applied biosystems steponeplus device  (Thermo Fisher)


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    Structured Review

    Thermo Fisher applied biosystems steponeplus device
    Applied Biosystems Steponeplus Device, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/applied biosystems steponeplus device/product/Thermo Fisher
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    applied biosystems steponeplus device - by Bioz Stars, 2020-04
    90/100 stars

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    Related Articles

    Amplification:

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: 9 Although this qRT-PCR method employs SYBR® green to quantify PCR amplification, the use of an alternate fluorogenic strategy (i.e., TaqMan) may also be readily adopted, assuming that the appropriate primer/probe sets are designed/utilized [ , ]. .. We use the StepOnePlus™ Real-Time PCR System (Life Technologies) and incorporate the “Fast” setting for all qRT-PCR runs.

    Article Title: Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation
    Article Snippet: RT-PCR reactions were conducted using 35 cycles at 95°C for 30 s, 55°C for 30 s, then 72°C for 60 s. PCR products were run on 1.5% agarose gel for 30 min at 100 V. qPCR was carried out using RealAmp SYBR qPCR master mix (GeneAll Biotechnology Co., LTD, Seoul, Korea) and a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA) according to the manufacturers’ instructions. .. The relative transcript quantities were calculated using the 2-ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) as the endogenous reference gene amplified from the samples.

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: .. We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). .. The assay amplification efficiency was calculated by the standard curve method ( ) using a 10-fold, 8-log, serial dilution starting at 2 × 108 RNA copies/µL, in duplicate.

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor
    Article Snippet: .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers for each gene amplicon ( ). ..

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: .. The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C. .. Using specific software from the qRT-PCR system, assign a baseline range that demonstrates little to no fluorescent activity up to the cycle before amplification becomes evident (usually cycles 3–15).

    Polymerase Chain Reaction:

    Article Title: Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways
    Article Snippet: .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus ™Real-Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers. ..

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: 9 Although this qRT-PCR method employs SYBR® green to quantify PCR amplification, the use of an alternate fluorogenic strategy (i.e., TaqMan) may also be readily adopted, assuming that the appropriate primer/probe sets are designed/utilized [ , ]. .. We use the StepOnePlus™ Real-Time PCR System (Life Technologies) and incorporate the “Fast” setting for all qRT-PCR runs.

    Article Title: Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation
    Article Snippet: .. RT-PCR reactions were conducted using 35 cycles at 95°C for 30 s, 55°C for 30 s, then 72°C for 60 s. PCR products were run on 1.5% agarose gel for 30 min at 100 V. qPCR was carried out using RealAmp SYBR qPCR master mix (GeneAll Biotechnology Co., LTD, Seoul, Korea) and a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA) according to the manufacturers’ instructions. ..

    Article Title: Stretch-Sensitive Down-Regulation of the miR-144/451 Cluster in Vascular Smooth Muscle and Its Role in AMP-Activated Protein Kinase Signaling
    Article Snippet: .. The relative expression of miRNAs was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using miScript SYBR Green PCR Kit (Qiagen, # 218073) and miScript Primer Assays (Mm_miR-144_3, #MS00032326; Mm_miR-451_1 # MS00002408, Hs_RNU6-2_1, #MS00033740). .. For mRNA analysis, the same isolation kit, including on-column DNAse I digestion, and qPCR equipment as for miRNA was used, but mRNA expression was detected using QuantiFast SYBR Green RT-PCR Kit Qiagen, #204156) and QuantiTect primer assays (Qiagen, Mm_Kcnmb1, #QT00101500, Mm_Tagln, QT00165179, Mm_Cnn1, QT00105420, Mm_Des, #QT00102333).

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: Before the in vitro transcription of RNA, the insert and the T7 promoter site were PCR amplified (amplicon size, 925 bp) to limit the template size, for enhancement of in vitro RNA transcription. .. We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems).

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor
    Article Snippet: .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers for each gene amplicon ( ). ..

    Quantitative RT-PCR:

    Article Title: Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways
    Article Snippet: .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus ™Real-Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers. ..

    Article Title: Synergistic effects on mesenchymal stem cell-based cartilage regeneration by chondrogenic preconditioning and mechanical stimulation
    Article Snippet: .. Complementary DNA quantification was performed in duplicate by qRT-PCR using Step-One-Plus Real Time PCR Systems (Applied Biosystems) and normalized against GAPDH expression. ..

