steponeplus cycler  (Thermo Fisher)


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    Structured Review

    Thermo Fisher steponeplus cycler
    Correction of exogenous (spiked) gDNA with ValidPrime. The data are presented in linear scale as fold ratio (2 - Cq /2 - Cq ref ), where Cq ref is the Cq NA measured on non-spiked controls and Cq refers to Cq RNA (light bars) or Cq NA (dark bars) depending on whether or not ValidPrime correction was applied (VP − /VP + ). The data are grouped based on the impact of exogenous DNA, expressed as percentage of the total signal (%DNA) in each sample. Data were collected with either 17 GOI assays on a <t>StepOnePlus</t> (Applied Biosystems) using mVPA1 and mVPA5 ( A ), or with 19 assays on a BioMark (Fluidigm) using mVPA1 ( B ). All assays passed the high confidence ValidPrime criteria ( Supplementary Figure S3 ). Data are presented as the mean ± SD, with ( n ) designating the number of samples in each group. cDNAs were from mouse kidney or liver for the StepOnePlus studies and mouse uterus for the BioMark study.
    Steponeplus Cycler, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/steponeplus cycler/product/Thermo Fisher
    Average 94 stars, based on 31 article reviews
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    steponeplus cycler - by Bioz Stars, 2020-04
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    1) Product Images from "Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime"

    Article Title: Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1259

    Correction of exogenous (spiked) gDNA with ValidPrime. The data are presented in linear scale as fold ratio (2 - Cq /2 - Cq ref ), where Cq ref is the Cq NA measured on non-spiked controls and Cq refers to Cq RNA (light bars) or Cq NA (dark bars) depending on whether or not ValidPrime correction was applied (VP − /VP + ). The data are grouped based on the impact of exogenous DNA, expressed as percentage of the total signal (%DNA) in each sample. Data were collected with either 17 GOI assays on a StepOnePlus (Applied Biosystems) using mVPA1 and mVPA5 ( A ), or with 19 assays on a BioMark (Fluidigm) using mVPA1 ( B ). All assays passed the high confidence ValidPrime criteria ( Supplementary Figure S3 ). Data are presented as the mean ± SD, with ( n ) designating the number of samples in each group. cDNAs were from mouse kidney or liver for the StepOnePlus studies and mouse uterus for the BioMark study.
    Figure Legend Snippet: Correction of exogenous (spiked) gDNA with ValidPrime. The data are presented in linear scale as fold ratio (2 - Cq /2 - Cq ref ), where Cq ref is the Cq NA measured on non-spiked controls and Cq refers to Cq RNA (light bars) or Cq NA (dark bars) depending on whether or not ValidPrime correction was applied (VP − /VP + ). The data are grouped based on the impact of exogenous DNA, expressed as percentage of the total signal (%DNA) in each sample. Data were collected with either 17 GOI assays on a StepOnePlus (Applied Biosystems) using mVPA1 and mVPA5 ( A ), or with 19 assays on a BioMark (Fluidigm) using mVPA1 ( B ). All assays passed the high confidence ValidPrime criteria ( Supplementary Figure S3 ). Data are presented as the mean ± SD, with ( n ) designating the number of samples in each group. cDNAs were from mouse kidney or liver for the StepOnePlus studies and mouse uterus for the BioMark study.

    Techniques Used:

    Equivalence between Cq DNA calculated by ValidPrime and RT(−) measurements. Fold ratios in linear scale (2 - Cq (RT+) /2 - Cq (RT−) ) between either the total signal (NA) measured in spiked RT(+) reactions (dark bars) or the gDNA signal (DNA) estimated by ValidPrime (VP) from RT(+) reactions (light bars) compared to the signal in RT(−) reactions. A quantity of 20 ng of cDNA from adipose tissue (hatched bars) or from kidney, were spiked with 0.30 ng gDNA to decrease the variability due to stochastic amplification observed in RT(−) reactions. Independently of the expression level of the three genes studied in RT(+) samples, the estimations by ValidPrime of the gDNA-derived signals in RT(+) were very similar to the signals measured in RT(−) reactions, as the ratio was close to 1 (illustrated by the red dashed line; mean 1.20 ± 0.29). Data are mean ± SD from two experiments in duplicate on the StepOnePlus.
    Figure Legend Snippet: Equivalence between Cq DNA calculated by ValidPrime and RT(−) measurements. Fold ratios in linear scale (2 - Cq (RT+) /2 - Cq (RT−) ) between either the total signal (NA) measured in spiked RT(+) reactions (dark bars) or the gDNA signal (DNA) estimated by ValidPrime (VP) from RT(+) reactions (light bars) compared to the signal in RT(−) reactions. A quantity of 20 ng of cDNA from adipose tissue (hatched bars) or from kidney, were spiked with 0.30 ng gDNA to decrease the variability due to stochastic amplification observed in RT(−) reactions. Independently of the expression level of the three genes studied in RT(+) samples, the estimations by ValidPrime of the gDNA-derived signals in RT(+) were very similar to the signals measured in RT(−) reactions, as the ratio was close to 1 (illustrated by the red dashed line; mean 1.20 ± 0.29). Data are mean ± SD from two experiments in duplicate on the StepOnePlus.

