, & Immunity - Health, 26, 100544.

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Quantifying Circulating Human DNA in Xenograft Mice

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Peripheral blood from orthotopic xenograft mouse models was taken biweekly. Circulating DNA was isolated from 100µL of each plasma sample using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Cat. 55114.). The amount of human derived DNA in each sample was quantified via real time real-time PCR (QuantStudio 12K Flex) using a human specific FOXP2 gene primer/probe set (Life Technologies, cat#4400291). ULK1 gene expression was assessed in MDA-MB-231 cells using ULK1 gene primer/probe set (Life Technologies, cat#4331182)

Dower C.M., Bhat N., Wang E.W, & Wang H.G. (2016). Selective reversible inhibition of autophagy in hypoxic breast cancer cells promotes pulmonary metastasis. Cancer research, 77(3), 646-657.

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Quantifying CHL1 Expression in Breast Cancer

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qRT-PCR was performed to measure the levels of CHL1 expression in BC-derived cell lines and to check the restoration of gene expression by AZA+TSA treatment. To this end, first, total RNA was extracted and purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. 500 ng of total RNA were retrotranscribed using a PrimeScript™ RT Reagent Kit (TaKaRa, Otsu, Japan) at 37°C for 15 min and 85°C for 5 s. 1 μl of the resulting cDNA was placed in a 96-well plate with 0.5 μl TaqMan probes (CHL1: Hs00544069_m1 from Life Technologies, Carlsbad, CA, USA; and GAPDH: Hs.PT.39a.22214836, from IDT, Coralville, Iowa, USA) and 19 μl of mix were included in the Premix Ex Taq™ kit (TaKaRa, Otsu, Japan). PCR amplification was performed in triplicate using the Quant Studio 12K Flex (Life Technologies, Carlsbad, CA, USA) under thermal cycler conditions of 95°C for 30 s and 40 cycles at 95°C for 5 sec and 60°C for 34 s. The cycle threshold (Ct) values were calculated using Quant Studio software (Life Technologies, Carlsbad, CA, USA), and the relative quantification (RQ) was calculated by the ΔCt method (RQ = 2^−ΔCt), using GAPDH as the endogenous control gene.

Martín-Sánchez E., Mendaza S., Ulazia-Garmendia A., Monreal-Santesteban I., Blanco-Luquin I., Córdoba A., Vicente-García F., Pérez-Janices N., Escors D., Megías D., López-Serra P., Esteller M., Illarramendi J.J, & Guerrero-Setas D. (2017). CHL1 hypermethylation as a potential biomarker of poor prognosis in breast cancer. Oncotarget, 8(9), 15789-15801.

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Quantifying DNA Release from Phages

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For fluorescent assays, T5 wt or T5Δdec phages were diluted to a final concentration of 5 × 10⁹ pfu/mL in buffer solutions containing SYBR-Gold (diluted 3,000X from a 10,000X stock solution of SYBR^® Gold in DMSO, Invitrogen). SYBR-Gold exhibits a 1,000-fold fluorescence enhancement upon binding to nucleic acids, which allows to quantify free DNA in solution. Reaction mixtures were made in quadruplicate in a 384-well fast PCR plate at a final volume of 10 μL. Temperature was increased from 4 °C to 99 °C at 3 °C/min with a QuantStudio 12 K Flex instrument (LifeTechnologies). Fluorescence was recorded as a function of temperature in real time (excitation with a white LED source and emission filtered through a Vic emission filter). The DNA exit temperature (T_ex), assessed by the release of DNA, was calculated with QuantStudio 12 K Flex software v1.2.2 as the maximum of the derivative of the resulting SYBR-Gold fluorescence curves. For EM imaging of DNA-free phages, particles heated at 1 °C/min up to 78 °C were cooled at room temperature and stained with 1% uranyl acetate as described in SI.

Vernhes E., Renouard M., Gilquin B., Cuniasse P., Durand D., England P., Hoos S., Huet A., Conway J.F., Glukhov A., Ksenzenko V., Jacquet E., Nhiri N., Zinn-Justin S, & Boulanger P. (2017). High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid. Scientific Reports, 7, 41662.

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Quantifying HDAC-modulated Genes in VOR

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HDACi-modulated genes identified in previous studies and in the transcriptome analyses were also evaluated in RNA samples from in vitro VOR simulation studies. RNA previously isolated for each study (see above) was converted to cDNA with the high-capacity cDNA reverse transcription kit (Life Technologies, Carlsbad, CA) per the manufacturer’s protocol at a concentration of 20 ng/μl. Twelve HDAC-modulated genes (Table 1) and Human GAPD (GAPDH) endogenous control (catalog no. 4310884E) from Life Technologies (Carlsbad, CA) were evaluated in samples from in vitro VOR studies using the TaqMan Fast advanced master mix and analyzed according to the manufacturer’s instructions on the QuantStudio 12K Flex instrument (Life Technologies, Carlsbad, CA). Changes in gene expression for VOR-treated samples were compared to gene expression in DMSO controls at the same time point using the threshold cycle (2^−ΔΔ^CT) method (27 (link)).