    Article Title: The RBP-J? Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation
    Article Snippet: .. RT-qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems Inc, Carlsbad, CA) or Opticon 2 Real-Time PCR System. ..

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: .. We use the StepOnePlus™ Real-Time PCR System (Life Technologies) and incorporate the “Fast” setting for all qRT-PCR runs. .. As such, our laboratory incorporates the Fast SYBR® Green Master Mix along with MicroAmp® Fast tubes (all from Life Technologies) for Subheading 3.4, step 1 .

    Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation
    Article Snippet: .. RT-qPCR was performed on a StepOnePlus real-time PCR System (Applied Biosystems Inc, Carlsbad, CA). ..

    Article Title: Stretch-Sensitive Down-Regulation of the miR-144/451 Cluster in Vascular Smooth Muscle and Its Role in AMP-Activated Protein Kinase Signaling
    Article Snippet: Paragraph title: Quantitative real-time PCR (qRT-PCR) ... The relative expression of miRNAs was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using miScript SYBR Green PCR Kit (Qiagen, # 218073) and miScript Primer Assays (Mm_miR-144_3, #MS00032326; Mm_miR-451_1 # MS00002408, Hs_RNU6-2_1, #MS00033740).

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: .. We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). .. The assay amplification efficiency was calculated by the standard curve method ( ) using a 10-fold, 8-log, serial dilution starting at 2 × 108 RNA copies/µL, in duplicate.

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor
    Article Snippet: .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers for each gene amplicon ( ). ..

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: Paragraph title: 3.4 qRT-PCR Performance and Analysis ... The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C.

    SYBR Green Assay:

    Article Title: Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways
    Article Snippet: .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus ™Real-Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers. ..

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: 9 Although this qRT-PCR method employs SYBR® green to quantify PCR amplification, the use of an alternate fluorogenic strategy (i.e., TaqMan) may also be readily adopted, assuming that the appropriate primer/probe sets are designed/utilized [ , ]. .. We use the StepOnePlus™ Real-Time PCR System (Life Technologies) and incorporate the “Fast” setting for all qRT-PCR runs.

    Article Title: Stretch-Sensitive Down-Regulation of the miR-144/451 Cluster in Vascular Smooth Muscle and Its Role in AMP-Activated Protein Kinase Signaling
    Article Snippet: .. The relative expression of miRNAs was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using miScript SYBR Green PCR Kit (Qiagen, # 218073) and miScript Primer Assays (Mm_miR-144_3, #MS00032326; Mm_miR-451_1 # MS00002408, Hs_RNU6-2_1, #MS00033740). .. For mRNA analysis, the same isolation kit, including on-column DNAse I digestion, and qPCR equipment as for miRNA was used, but mRNA expression was detected using QuantiFast SYBR Green RT-PCR Kit Qiagen, #204156) and QuantiTect primer assays (Qiagen, Mm_Kcnmb1, #QT00101500, Mm_Tagln, QT00165179, Mm_Cnn1, QT00105420, Mm_Des, #QT00102333).

    Article Title: Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization *
    Article Snippet: .. The expression of mRNA was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using the QuantiFast SYBR Green RT-PCR kit (Qiagen, catalog no. 204156) and QuantiTect primer assays: mouse, Mm_Kcnmb1, QT00101500; Mm_Tagln, QT00165179; Mm_Cnn1, QT00105420, Mm_GADPH, QT01658692; Mm_Dmd, QT00161336; Mm_Des, QT00102333; Mm_Dcn, QT00131068, Mm_Ptgs2 (COX-2), QT00165347; Mm_Tnf, QT00165347; Mm_Mglap, QT00145775; Mm_Spp1, QT02524536. .. Primer sequences are proprietary of Qiagen.

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor
    Article Snippet: .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers for each gene amplicon ( ). ..

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: Mix the SYBR® Green Master Mix, IFN-γ or CD8 forward/reverse primers, H2 O, and cDNA together in the appropriate tube on a cold block following the specific guidelines of a qRT-PCR system as diagramed in . .. The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C.