    Techniques Used: Amplification, Expressing, Derivative Assay

    Related Articles

    Amplification:

    Article Title: Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy
    Article Snippet: A list of lncRNA including primer sequences and amplicon sizes is given in Table . .. Expression analysis was performed by means of SYBR Green detection chemistry using the SensiMix SYBR Hi-ROX Kit (Bioline GmbH) and the StepOnePlus Cycler (Life Technologies).

    Article Title: RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome
    Article Snippet: .. For quantitative real-time RT-PCR, cDNA was synthesized from 1 μg of total RNA with the Reverse-iT First-Strand Synthesis Kit (ABgene), oligo dT, and random hexamers in a 3:1 ratio. cDNA PCR amplification was carried out with the use of SYBR Green JumpStart Taq ReadyMix (Sigma) with ROX reference dye (Sigma) on a StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA), with gene-specific intron-crossing oligonucleotide pairs: 5′-CACAAACGGAGAACCACCAA-3′ and 5′-CTGAAATGCAACTCGGAGGTAAT-3′. .. For quantification, standard curves were obtained with the use of serially diluted cDNA amplified in the same real-time PCR run.

    Article Title: Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime
    Article Snippet: .. Specific target amplification Pre-amplification of cDNA (produced from 25 to 65 ng of total RNA) was performed in the StepOnePlus cycler (Applied Biosystems) [at 95°C for 10 min activation step followed by 14 cycles: 95°C, (15 s), 60°C, (4 min)] in a total volume of 5 µl in the presence of all primers at a concentration of 50 nM. .. After pre-amplification, 20 µl Low EDTA TE Buffer [10 mM Tris pH8 (Ambion), 0.1 mM EDTA pH8 (Sigma)] was added to each sample.

    Article Title: Precursor Cells in Mouse Islets Generate New ?-Cells in Vivo during Aging and after Islet Injury
    Article Snippet: Information about genes is as follows: human (h) PLAP (35 cycles, annealing: 62 C) (GenBank accession no. ), amplicon size 205 bp, sense, GAA ACG GTC CAG GCT ATG TG and antisense, ATG ACG TGC GCT ATG AAG GT; 18s rRNA, GenBank accession no. , amplicon size 68 bp, forward primer, AGT CCC TGC CCT TTG TAC ACA, reverse primer, GAT CCG AGG GCC TCA CTA AAC; cyclophylin A (GenBank accession no. , band 150 bp), 5′-GGT GGA GAG CAC CAAG ACA GA-3′ (forward), 5′-GCC GGA GTC GAC AAT GAT G-3′ (reverse). .. All semiquantitative RT-PCR experiments were confirmed by quantitative real-time PCR using SybrGreen mix (SA Biosciences, Frederick, MD) run on a StepOnePlus cycler (Applied Biosystems, Foster City, CA).

    Synthesized:

    Article Title: Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells
    Article Snippet: .. Quantitative real time PCR (qRT-PCR) Total mRNA was isolated (GenElute Mammalian Total RNA Purification Kit, Sigma-Aldrich) following the manufacturer's instructions. cDNA was synthesized using the SuperScript VILO kit (Life Technologies), and qRT-PCR was carried out using TaqMan gene expression master mix (Life Technologies) according to the manufacturer's instructions on a StepOnePlus cycler (Life Technologies). .. GAPDH was used to normalize the amount of cDNA in each sample and to guarantee the comparability of the calculated mRNA expression in all samples analyzed.

    Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype
    Article Snippet: For mRNA expression in murine livers, snap-frozen specimens were homogenized with trizole, and RNA was extracted using the Qiagen RNeasy plus mini kit (Qiagen, Germany), according to the manufacturer’s instructions. cDNA was synthesized using qScript cDNA SuperMix (Quanta Biosciences), following the manufacturer’s instructions, from 1 μg of RNA in a 40-μl reaction. qRT-PCR was performed using 1 μl of cDNA as a template per reaction well and SYBR Select Master Mix (Life Technologies) using 200 nM of forward and reverse primers in 20 μl. .. Samples were run in triplicate on a StepOnePlus Cycler (Thermo Fisher Scientific).

    Article Title: Blockade of MCAM/CD146 impedes CNS infiltration of T cells over the choroid plexus
    Article Snippet: Real-time quantitative PCR RNA was extracted from confluent monolayers of HCPEpiC-derived fibroblasts or HBMEC using TRIzol reagent (Invitrogen). cDNA was synthesized from 1 μg of total RNA using a standard protocol with random hexamer primers (ThermoScientifc). .. Real-time qPCR was performed in a StepOnePlus cycler (Applied Biosystems, Darmstadt, Germany) employing endogen control primers for 18sRNA as well as a TaqMan Gene Expression Assays specific for human laminin α4, cytokeratin 18, VE-cadherin, vimentin, or PECAM1 (Applied Biosystems, Darmstadt, Germany).

