Steponeplus machine

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The StepOnePlus is a Real-Time PCR System designed for gene expression analysis, genotyping, copy number variation studies, and other real-time PCR applications. It features 96-well block format, a 7-inch color touchscreen display, and supports a wide range of fluorescent chemistries and detection channels.

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StepOnePlus (2314 citations) StepOnePlus real-time PCR machine (381 citations) StepOnePlus qPCR machine (71 citations) StepOnePlus PCR machine (42 citations) StepOnePlus RT-PCR machine (32 citations) ABI StepOnePlus Real-Time PCR machine (26 citations) ABI StepOnePlus machine (26 citations) StepOnePlus Real-Time PCR System machine (12 citations) StepOnePlus qRT-PCR machine (8 citations) StepOnePlus RT-qPCR machine (6 citations)

194 protocols using steponeplus machine

Gene Expression Quantification by RT-qPCR

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Total RNA and small RNA were extracted by mirVana miRNA isolation kit (Ambion, Thermo Fisher Scientific) according to the manufacturer’s protocol. Isolated RNA was treated with DNase to remove DNA (Turbo DNA-free kit, Ambion). One µg of isolated total RNA was then immediately reverse-transcribed into cDNA using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Thermo Fisher Scientific). Quantitative PCR was performed with a StepOne Plus machine (Applied Biosystem, Thermo Fisher Scientific) using SYBR green ROX qPCR mastermix in a 96-well optical plate. PCR cycling consists of 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 64°C for 30 s and 72°C for 30 s. A melt curve was conducted to determine the specificity of PCR amplification. Gapdh was served as an internal control to nomalize data. To assay the direct transcripts pre-mRNA, primers were designed to bind with the intron of genes.
For analysis of miRNA abundance, 30 ng of isolated samll RNA was reverse-transcribed using Taqman micoRNA reverse transcription kit (Applied Biosystems), and then subjected to Taqman microRNA assays according to manufacturer’s protocol (Applied Biosystems) in StepOne Plus machine. PCR cycling consists of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s. RNU48 served as an internal control to nomalize data.

Xiao M.F., Xu D., Craig M.T., Pelkey K.A., Chien C.C., Shi Y., Zhang J., Resnick S., Pletnikova O., Salmon D., Brewer J., Edland S., Wegiel J., Tycko B., Savonenko A., Reeves R.H., Troncoso J.C., McBain C.J., Galasko D, & Worley P.F. (2017). NPTX2 and cognitive dysfunction in Alzheimer’s Disease. eLife, 6, e23798.

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Quantitative RT-PCR for Gene Expression

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Total RNA was extracted from cells using the RNeasy micro kit (Qiagen, Hilden, Germany). RNA was converted into cDNA using the Superscript III reverse transcription system (Thermo-Fisher, Waltham, MA). Quantitative polymerase chain reaction (qPCR) was performed using Sybr green PCR mix (Bio-rad, Hercules, CA) and StepOnePlus machine (Applied Biosystems). PCR primers were Gpr109a; forward 5’-ATGGCGAGGCATATCTGTGTAGCA-3’, reverse 5’-TCCTGCCTGAGCAGAACAAGATGA-3’ Gapdh; forward 5’AGGTCGGTGAACGGATTTG-3’, reverse 5’-TGTAGACCATGTAGTTGAGGTCA-3’ Hprt; forward 5’-GCGTCGTGATTAGCGATGAAC-3’ reverse 5’CCTCCCATCTCCTTCATGACATCT-3’, Il23P19; forward 5’-GACCCACAAGGACTCAAGGA-3’, reverse 5’-CATGGGGCTATCAGGGAGTA-3’.

Bhatt B., Zeng P., Zhu H., Sivaprakasam S., Li S., Xiao H., Dong L., Shiao P., Kolhe R., Patel N., Li H., Levy-Bercowski D., Ganapathy V, & Singh N. (2018). Gpr109a limits microbiota-induced IL-23 production to constrain ILC3-mediated colonic inflammation. Journal of immunology (Baltimore, Md. : 1950), 200(8), 2905-2914.

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Quantitative Expression Analysis of ApoE Transcript

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RNA was extracted from frozen cortical tissue using Trizol (Life Technologies # 15596026) and purified using the RNeasy mine kit (Qiagen # 71404). Reverse transcription was performed using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Real-time qPCR was conducted with TaqMan primers (Life Technologies) and the TaqMan Universal PCR Master Mix (Thermo Fisher Scientific # 4304437) using the StepOnePlus machine (Applied Biosystems). Relative gene expression levels in ASO- and control-treated mice were compared using the ΔΔC_t method with Taqman probe for human apoE (Hs00171168_m1). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level was used as a reference (Mm99999915_g1 Gapdh).

Huynh T.P., Liao F., Francis C.M., Robinson G.O., Serrano J.R., Jiang H., Roh J., Finn M.B., Sullivan P.M., Esparza T.J., Stewart F.R., Mahan T.E., Ulrich J.D., Cole T, & Holtzman D.M. (2017). Age-dependent effects of apoE reduction using antisense oligonucleotides in a model of β-amyloidosis. Neuron, 96(5), 1013-1023.e4.