    Incubation:

    Article Title: Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation
    Article Snippet: For reverse transcription polymerase chain reaction (RT-PCR) and quantitative polymerase chain reaction (qPCR) assay, the cell-seeded hydrogels were incubated in the chondrocyte maintenance medium for 14 days. .. RT-PCR reactions were conducted using 35 cycles at 95°C for 30 s, 55°C for 30 s, then 72°C for 60 s. PCR products were run on 1.5% agarose gel for 30 min at 100 V. qPCR was carried out using RealAmp SYBR qPCR master mix (GeneAll Biotechnology Co., LTD, Seoul, Korea) and a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA) according to the manufacturers’ instructions.

    Activity Assay:

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C. .. Using specific software from the qRT-PCR system, assign a baseline range that demonstrates little to no fluorescent activity up to the cycle before amplification becomes evident (usually cycles 3–15).

    Cell Culture:

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor
    Article Snippet: RNA extraction and gene expression assays For the RT-qPCR analysis, total RNA from the wild-type strain and the Δpac-3 , Δpal-1 , Δpal-2 , Δpal-3 , Δpal-6 , Δpal-8 and Δpal-9 mutants was prepared using mycelia samples cultured at pH 5.8 for 24 h at 30°C and mycelia grown at pH 5.8 and shifted to pH 7.8 for 1 h, according to Sokolovsky et al. [ ]. .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers for each gene amplicon ( ).

    Expressing:

    Article Title: Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways
    Article Snippet: Paragraph title: RNA isolation and gene expression analysis ... The cDNA libraries were subjected to RT-qPCR on a StepOnePlus ™Real-Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers.

    Article Title: Synergistic effects on mesenchymal stem cell-based cartilage regeneration by chondrogenic preconditioning and mechanical stimulation
    Article Snippet: .. Complementary DNA quantification was performed in duplicate by qRT-PCR using Step-One-Plus Real Time PCR Systems (Applied Biosystems) and normalized against GAPDH expression. ..

    Article Title: Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation
    Article Snippet: Paragraph title: Expression pattern of chondrogenic genes ... RT-PCR reactions were conducted using 35 cycles at 95°C for 30 s, 55°C for 30 s, then 72°C for 60 s. PCR products were run on 1.5% agarose gel for 30 min at 100 V. qPCR was carried out using RealAmp SYBR qPCR master mix (GeneAll Biotechnology Co., LTD, Seoul, Korea) and a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA) according to the manufacturers’ instructions.

    Article Title: Stretch-Sensitive Down-Regulation of the miR-144/451 Cluster in Vascular Smooth Muscle and Its Role in AMP-Activated Protein Kinase Signaling
    Article Snippet: .. The relative expression of miRNAs was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using miScript SYBR Green PCR Kit (Qiagen, # 218073) and miScript Primer Assays (Mm_miR-144_3, #MS00032326; Mm_miR-451_1 # MS00002408, Hs_RNU6-2_1, #MS00033740). .. For mRNA analysis, the same isolation kit, including on-column DNAse I digestion, and qPCR equipment as for miRNA was used, but mRNA expression was detected using QuantiFast SYBR Green RT-PCR Kit Qiagen, #204156) and QuantiTect primer assays (Qiagen, Mm_Kcnmb1, #QT00101500, Mm_Tagln, QT00165179, Mm_Cnn1, QT00105420, Mm_Des, #QT00102333).

    Article Title: Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization *
    Article Snippet: .. The expression of mRNA was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using the QuantiFast SYBR Green RT-PCR kit (Qiagen, catalog no. 204156) and QuantiTect primer assays: mouse, Mm_Kcnmb1, QT00101500; Mm_Tagln, QT00165179; Mm_Cnn1, QT00105420, Mm_GADPH, QT01658692; Mm_Dmd, QT00161336; Mm_Des, QT00102333; Mm_Dcn, QT00131068, Mm_Ptgs2 (COX-2), QT00165347; Mm_Tnf, QT00165347; Mm_Mglap, QT00145775; Mm_Spp1, QT02524536. .. Primer sequences are proprietary of Qiagen.

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor
    Article Snippet: Paragraph title: RNA extraction and gene expression assays ... The cDNA libraries were subjected to RT-qPCR on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers for each gene amplicon ( ).