    Article Title: VLA-4 blockade promotes differential routes into human CNS involving PSGL-1 rolling of T cells and MCAM-adhesion of TH17 cells
    Article Snippet: In short, RNA was extracted from primary human brain microvascular endothelial cells using TRIzol reagent (Invitrogen). cDNA was synthesized from 1 µg of total RNA using a standard protocol with random hexamer primers (Thermo Fisher Scientific). .. Real-time quantitative PCR (qPCR) was performed in a StepOnePlus cycler (Applied Biosystems) using endogen control primers for 18sRNA as well as TaqMan Gene Expression Assays specific for human P-selectin (Applied Biosystems).

    Article Title: RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome
    Article Snippet: .. For quantitative real-time RT-PCR, cDNA was synthesized from 1 μg of total RNA with the Reverse-iT First-Strand Synthesis Kit (ABgene), oligo dT, and random hexamers in a 3:1 ratio. cDNA PCR amplification was carried out with the use of SYBR Green JumpStart Taq ReadyMix (Sigma) with ROX reference dye (Sigma) on a StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA), with gene-specific intron-crossing oligonucleotide pairs: 5′-CACAAACGGAGAACCACCAA-3′ and 5′-CTGAAATGCAACTCGGAGGTAAT-3′. .. For quantification, standard curves were obtained with the use of serially diluted cDNA amplified in the same real-time PCR run.

    Cytometry:

    Article Title: Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s
    Article Snippet: Quantitative RT-PCR ILCs and CD4+ cells were sorted from the lungs and eosinophils from the blood of naive mice, as described in the Flow cytometry and cell sorting section. .. The resulting cDNA was used as a template for quantitative PCR with the Power SYBR green reagent on a StepOnePlus cycler (Applied Biosystems).

    Quantitative RT-PCR:

    Article Title: Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy
    Article Snippet: Paragraph title: Selection of lncRNA, oligonucleotides and RT-qPCR ... Expression analysis was performed by means of SYBR Green detection chemistry using the SensiMix SYBR Hi-ROX Kit (Bioline GmbH) and the StepOnePlus Cycler (Life Technologies).

    Article Title: Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells
    Article Snippet: .. Quantitative real time PCR (qRT-PCR) Total mRNA was isolated (GenElute Mammalian Total RNA Purification Kit, Sigma-Aldrich) following the manufacturer's instructions. cDNA was synthesized using the SuperScript VILO kit (Life Technologies), and qRT-PCR was carried out using TaqMan gene expression master mix (Life Technologies) according to the manufacturer's instructions on a StepOnePlus cycler (Life Technologies). .. GAPDH was used to normalize the amount of cDNA in each sample and to guarantee the comparability of the calculated mRNA expression in all samples analyzed.

    Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype
    Article Snippet: Paragraph title: Total RNA preparation and quantitative reverse transcription polymerase chain reaction (qRT-PCR) ... Samples were run in triplicate on a StepOnePlus Cycler (Thermo Fisher Scientific).

    Article Title: A metabolite-triggered tuft cell-ILC2 circuit drives small intestinal remodeling
    Article Snippet: Paragraph title: Quantitative RT–PCR ... The resulting cDNA was used as template for quantitative PCR (qPCR) with the Power SYBR Green reagent on a StepOnePlus cycler (Applied Biosystems).

    Article Title: Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s
    Article Snippet: Paragraph title: Quantitative RT-PCR ... The resulting cDNA was used as a template for quantitative PCR with the Power SYBR green reagent on a StepOnePlus cycler (Applied Biosystems).

    Article Title: RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome
    Article Snippet: .. For quantitative real-time RT-PCR, cDNA was synthesized from 1 μg of total RNA with the Reverse-iT First-Strand Synthesis Kit (ABgene), oligo dT, and random hexamers in a 3:1 ratio. cDNA PCR amplification was carried out with the use of SYBR Green JumpStart Taq ReadyMix (Sigma) with ROX reference dye (Sigma) on a StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA), with gene-specific intron-crossing oligonucleotide pairs: 5′-CACAAACGGAGAACCACCAA-3′ and 5′-CTGAAATGCAACTCGGAGGTAAT-3′. .. For quantification, standard curves were obtained with the use of serially diluted cDNA amplified in the same real-time PCR run.

    Article Title: Cancer-Predicting Gene Expression Changes in Colonic Mucosa of Western Diet Fed Mlh1+/- Mice
    Article Snippet: In contrast to StellARray RT-qPCR, the TaqMan RT-qPCR analysis was done from pooled samples. .. Thermal cycling and fluorescence data acquisition were performed with a StepOnePlus cycler (Life Technologies, Carlsbad, CA) and Cq values were called using the Data-assist v2.0 software (Life Technologies, Carlsbad, CA).