Maxwell J.W., Falcinelli S.D., Nefedov A., Dorfmeier C., Wu G., Dewey M., Webber A.L., Archin N.M., Margolis D.M., Hazuda D.J., Barnard R.J, & Howell B.J. (2020). Cellular Gene Modulation of HIV-Infected CD4 T Cells in Response to Serial Treatment with the Histone Deacetylase Inhibitor Vorinostat. Journal of Virology, 94(13), e00351-20.

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Cardiac Tissue Gene Expression Analysis

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Cardiac tissue samples collected in RNAlater stabilization solution were washed with DEPC water. Total RNA was isolated using TRIzol reagent. cDNA was synthesized by reverse transcription of total RNA (1 μg) using iScript cDNA Synthesis kit. Further, mRNA levels of enzymatic antioxidants (superoxide dismutase; sod and catalase), apoptotic genes (bax and bcl-2), myocardium specific caveolae protein (caveolin-3), and sarco/endopalsmic reticulum calcium ATPases (SERCA2a) with GAPDH as an internal control were evaluated by quantitative PCR as elucidated herein.
Quantitative PCR analysis (QuantStudio 12K Flex, Life Technologies, CA, USA) was performed using SYBR Select Master Mix. The reaction mixture consisted of cDNA (0.8 μl), forward and reverse primers (0.4 μl each), SYBR green master mix (5 μl) and ultrapure water (3.4 μl). Melting curve of each sample was measured to ensure the specificity of the products. The data were normalized to the internal control GAPDH and analysed using 2^-ΔΔCTmethod [25 (link)]. Primers used for this study are listed in Table 1.

Jana S., Patel D., Patel S., Upadhyay K., Thadani J., Mandal R., Das S, & Devkar R. (2017). Anthocyanin rich extract of Brassica oleracea L. alleviates experimentally induced myocardial infarction. PLoS ONE, 12(8), e0182137.

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Quantitative PCR Genotyping and Copy Number Analysis

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Specimens were analyzed using the LifeTech QuantStudio 12K Flex (software v1.2.2; Grand Island, NY) and subjected to Taqman® allele discrimination using LifeTech (Grand Island, NY) reagents in a custom-designed open array. Genomic DNA was amplified and mixed with dual-labeled oligonucleotides that hybridize to a specific target sequence. Hydrolysis by the 5′-3′ exonuclease activity of Taq polymerase releases the fluorescent reporter signal, permitting quantitative measurement of the accumulation of the PCR product via the fluorophore signal.^{18 (link)} Software utilized were Genotyper (v1.3) (LifeTech, Grand Island, NY) and Alleletyper (1.0) (LifeTech, Grand Island, NY).
For copy number analysis, CYP2D6 and a reference gene were compared using commercially available reagents from LifeTech (Grand Island, NY). Individual samples were run in quadruplicate. Each replicate was normalized to the reference gene to obtain a ΔCt (FAM dye Ct, VIC dye Ct), and then an average ΔCt for each sample (from the 4 replicates) was calculated. All samples were then normalized to a calibrator sample to determine ΔΔCt. Relative quantity (RQ) is 2-ΔΔCt, and copy number is 2 X RQ. Copy number was assigned using CopyCaller®Software (v 2.0) (LifeTech, Grand Island, NY).

Pratt V.M., Everts R.E., Aggarwal P., Beyer B.N., Broeckel U., Epstein-Baak R., Hujsak P., Kornreich R., Liao J., Lorier R., Scott S.A., Smith C.H., Toji L.H., Turner A, & Kalman L.V. (2016). Characterization of 137 Genomic DNA Reference Materials for 28 Pharmacogenetic Genes: A GeT-RM Collaborative Project. The Journal of Molecular Diagnostics : JMD, 18(1), 109-123.

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Ear RNA Extraction and qPCR Analysis

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Total RNA was extracted from the ears using RNeasy plus mini kit (Qiagen). The concentration was measured using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). RNA was converted to cDNA using high capacity cDNA Reverse Transcription kit (Life technologies, Foster City, CA) and real time PCR reaction was performed using Quant Studio 12k Flex (Life technologies). All Taqman probes were purchased from Life technologies, mIL-17A Mm00439619_m1; mIL-17F Mm00521423_m1; mIL-22 Mm00444241_m1, IFN-γ Mm00801778_m1; IL6 Mm00446191_m1; TNFα Mm00443260_g1; B2M Mm00437762_m1.

Xue X., Soroosh P., De Leon-Tabaldo A., Luna-Roman R., Sablad M., Rozenkrants N., Yu J., Castro G., Banie H., Fung-Leung W.P., Santamaria-Babi L., Schlueter T., Albers M., Leonard K., Budelsky A.L, & Fourie A.M. (2016). Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis. Scientific Reports, 6, 37977.

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Quantitative Analysis of Pluripotency Markers

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Total RNA was extracted using an RNeasy micro kit (74004, QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. One μg of total RNA was used to synthesize cDNA with the QuantiTect Reverse Transcription Kit (205311, QIAGEN). Quantitative PCR (qPCR) was performed with TaqMan hPSC Scorecard Panel (A15870, Life Technologies)[19 (link)], using a qRT-PCR device (QuantStudio 12K Flex, Life Technologies).

Takenaka C., Miyajima H., Yoda Y., Imazato H., Yamamoto T., Gomi S., Ohshima Y., Kagawa K., Sasaki T, & Kawamata S. (2015). Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes. PLoS ONE, 10(6), e0129855.

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