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C. .. Following a successful qRT-PCR run, calculate IFN-γ gene expression values using an appropriate quantitation method (i.e., absolute or relative quantitation).

    Blocking Assay:

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: Mix the SYBR® Green Master Mix, IFN-γ or CD8 forward/reverse primers, H2 O, and cDNA together in the appropriate tube on a cold block following the specific guidelines of a qRT-PCR system as diagramed in . .. The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C.

    Hybridization:

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: Additionally, primer pairs must be designed to avoid hybridization (i.e., primer-dimer amplification) and either anneal to exons with an internal intron region or span an exon/exon boundary. .. The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C.

    Serial Dilution:

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). .. The assay amplification efficiency was calculated by the standard curve method ( ) using a 10-fold, 8-log, serial dilution starting at 2 × 108 RNA copies/µL, in duplicate.

    Infection:

    Article Title: The RBP-J? Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation
    Article Snippet: Quantitation of KSHV RNA Total RNA from infected PBMCs were extracted by using TRIzol (Invitrogen, Inc., Carlsbad, CA) and 1 µg DNase-treated total RNA were used to generate cDNA using the High capacity RNA-to-cDNA kit (Applied Biosystems Inc., Foster City, CA) according to manufacturer's instructions. .. RT-qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems Inc, Carlsbad, CA) or Opticon 2 Real-Time PCR System.

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). .. Viral RNA from MAYV and OROV, as well as 15 other arboviruses, from three different families were extracted from previously infected suckling mouse brain or liver with Trizol LS (Invitrogen), as recommended by the manufacturer.

    Generated:

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: The generated amplicons were precipitated with molecular biology grade polyethylene glycol 8000 (20% w/v; Promega, WI, USA) for primers and dNTP removal. .. We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation
    Article Snippet: Paragraph title: RT-PCR ... RT-qPCR was performed on a StepOnePlus real-time PCR System (Applied Biosystems Inc, Carlsbad, CA).

    Article Title: Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation
    Article Snippet: .. RT-PCR reactions were conducted using 35 cycles at 95°C for 30 s, 55°C for 30 s, then 72°C for 60 s. PCR products were run on 1.5% agarose gel for 30 min at 100 V. qPCR was carried out using RealAmp SYBR qPCR master mix (GeneAll Biotechnology Co., LTD, Seoul, Korea) and a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA) according to the manufacturers’ instructions. ..

    Article Title: Stretch-Sensitive Down-Regulation of the miR-144/451 Cluster in Vascular Smooth Muscle and Its Role in AMP-Activated Protein Kinase Signaling
    Article Snippet: The relative expression of miRNAs was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using miScript SYBR Green PCR Kit (Qiagen, # 218073) and miScript Primer Assays (Mm_miR-144_3, #MS00032326; Mm_miR-451_1 # MS00002408, Hs_RNU6-2_1, #MS00033740). .. For mRNA analysis, the same isolation kit, including on-column DNAse I digestion, and qPCR equipment as for miRNA was used, but mRNA expression was detected using QuantiFast SYBR Green RT-PCR Kit Qiagen, #204156) and QuantiTect primer assays (Qiagen, Mm_Kcnmb1, #QT00101500, Mm_Tagln, QT00165179, Mm_Cnn1, QT00105420, Mm_Des, #QT00102333).

    Article Title: Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization *
    Article Snippet: .. The expression of mRNA was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using the QuantiFast SYBR Green RT-PCR kit (Qiagen, catalog no. 204156) and QuantiTect primer assays: mouse, Mm_Kcnmb1, QT00101500; Mm_Tagln, QT00165179; Mm_Cnn1, QT00105420, Mm_GADPH, QT01658692; Mm_Dmd, QT00161336; Mm_Des, QT00102333; Mm_Dcn, QT00131068, Mm_Ptgs2 (COX-2), QT00165347; Mm_Tnf, QT00165347; Mm_Mglap, QT00145775; Mm_Spp1, QT02524536. .. Primer sequences are proprietary of Qiagen.

    RNA HS Assay:

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: DNase digested in vitro transcribed RNA was quantified with a Qubit® RNA HS Assay Kit using a Qubit® 2.0 Fluorometer (Invitrogen, Thermo Scientific, CA, USA). .. We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems).