    Real-time Polymerase Chain Reaction:

    Article Title: Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells
    Article Snippet: .. Quantitative real time PCR (qRT-PCR) Total mRNA was isolated (GenElute Mammalian Total RNA Purification Kit, Sigma-Aldrich) following the manufacturer's instructions. cDNA was synthesized using the SuperScript VILO kit (Life Technologies), and qRT-PCR was carried out using TaqMan gene expression master mix (Life Technologies) according to the manufacturer's instructions on a StepOnePlus cycler (Life Technologies). .. GAPDH was used to normalize the amount of cDNA in each sample and to guarantee the comparability of the calculated mRNA expression in all samples analyzed.

    Article Title: Blockade of MCAM/CD146 impedes CNS infiltration of T cells over the choroid plexus
    Article Snippet: .. Real-time qPCR was performed in a StepOnePlus cycler (Applied Biosystems, Darmstadt, Germany) employing endogen control primers for 18sRNA as well as a TaqMan Gene Expression Assays specific for human laminin α4, cytokeratin 18, VE-cadherin, vimentin, or PECAM1 (Applied Biosystems, Darmstadt, Germany). .. Immunofluorescence staining For immunofluorescence studies, 4-μm-thick formalin-fixed paraffin embedded (FFPE) murine brain sections were stained for MCAM (CD146, rabbit monoclonal IgG, clone: EPR3208; Abcam, Cambridge, UK).

    Article Title: A metabolite-triggered tuft cell-ILC2 circuit drives small intestinal remodeling
    Article Snippet: .. The resulting cDNA was used as template for quantitative PCR (qPCR) with the Power SYBR Green reagent on a StepOnePlus cycler (Applied Biosystems). .. Transcripts were normalized to Rps17 (40S ribosomal protein S17) expression and relative expression shown as 2−ΔCt .

    Article Title: VLA-4 blockade promotes differential routes into human CNS involving PSGL-1 rolling of T cells and MCAM-adhesion of TH17 cells
    Article Snippet: .. Real-time quantitative PCR (qPCR) was performed in a StepOnePlus cycler (Applied Biosystems) using endogen control primers for 18sRNA as well as TaqMan Gene Expression Assays specific for human P-selectin (Applied Biosystems). .. Parallel plate flow chamber.

    Article Title: Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)
    Article Snippet: .. Real-time PCR was operated on StepOnePlus cycler (Applied Biosystems, Life technology) using 2X TaqMan® Universal PCR Master Mix. ..

    Article Title: Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s
    Article Snippet: .. The resulting cDNA was used as a template for quantitative PCR with the Power SYBR green reagent on a StepOnePlus cycler (Applied Biosystems). .. Transcripts were normalized to Rps17 (40S ribosomal protein S17) expression.

    Article Title: RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome
    Article Snippet: For quantitative real-time RT-PCR, cDNA was synthesized from 1 μg of total RNA with the Reverse-iT First-Strand Synthesis Kit (ABgene), oligo dT, and random hexamers in a 3:1 ratio. cDNA PCR amplification was carried out with the use of SYBR Green JumpStart Taq ReadyMix (Sigma) with ROX reference dye (Sigma) on a StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA), with gene-specific intron-crossing oligonucleotide pairs: 5′-CACAAACGGAGAACCACCAA-3′ and 5′-CTGAAATGCAACTCGGAGGTAAT-3′. .. For quantification, standard curves were obtained with the use of serially diluted cDNA amplified in the same real-time PCR run.

    Article Title: Precursor Cells in Mouse Islets Generate New ?-Cells in Vivo during Aging and after Islet Injury
    Article Snippet: .. All semiquantitative RT-PCR experiments were confirmed by quantitative real-time PCR using SybrGreen mix (SA Biosciences, Frederick, MD) run on a StepOnePlus cycler (Applied Biosystems, Foster City, CA). .. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick-end labeling (TUNEL) staining was carried out as indicated in the manufacturer’s instructions (Chemicon, Temecula, CA).

    Random Hexamer Labeling:

    Article Title: Blockade of MCAM/CD146 impedes CNS infiltration of T cells over the choroid plexus
    Article Snippet: Real-time quantitative PCR RNA was extracted from confluent monolayers of HCPEpiC-derived fibroblasts or HBMEC using TRIzol reagent (Invitrogen). cDNA was synthesized from 1 μg of total RNA using a standard protocol with random hexamer primers (ThermoScientifc). .. Real-time qPCR was performed in a StepOnePlus cycler (Applied Biosystems, Darmstadt, Germany) employing endogen control primers for 18sRNA as well as a TaqMan Gene Expression Assays specific for human laminin α4, cytokeratin 18, VE-cadherin, vimentin, or PECAM1 (Applied Biosystems, Darmstadt, Germany).