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Quantitative RT-PCR Analysis of LCM Samples

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Total RNA was purified from LCM-isolated samples of E14.5 mouse forelimbs using RNeasy FFPE Kit (Qiagen). Reverse transcription was performed with High Capacity Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. Analysis of Col2a1 and Scx was performed to monitor RNA quality during LCM calibrations, whereas RNA quantity was monitored by analysis of β–actin. RT-PCR was performed using Fast SYBR Green master mix (Applied Biosystems) on the StepOnePlus machine (Applied Biosystems). Values were calculated using the StepOne software. Data were normalized to 18S rRNA or β-actin in all cases.

Kult S., Olender T., Osterwalder M., Markman S., Leshkowitz D., Krief S., Blecher-Gonen R., Ben-Moshe S., Farack L., Keren-Shaul H., Salame T.M., Capellini T.D., Itzkovitz S., Amit I., Visel A, & Zelzer E. (2021). Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors. eLife, 10, e55361.

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RNA Extraction and Real-Time PCR Analysis of 3D Spheroids

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Total RNA was extracted from 3D n-HOF spheroids under several conditions as above using a RNeasy mini kit (Qiagen, Valencia, CA, USA) and following reverse transcription by the SuperScript IV kit (Invitrogen, Carlsbad, CA, USA), were processed according to the manufacturer’s instructions. In addition to these n-HOF-related cDNAs, previously prepared cDNAs obtained from GHOF spheroids [11 (link)] were subjected to real-time PCR with the Universal Taqman Master mix using a StepOnePlus machine (Applied Biosystems/Thermo Fisher Scientific, Carlsbad, MA, USA). cDNA levels expressed as fold-change relative to the expression of a housekeeping 36B4 (Rplp0) gene were then calculated. The sequences of the primers and Taqman probes used are shown in Table 1.

Ida Y., Ichioka H., Furuhashi M., Hikage F., Watanabe M., Umetsu A, & Ohguro H. (2021). Reactivities of a Prostanoid EP2 Agonist, Omidenepag, Are Useful for Distinguishing between 3D Spheroids of Human Orbital Fibroblasts without or with Graves’ Orbitopathy. Cells, 10(11), 3196.

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Quantification of Cellular and Extracellular RNA

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Total RNA was extracted from cultured cells and EVs using TRIzol and TRIzolLS Reagents (Life Technologies). cDNA was prepared using an oligo (dT) primer and reverse transcriptase (Takara, Shiga, Japan) following standard protocols. miRNAs were reverse transcribed using the Mir-X miRNA First Strand Synthesis Kit (Clontec). mRNA levels were performed using real-time analysis with SYBR Green on a StepOne-Plus machine (Applied Biosystems, Foster City, CA, USA). The relative expression levels of the cellular mRNA and miRNA were normalized to glyceraldehyde 3-phosphate dehydrogenase or U6 small nuclear RNA (snRNA), respectively. EV miRNA purified from serum was normalized with cel-miR-39 (Exiqon, Vedbaek, Denmark) as a spike-in control, and compared with a reference sample. The primers used are listed in Table S2. The relative standard curve method (2^−ΔΔCt) was used to determine relative mRNA or miRNA expression.

Hsu Y.L., Hung J.Y., Chang W.A., Jian S.F., Lin Y.S., Pan Y.C., Wu C.Y, & Kuo P.L. (2017). Hypoxic Lung-Cancer-Derived Extracellular Vesicle MicroRNA-103a Increases the Oncogenic Effects of Macrophages by Targeting PTEN. Molecular Therapy, 26(2), 568-581.

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Quantitative Real-Time PCR Analysis

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Total RNA was isolated from cells by using Total RNA preparation kits (easy-Blue Total RNA Extraction kit, iNtRON Biotechnology, Seongnam, Korea) following the manufacturer’s protocol. The RNA was reverse transcribed to cDNA by reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR (qPCR) was used to determine the mRNA levels of target genes by running on a StepOnePlus machine (Applied Biosystems, Foster City, CA, USA). The SYBR Green fluorescence probe system (KAPA Biosystems, Woburn, MA, USA) was used for determining the threshold cycle (C_T) of target genes. Primers of human VCAM-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Sigma-Aldrich. The expression levels of the target genes were normalized to GAPDH levels and the formula of level ratio = 2^−ΔΔCt, where ΔΔCt = (Ct _target−Ct _GADPH)_Sample−(Ct _target−Ct _GADPH)_Control, was used for calculation. The data were represented with three independent experiments with triplicate of each sample.

Lee C.W., Chiang Y.C., Yu P.A., Peng K.T., Chi M.C., Lee M.H., Fang M.L., Lee K.H., Hsu L.F, & Liu J.F. (2021). A Role of CXCL1 Drives Osteosarcoma Lung Metastasis via VCAM-1 Production. Frontiers in Oncology, 11, 735277.