    Fluorescence:

    Article Title: Contribution of histone N-terminal tails to the structure and stability of nucleosomes
    Article Snippet: .. The sample temperature was increased by the StepOnePlus™ Real-Time PCR unit (Applied Biosystems), and the fluorescence signals were measured with this system. .. Since the wavelength at the fluorescence emission maximum of SYPRO Orange is 570 nm, the fluorescence filter ‘filter 3’, which covers the emission wavelength ranges of the TAMRA (580 nm) and NED (575 nm) dyes, was used for detecting the fluorescence of SYPRO Orange bound to the denatured histones.

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor
    Article Snippet: The cDNA libraries were subjected to RT-qPCR on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers for each gene amplicon ( ). .. The fluorescent dye ROX™ was used as the passive reference to normalize the SYBR green reporter dye fluorescence signal.

    Isolation:

    Article Title: Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways
    Article Snippet: Paragraph title: RNA isolation and gene expression analysis ... The cDNA libraries were subjected to RT-qPCR on a StepOnePlus ™Real-Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers.

    Article Title: Stretch-Sensitive Down-Regulation of the miR-144/451 Cluster in Vascular Smooth Muscle and Its Role in AMP-Activated Protein Kinase Signaling
    Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNA was isolated using miRNeasy mini kit (Qiagen, #217004) according to the manufacturer's instructions. .. The relative expression of miRNAs was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using miScript SYBR Green PCR Kit (Qiagen, # 218073) and miScript Primer Assays (Mm_miR-144_3, #MS00032326; Mm_miR-451_1 # MS00002408, Hs_RNU6-2_1, #MS00033740).

    Article Title: Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization *
    Article Snippet: Total RNA was isolated using the miRNeasy minikit (Qiagen, catalog no. 217004), as described previously ( ). .. The expression of mRNA was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using the QuantiFast SYBR Green RT-PCR kit (Qiagen, catalog no. 204156) and QuantiTect primer assays: mouse, Mm_Kcnmb1, QT00101500; Mm_Tagln, QT00165179; Mm_Cnn1, QT00105420, Mm_GADPH, QT01658692; Mm_Dmd, QT00161336; Mm_Des, QT00102333; Mm_Dcn, QT00131068, Mm_Ptgs2 (COX-2), QT00165347; Mm_Tnf, QT00165347; Mm_Mglap, QT00145775; Mm_Spp1, QT02524536.

    Stability Assay:

    Article Title: Contribution of histone N-terminal tails to the structure and stability of nucleosomes
    Article Snippet: Paragraph title: Thermal stability assay ... The sample temperature was increased by the StepOnePlus™ Real-Time PCR unit (Applied Biosystems), and the fluorescence signals were measured with this system.

    Titration:

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: The in vitro transcribed RNA was used in all optimisation steps of this protocol, including primer and probe titration. .. We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems).

    Sequencing:

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: RNA quantitation and its sequence were used to calculate the exact target copy numbers with the aid of the endmemo server (endmemo.com/bio/dnacopynum.php). .. We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems).

    Construct:

    Article Title: Synergistic effects on mesenchymal stem cell-based cartilage regeneration by chondrogenic preconditioning and mechanical stimulation
    Article Snippet: Gene expression Total RNA extracted from cell pellets (n = 3 replicates from each donors) or hydrogel constructs (n = 4 per group) were used to perform qRT-PCR. .. Complementary DNA quantification was performed in duplicate by qRT-PCR using Step-One-Plus Real Time PCR Systems (Applied Biosystems) and normalized against GAPDH expression.

    Software:

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor
    Article Snippet: The cDNA libraries were subjected to RT-qPCR on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers for each gene amplicon ( ). .. Data analysis was performed by the StepOne Software (Applied Biosystems) using the comparative CT (ΔΔCT) method [ ].

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C. .. Using specific software from the qRT-PCR system, assign a baseline range that demonstrates little to no fluorescent activity up to the cycle before amplification becomes evident (usually cycles 3–15).

    Real-time Polymerase Chain Reaction:

    Article Title: Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways
    Article Snippet: .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus ™Real-Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers. ..

    Article Title: Synergistic effects on mesenchymal stem cell-based cartilage regeneration by chondrogenic preconditioning and mechanical stimulation
    Article Snippet: .. Complementary DNA quantification was performed in duplicate by qRT-PCR using Step-One-Plus Real Time PCR Systems (Applied Biosystems) and normalized against GAPDH expression. ..