    Article Title: VLA-4 blockade promotes differential routes into human CNS involving PSGL-1 rolling of T cells and MCAM-adhesion of TH17 cells
    Article Snippet: In short, RNA was extracted from primary human brain microvascular endothelial cells using TRIzol reagent (Invitrogen). cDNA was synthesized from 1 µg of total RNA using a standard protocol with random hexamer primers (Thermo Fisher Scientific). .. Real-time quantitative PCR (qPCR) was performed in a StepOnePlus cycler (Applied Biosystems) using endogen control primers for 18sRNA as well as TaqMan Gene Expression Assays specific for human P-selectin (Applied Biosystems).

    Expressing:

    Article Title: Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy
    Article Snippet: .. Expression analysis was performed by means of SYBR Green detection chemistry using the SensiMix SYBR Hi-ROX Kit (Bioline GmbH) and the StepOnePlus Cycler (Life Technologies). .. All reactions were carried out using clear MicroAmp® Fast 96-Well Reaction Plates (Life Technologies) that were sealed with adhesive films.

    Article Title: Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells
    Article Snippet: .. Quantitative real time PCR (qRT-PCR) Total mRNA was isolated (GenElute Mammalian Total RNA Purification Kit, Sigma-Aldrich) following the manufacturer's instructions. cDNA was synthesized using the SuperScript VILO kit (Life Technologies), and qRT-PCR was carried out using TaqMan gene expression master mix (Life Technologies) according to the manufacturer's instructions on a StepOnePlus cycler (Life Technologies). .. GAPDH was used to normalize the amount of cDNA in each sample and to guarantee the comparability of the calculated mRNA expression in all samples analyzed.

    Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype
    Article Snippet: For mRNA expression in murine livers, snap-frozen specimens were homogenized with trizole, and RNA was extracted using the Qiagen RNeasy plus mini kit (Qiagen, Germany), according to the manufacturer’s instructions. cDNA was synthesized using qScript cDNA SuperMix (Quanta Biosciences), following the manufacturer’s instructions, from 1 μg of RNA in a 40-μl reaction. qRT-PCR was performed using 1 μl of cDNA as a template per reaction well and SYBR Select Master Mix (Life Technologies) using 200 nM of forward and reverse primers in 20 μl. .. Samples were run in triplicate on a StepOnePlus Cycler (Thermo Fisher Scientific).

    Article Title: Blockade of MCAM/CD146 impedes CNS infiltration of T cells over the choroid plexus
    Article Snippet: .. Real-time qPCR was performed in a StepOnePlus cycler (Applied Biosystems, Darmstadt, Germany) employing endogen control primers for 18sRNA as well as a TaqMan Gene Expression Assays specific for human laminin α4, cytokeratin 18, VE-cadherin, vimentin, or PECAM1 (Applied Biosystems, Darmstadt, Germany). .. Immunofluorescence staining For immunofluorescence studies, 4-μm-thick formalin-fixed paraffin embedded (FFPE) murine brain sections were stained for MCAM (CD146, rabbit monoclonal IgG, clone: EPR3208; Abcam, Cambridge, UK).

    Article Title: A metabolite-triggered tuft cell-ILC2 circuit drives small intestinal remodeling
    Article Snippet: The resulting cDNA was used as template for quantitative PCR (qPCR) with the Power SYBR Green reagent on a StepOnePlus cycler (Applied Biosystems). .. Transcripts were normalized to Rps17 (40S ribosomal protein S17) expression and relative expression shown as 2−ΔCt .

    Article Title: VLA-4 blockade promotes differential routes into human CNS involving PSGL-1 rolling of T cells and MCAM-adhesion of TH17 cells
    Article Snippet: .. Real-time quantitative PCR (qPCR) was performed in a StepOnePlus cycler (Applied Biosystems) using endogen control primers for 18sRNA as well as TaqMan Gene Expression Assays specific for human P-selectin (Applied Biosystems). .. Parallel plate flow chamber.

    Article Title: Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s
    Article Snippet: The resulting cDNA was used as a template for quantitative PCR with the Power SYBR green reagent on a StepOnePlus cycler (Applied Biosystems). .. Transcripts were normalized to Rps17 (40S ribosomal protein S17) expression.

    Article Title: Cancer-Predicting Gene Expression Changes in Colonic Mucosa of Western Diet Fed Mlh1+/- Mice
    Article Snippet: The expression differences between different mouse groups were analyzed using the Global Pattern Recognition™ (GPR) software (Bar Harbor Biotechnology, Trenton, ME) The results of statistically significant expression changes in relation to WD* and/or inherited cancer predisposition were validated at tp1 using TaqMan assays ( ). .. Thermal cycling and fluorescence data acquisition were performed with a StepOnePlus cycler (Life Technologies, Carlsbad, CA) and Cq values were called using the Data-assist v2.0 software (Life Technologies, Carlsbad, CA).