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Comprehensive RNA Isolation and Analysis

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Total RNA was isolated from freshly picked mouse islets or transfected INS-1 832/13 cells using the RNeasy mini kit (Qiagen, Hilden, Germany). For quantitative real-time PCR, mRNA was converted into complementary DNA (cDNA) using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA), and qRT-PCR was performed on the StepOnePlus machine (Applied Biosystems, Carlsbad, CA) under default thermal cycling conditions. Data were analyzed using the 2^−ΔΔCT method with mRNA levels normalized to genes β-actin for every sample. For RNA-seq, RNA size and quality were assessed using the Tape Station Bionalyzer (Agilent, Santa Clara, CA), and concentration was determined by Qubit fluorometry. All samples had DV₂₀₀ scores (percentage of RNA fragments >200 nucleotides) ≥70% and were subsequently approved for library preparation and sequencing by GENEWIZ (South Plainfield, NJ). RNA libraries were prepared using the polyA selection method (Illumina, San Diego, CA) from 100 ng of total RNA. Paired-end, 150–base pair (bp) sequencing of the libraries (>25 million reads per sample) was performed using an Illumina HiSeq instrument according to the manufacturer’s instructions. All sequenced samples had a Q₃₀ score (percentage of bases with a quality score > 30) ≥93% with a mean quality score ≥35. RNA-seq bioinformatic analysis was performed as described in the Supplementary Materials.

Brown M.R., Sen S.K., Mazzone A., Her T.K., Xiong Y., Lee J.H., Javeed N., Colwell C.S., Rakshit K., LeBrasseur N.K., Gaspar-Maia A., Ordog T, & Matveyenko A.V. (2021). Time-restricted feeding prevents deleterious metabolic effects of circadian disruption through epigenetic control of β cell function. Science Advances, 7(51), eabg6856.

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Quantification of HO-1 Expression in LDL-Treated Cells

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Cells were either treated or not with native LDL or Mox-LDL at a final concentration of 100 μg/mL for 24 hours. Total RNA was extracted from cells using TRIzol total RNA isolation reagent (Roche Applied Science) and reverse-transcribed using Reverse Transcriptase Core Kit (Eurogentec) according to the manufacturer's instructions. qRT-PCR was performed in 96-well plate format using SYBR Green-based detection on a Step-One-Plus machine (Applied Biosystems) with each 20 μL reaction containing ~50 ng cDNA, 0.3 μM sense and antisense primers, and ABsolute qPCR SYBR Green ROX Mix (Thermo Scientific). The plate was sealed and cycled under the following conditions: 95°C for 15 min, 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Each reaction was performed in triplicate and mRNA levels of RPL27 were used for normalization. The relative expression levels of mRNAs were calculated using the comparative ΔΔCt method. PCR primers used for the quantification of HO-1 and RPL27 were as follows:

HO-1 forward primer 5′-AAGACTGCGTTCCTGCTCAAC-3′;

HO-1 reverse primer 5′-AAAGCCCTACAGCAACTGTCG-3′;

RPL27 forward primer 5′-ATCGCCAAGAGATCAAAGATAA-3′;

RPL27 reverse primer 5′-TCTGAAGACATCCTTATGACG-3′.

Daher J., Martin M., Rousseau A., Nuyens V., Fayyad-Kazan H., Van Antwerpen P., Courbebaisse G., Martiat P., Badran B., Dequiedt F., Zouaoui Boudjeltia K, & Vanhamme L. (2014). Myeloperoxidase Oxidized LDL Interferes with Endothelial Cell Motility through miR-22 and Heme Oxygenase 1 Induction: Possible Involvement in Reendothelialization of Vascular Injuries. Mediators of Inflammation, 2014, 134635.

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RNA Isolation and RT-PCR Analysis

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RNA was isolated using TRIzol reagent (Life Technologies) and cDNA was prepared (cDNA synthesis kit; Life Technologies). RT-PCR was performed using Thermal Cycler (Eppendorf, Hamberg, Germany). Each cycle of RT-PCR consisted of 35 cycles of 30 seconds of denaturation at 94 °C, 30 seconds of annealing at 60 °C and 30 seconds of extension at 72 °C. RT-PCR primers used were IL-3Rα F-5′-GGAGAATCTGACCTGCTGGA-3′ and R-5′-ACTTTGAGAACCGCTGGAGA-3′, and β-actin F-5′-CGGGAAATCGTGCGTGACAT-3′ and R-5′-ATCTTCATTGTGCTGGGTGCC-3′. Quantitative real-time PCR was performed using Universal PCR mix and Taqman primers and probe (Applied Biosystems, CA, USA) for human CXCR4 (Hs00607978_s1) and β-actin (Hs00664172_s1) on a StepOnePlus machine (Applied Biosystems). The reaction consisted of 30 seconds of annealing at 60 °C for 40 cycles. Fold change in expression of genes was calculated relative to β-actin levels by the comparative ∆Ct method.

Barhanpurkar-Naik A., Mhaske S.T., Pote S.T., Singh K, & Wani M.R. (2017). Interleukin-3 enhances the migration of human mesenchymal stem cells by regulating expression of CXCR4. Stem Cell Research & Therapy, 8, 168.

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