    Article Title: Contribution of histone N-terminal tails to the structure and stability of nucleosomes
    Article Snippet: .. The sample temperature was increased by the StepOnePlus™ Real-Time PCR unit (Applied Biosystems), and the fluorescence signals were measured with this system. .. Since the wavelength at the fluorescence emission maximum of SYPRO Orange is 570 nm, the fluorescence filter ‘filter 3’, which covers the emission wavelength ranges of the TAMRA (580 nm) and NED (575 nm) dyes, was used for detecting the fluorescence of SYPRO Orange bound to the denatured histones.

    Article Title: The RBP-J? Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation
    Article Snippet: .. RT-qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems Inc, Carlsbad, CA) or Opticon 2 Real-Time PCR System. ..

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: .. We use the StepOnePlus™ Real-Time PCR System (Life Technologies) and incorporate the “Fast” setting for all qRT-PCR runs. .. As such, our laboratory incorporates the Fast SYBR® Green Master Mix along with MicroAmp® Fast tubes (all from Life Technologies) for Subheading 3.4, step 1 .

    Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation
    Article Snippet: .. RT-qPCR was performed on a StepOnePlus real-time PCR System (Applied Biosystems Inc, Carlsbad, CA). ..

    Article Title: Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation
    Article Snippet: .. RT-PCR reactions were conducted using 35 cycles at 95°C for 30 s, 55°C for 30 s, then 72°C for 60 s. PCR products were run on 1.5% agarose gel for 30 min at 100 V. qPCR was carried out using RealAmp SYBR qPCR master mix (GeneAll Biotechnology Co., LTD, Seoul, Korea) and a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA) according to the manufacturers’ instructions. ..

    Article Title: Stretch-Sensitive Down-Regulation of the miR-144/451 Cluster in Vascular Smooth Muscle and Its Role in AMP-Activated Protein Kinase Signaling
    Article Snippet: .. The relative expression of miRNAs was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using miScript SYBR Green PCR Kit (Qiagen, # 218073) and miScript Primer Assays (Mm_miR-144_3, #MS00032326; Mm_miR-451_1 # MS00002408, Hs_RNU6-2_1, #MS00033740). .. For mRNA analysis, the same isolation kit, including on-column DNAse I digestion, and qPCR equipment as for miRNA was used, but mRNA expression was detected using QuantiFast SYBR Green RT-PCR Kit Qiagen, #204156) and QuantiTect primer assays (Qiagen, Mm_Kcnmb1, #QT00101500, Mm_Tagln, QT00165179, Mm_Cnn1, QT00105420, Mm_Des, #QT00102333).

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: .. We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). .. The assay amplification efficiency was calculated by the standard curve method ( ) using a 10-fold, 8-log, serial dilution starting at 2 × 108 RNA copies/µL, in duplicate.

    Article Title: Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization *
    Article Snippet: .. The expression of mRNA was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using the QuantiFast SYBR Green RT-PCR kit (Qiagen, catalog no. 204156) and QuantiTect primer assays: mouse, Mm_Kcnmb1, QT00101500; Mm_Tagln, QT00165179; Mm_Cnn1, QT00105420, Mm_GADPH, QT01658692; Mm_Dmd, QT00161336; Mm_Des, QT00102333; Mm_Dcn, QT00131068, Mm_Ptgs2 (COX-2), QT00165347; Mm_Tnf, QT00165347; Mm_Mglap, QT00145775; Mm_Spp1, QT02524536. .. Primer sequences are proprietary of Qiagen.

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor
    Article Snippet: .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers for each gene amplicon ( ). ..

    RNA Extraction:

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor
    Article Snippet: Paragraph title: RNA extraction and gene expression assays ... The cDNA libraries were subjected to RT-qPCR on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers for each gene amplicon ( ).

    Agarose Gel Electrophoresis:

    Article Title: Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways
    Article Snippet: RNA (10 μg) from each sample was fractionated on agarose gel to assess the rRNAs integrity. .. The cDNA libraries were subjected to RT-qPCR on a StepOnePlus ™Real-Time PCR System (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and specific primers.