    Derivative Assay:

    Article Title: RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome
    Article Snippet: For quantitative real-time RT-PCR, cDNA was synthesized from 1 μg of total RNA with the Reverse-iT First-Strand Synthesis Kit (ABgene), oligo dT, and random hexamers in a 3:1 ratio. cDNA PCR amplification was carried out with the use of SYBR Green JumpStart Taq ReadyMix (Sigma) with ROX reference dye (Sigma) on a StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA), with gene-specific intron-crossing oligonucleotide pairs: 5′-CACAAACGGAGAACCACCAA-3′ and 5′-CTGAAATGCAACTCGGAGGTAAT-3′. .. We found that the identified mutation resulted in markedly decreased RIN2 mRNA levels in RNA derived from patient skin biopsies as compared with those from normal skin biopsies ( A), suggesting that the mutation induces nonsense-mRNA decay.

    Countercurrent Chromatography:

    Article Title: Precursor Cells in Mouse Islets Generate New ?-Cells in Vivo during Aging and after Islet Injury
    Article Snippet: Information about genes is as follows: human (h) PLAP (35 cycles, annealing: 62 C) (GenBank accession no. ), amplicon size 205 bp, sense, GAA ACG GTC CAG GCT ATG TG and antisense, ATG ACG TGC GCT ATG AAG GT; 18s rRNA, GenBank accession no. , amplicon size 68 bp, forward primer, AGT CCC TGC CCT TTG TAC ACA, reverse primer, GAT CCG AGG GCC TCA CTA AAC; cyclophylin A (GenBank accession no. , band 150 bp), 5′-GGT GGA GAG CAC CAAG ACA GA-3′ (forward), 5′-GCC GGA GTC GAC AAT GAT G-3′ (reverse). .. All semiquantitative RT-PCR experiments were confirmed by quantitative real-time PCR using SybrGreen mix (SA Biosciences, Frederick, MD) run on a StepOnePlus cycler (Applied Biosystems, Foster City, CA).

    Flow Cytometry:

    Article Title: Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s
    Article Snippet: Quantitative RT-PCR ILCs and CD4+ cells were sorted from the lungs and eosinophils from the blood of naive mice, as described in the Flow cytometry and cell sorting section. .. The resulting cDNA was used as a template for quantitative PCR with the Power SYBR green reagent on a StepOnePlus cycler (Applied Biosystems).

    Sequencing:

    Article Title: RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome
    Article Snippet: Direct sequencing of PCR products encompassing the entire coding sequence of the gene revealed a homozygous C deletion at cDNA position 1731 (c.1731delC [p.Ile578SerfsX4]) ( C). .. For quantitative real-time RT-PCR, cDNA was synthesized from 1 μg of total RNA with the Reverse-iT First-Strand Synthesis Kit (ABgene), oligo dT, and random hexamers in a 3:1 ratio. cDNA PCR amplification was carried out with the use of SYBR Green JumpStart Taq ReadyMix (Sigma) with ROX reference dye (Sigma) on a StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA), with gene-specific intron-crossing oligonucleotide pairs: 5′-CACAAACGGAGAACCACCAA-3′ and 5′-CTGAAATGCAACTCGGAGGTAAT-3′.

    Concentration Assay:

    Article Title: Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)
    Article Snippet: DNA concentration in the extracts was quantified by spectrophotometry with A260/280 before adjustment to the concentration of 50 ng/μL in all assay. .. Real-time PCR was operated on StepOnePlus cycler (Applied Biosystems, Life technology) using 2X TaqMan® Universal PCR Master Mix.

    Article Title: Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime
    Article Snippet: .. Specific target amplification Pre-amplification of cDNA (produced from 25 to 65 ng of total RNA) was performed in the StepOnePlus cycler (Applied Biosystems) [at 95°C for 10 min activation step followed by 14 cycles: 95°C, (15 s), 60°C, (4 min)] in a total volume of 5 µl in the presence of all primers at a concentration of 50 nM. .. After pre-amplification, 20 µl Low EDTA TE Buffer [10 mM Tris pH8 (Ambion), 0.1 mM EDTA pH8 (Sigma)] was added to each sample.

    Serial Dilution:

    Article Title: Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)
    Article Snippet: The copy number of both IHHNV and WSSV were determined by using an external standard curve and plasmid DNA containing WSSV and IHHNV fragments as a standard (10-fold serial dilution, 107 -101 copies). .. Real-time PCR was operated on StepOnePlus cycler (Applied Biosystems, Life technology) using 2X TaqMan® Universal PCR Master Mix.

    Generated:

    Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype
    Article Snippet: Samples were run in triplicate on a StepOnePlus Cycler (Thermo Fisher Scientific). .. For the nfkb−/− aging experiment, RNA was extracted from solubilized lung tissue using the RNeasy Mini Kit (74106, Qiagen), according to the manufacturer’s instructions. cDNA was generated using the Omniscript RT Kit (205110, Qiagen) as per the user manual. qRT-PCR was performed using 4 μl of cDNA as a template per reaction well using a Power SYBR Green (4367659, Invitrogen) PCR Master Mix and 100 nM of forward and reverse primers to form a final reaction volume of 10 μl.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype
    Article Snippet: Paragraph title: Total RNA preparation and quantitative reverse transcription polymerase chain reaction (qRT-PCR) ... Samples were run in triplicate on a StepOnePlus Cycler (Thermo Fisher Scientific).