    Article Title: Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation
    Article Snippet: .. RT-PCR reactions were conducted using 35 cycles at 95°C for 30 s, 55°C for 30 s, then 72°C for 60 s. PCR products were run on 1.5% agarose gel for 30 min at 100 V. qPCR was carried out using RealAmp SYBR qPCR master mix (GeneAll Biotechnology Co., LTD, Seoul, Korea) and a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA) according to the manufacturers’ instructions. ..

    In Vitro:

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: The in vitro transcribed RNA was used in all optimisation steps of this protocol, including primer and probe titration. .. We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems).

    Quantitation Assay:

    Article Title: The RBP-J? Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation
    Article Snippet: Paragraph title: Quantitation of KSHV RNA ... RT-qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems Inc, Carlsbad, CA) or Opticon 2 Real-Time PCR System.

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
    Article Snippet: RNA quantitation and its sequence were used to calculate the exact target copy numbers with the aid of the endmemo server (endmemo.com/bio/dnacopynum.php). .. We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems).

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C. .. Following a successful qRT-PCR run, calculate IFN-γ gene expression values using an appropriate quantitation method (i.e., absolute or relative quantitation).

    Spectrophotometry:

    Article Title: Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization *
    Article Snippet: Purity and concentration were determined with an ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE). .. The expression of mRNA was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using the QuantiFast SYBR Green RT-PCR kit (Qiagen, catalog no. 204156) and QuantiTect primer assays: mouse, Mm_Kcnmb1, QT00101500; Mm_Tagln, QT00165179; Mm_Cnn1, QT00105420, Mm_GADPH, QT01658692; Mm_Dmd, QT00161336; Mm_Des, QT00102333; Mm_Dcn, QT00131068, Mm_Ptgs2 (COX-2), QT00165347; Mm_Tnf, QT00165347; Mm_Mglap, QT00145775; Mm_Spp1, QT02524536.

    Concentration Assay:

    Article Title: Contribution of histone N-terminal tails to the structure and stability of nucleosomes
    Article Snippet: Each nucleosome, containing tlH2A, tlH2B, tlH3, or tlH4 (final concentration, 2.25 μM), was prepared in 20 μl of a solution composed of 18 mM Tris–HCl (pH 7.5), 0.9 mM EDTA, 0.9 mM DTT, and SYPRO Orange (final concentration, 5×). .. The sample temperature was increased by the StepOnePlus™ Real-Time PCR unit (Applied Biosystems), and the fluorescence signals were measured with this system.

    Article Title: Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization *
    Article Snippet: Purity and concentration were determined with an ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE). .. The expression of mRNA was analyzed by real-time qPCR (StepOnePlus qPCR cycler, Applied Biosystems) using the QuantiFast SYBR Green RT-PCR kit (Qiagen, catalog no. 204156) and QuantiTect primer assays: mouse, Mm_Kcnmb1, QT00101500; Mm_Tagln, QT00165179; Mm_Cnn1, QT00105420, Mm_GADPH, QT01658692; Mm_Dmd, QT00161336; Mm_Des, QT00102333; Mm_Dcn, QT00131068, Mm_Ptgs2 (COX-2), QT00165347; Mm_Tnf, QT00165347; Mm_Mglap, QT00145775; Mm_Spp1, QT02524536.

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR
    Article Snippet: Primers are also usually dissolved in water at a working stock concentration of 20 μM, and qRT-PCR trial runs incorporating primers between 0.05 and 0.9 μM will typically yield optimum primer pair concentrations for qRT-PCR assay conditions. .. The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C.

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    Thermo Fisher steponeplus real time system
    p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems <t>StepOnePlus</t> Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p
    Steponeplus Real Time System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Azacitidine mitigates GvHD via differential effects on the proliferation of T effectors and nTregs in vivo

    doi: 10.4049/jimmunol.1502399

    Figure Lengend Snippet: p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p

    Article Snippet: QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System (Thermo fisher) using pre-designed TaqMan® Gene Expression Assays (Life Technologies) (18S RNA Mm03928990 and p21 Mm04205640) according to manufacturer's instructions.

    Techniques: Isolation, Cell Culture, Recombinant, FACS, RNA Extraction, Real-time Polymerase Chain Reaction, Expressing