    Article Title: Precursor Cells in Mouse Islets Generate New ?-Cells in Vivo during Aging and after Islet Injury
    Article Snippet: .. All semiquantitative RT-PCR experiments were confirmed by quantitative real-time PCR using SybrGreen mix (SA Biosciences, Frederick, MD) run on a StepOnePlus cycler (Applied Biosystems, Foster City, CA). .. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick-end labeling (TUNEL) staining was carried out as indicated in the manufacturer’s instructions (Chemicon, Temecula, CA).

    Software:

    Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype
    Article Snippet: Samples were run in triplicate on a StepOnePlus Cycler (Thermo Fisher Scientific). .. Samples were run in triplicate in a C1000TM Thermal Cycler, CFX96TM Real-Time System (Bio-Rad), and Bio-Rad CXF manager software. mRNA expression analysis was carried out using the change in Ct method and normalized to levels of the housekeeping gene actin or ribosomal protein 18S (nfkb−/− experiment only) to obtain relative mRNA expression.

    Article Title: Cancer-Predicting Gene Expression Changes in Colonic Mucosa of Western Diet Fed Mlh1+/- Mice
    Article Snippet: .. Thermal cycling and fluorescence data acquisition were performed with a StepOnePlus cycler (Life Technologies, Carlsbad, CA) and Cq values were called using the Data-assist v2.0 software (Life Technologies, Carlsbad, CA). ..

    DNA Extraction:

    Article Title: Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)
    Article Snippet: Determination of IHHNV and WSSV copy number in challenged shrimp All collected samples were subjected to genomic DNA extraction using the phenol-chloroform procedure as described previously [ ]. .. Real-time PCR was operated on StepOnePlus cycler (Applied Biosystems, Life technology) using 2X TaqMan® Universal PCR Master Mix.

    Fluorescence:

    Article Title: Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy
    Article Snippet: Expression analysis was performed by means of SYBR Green detection chemistry using the SensiMix SYBR Hi-ROX Kit (Bioline GmbH) and the StepOnePlus Cycler (Life Technologies). .. The fluorescence signal was acquired at 60 °C.

    Article Title: Cancer-Predicting Gene Expression Changes in Colonic Mucosa of Western Diet Fed Mlh1+/- Mice
    Article Snippet: .. Thermal cycling and fluorescence data acquisition were performed with a StepOnePlus cycler (Life Technologies, Carlsbad, CA) and Cq values were called using the Data-assist v2.0 software (Life Technologies, Carlsbad, CA). ..

    Mutagenesis:

    Article Title: RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome
    Article Snippet: We assessed the consequences of the c.1731delC mutation. .. For quantitative real-time RT-PCR, cDNA was synthesized from 1 μg of total RNA with the Reverse-iT First-Strand Synthesis Kit (ABgene), oligo dT, and random hexamers in a 3:1 ratio. cDNA PCR amplification was carried out with the use of SYBR Green JumpStart Taq ReadyMix (Sigma) with ROX reference dye (Sigma) on a StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA), with gene-specific intron-crossing oligonucleotide pairs: 5′-CACAAACGGAGAACCACCAA-3′ and 5′-CTGAAATGCAACTCGGAGGTAAT-3′.

    Isolation:

    Article Title: Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells
    Article Snippet: .. Quantitative real time PCR (qRT-PCR) Total mRNA was isolated (GenElute Mammalian Total RNA Purification Kit, Sigma-Aldrich) following the manufacturer's instructions. cDNA was synthesized using the SuperScript VILO kit (Life Technologies), and qRT-PCR was carried out using TaqMan gene expression master mix (Life Technologies) according to the manufacturer's instructions on a StepOnePlus cycler (Life Technologies). .. GAPDH was used to normalize the amount of cDNA in each sample and to guarantee the comparability of the calculated mRNA expression in all samples analyzed.

    Article Title: A metabolite-triggered tuft cell-ILC2 circuit drives small intestinal remodeling
    Article Snippet: RNA was isolated using the Micro Plus RNeasy kit (Qiagen) and reverse transcribed using the SuperScript Vilo Master Mix (Life Technologies). .. The resulting cDNA was used as template for quantitative PCR (qPCR) with the Power SYBR Green reagent on a StepOnePlus cycler (Applied Biosystems).

    Article Title: Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s
    Article Snippet: RNA was isolated using the Micro Plus RNeasy kit (QIAGEN) and subjected to reverse transcription using SuperScript Vilo Master Mix (Thermo Fisher Scientific). .. The resulting cDNA was used as a template for quantitative PCR with the Power SYBR green reagent on a StepOnePlus cycler (Applied Biosystems).

    Article Title: Precursor Cells in Mouse Islets Generate New ?-Cells in Vivo during Aging and after Islet Injury
    Article Snippet: Paragraph title: RNA isolation and semiquantitative RT-PCR ... All semiquantitative RT-PCR experiments were confirmed by quantitative real-time PCR using SybrGreen mix (SA Biosciences, Frederick, MD) run on a StepOnePlus cycler (Applied Biosystems, Foster City, CA).

    Purification:

    Article Title: Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells
    Article Snippet: .. Quantitative real time PCR (qRT-PCR) Total mRNA was isolated (GenElute Mammalian Total RNA Purification Kit, Sigma-Aldrich) following the manufacturer's instructions. cDNA was synthesized using the SuperScript VILO kit (Life Technologies), and qRT-PCR was carried out using TaqMan gene expression master mix (Life Technologies) according to the manufacturer's instructions on a StepOnePlus cycler (Life Technologies). .. GAPDH was used to normalize the amount of cDNA in each sample and to guarantee the comparability of the calculated mRNA expression in all samples analyzed.

    Polymerase Chain Reaction:

    Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype
    Article Snippet: Samples were run in triplicate on a StepOnePlus Cycler (Thermo Fisher Scientific). .. For the nfkb−/− aging experiment, RNA was extracted from solubilized lung tissue using the RNeasy Mini Kit (74106, Qiagen), according to the manufacturer’s instructions. cDNA was generated using the Omniscript RT Kit (205110, Qiagen) as per the user manual. qRT-PCR was performed using 4 μl of cDNA as a template per reaction well using a Power SYBR Green (4367659, Invitrogen) PCR Master Mix and 100 nM of forward and reverse primers to form a final reaction volume of 10 μl.

    Article Title: Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)
    Article Snippet: .. Real-time PCR was operated on StepOnePlus cycler (Applied Biosystems, Life technology) using 2X TaqMan® Universal PCR Master Mix. ..

    Article Title: RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome
    Article Snippet: .. For quantitative real-time RT-PCR, cDNA was synthesized from 1 μg of total RNA with the Reverse-iT First-Strand Synthesis Kit (ABgene), oligo dT, and random hexamers in a 3:1 ratio. cDNA PCR amplification was carried out with the use of SYBR Green JumpStart Taq ReadyMix (Sigma) with ROX reference dye (Sigma) on a StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA), with gene-specific intron-crossing oligonucleotide pairs: 5′-CACAAACGGAGAACCACCAA-3′ and 5′-CTGAAATGCAACTCGGAGGTAAT-3′. .. For quantification, standard curves were obtained with the use of serially diluted cDNA amplified in the same real-time PCR run.

    Article Title: Precursor Cells in Mouse Islets Generate New ?-Cells in Vivo during Aging and after Islet Injury
    Article Snippet: The number of cycles was optimized, depending on the particular mRNA abundance and chosen to select PCR amplification on the linear portion of the curve to avoid saturation effect. .. All semiquantitative RT-PCR experiments were confirmed by quantitative real-time PCR using SybrGreen mix (SA Biosciences, Frederick, MD) run on a StepOnePlus cycler (Applied Biosystems, Foster City, CA).

    Selection:

    Article Title: Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy
    Article Snippet: Paragraph title: Selection of lncRNA, oligonucleotides and RT-qPCR ... Expression analysis was performed by means of SYBR Green detection chemistry using the SensiMix SYBR Hi-ROX Kit (Bioline GmbH) and the StepOnePlus Cycler (Life Technologies).

    Staining:

    Article Title: A metabolite-triggered tuft cell-ILC2 circuit drives small intestinal remodeling
    Article Snippet: Single-cell epithelial suspensions were isolated, stained as described earlier and sorted using a MoFlo XDP (Beckman Coulter). .. The resulting cDNA was used as template for quantitative PCR (qPCR) with the Power SYBR Green reagent on a StepOnePlus cycler (Applied Biosystems).

    Mouse Assay:

    Article Title: Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s
    Article Snippet: Quantitative RT-PCR ILCs and CD4+ cells were sorted from the lungs and eosinophils from the blood of naive mice, as described in the Flow cytometry and cell sorting section. .. The resulting cDNA was used as a template for quantitative PCR with the Power SYBR green reagent on a StepOnePlus cycler (Applied Biosystems).

    Plasmid Preparation:

    Article Title: Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)
    Article Snippet: The copy number of both IHHNV and WSSV were determined by using an external standard curve and plasmid DNA containing WSSV and IHHNV fragments as a standard (10-fold serial dilution, 107 -101 copies). .. Real-time PCR was operated on StepOnePlus cycler (Applied Biosystems, Life technology) using 2X TaqMan® Universal PCR Master Mix.

    TaqMan Assay:

    Article Title: Cancer-Predicting Gene Expression Changes in Colonic Mucosa of Western Diet Fed Mlh1+/- Mice
    Article Snippet: Thermal cycling and fluorescence data acquisition were performed with a StepOnePlus cycler (Life Technologies, Carlsbad, CA) and Cq values were called using the Data-assist v2.0 software (Life Technologies, Carlsbad, CA). .. Mlh1 expression levels at tp0 were also quantitated using TaqMan assay.

    SYBR Green Assay:

    Article Title: Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy
    Article Snippet: .. Expression analysis was performed by means of SYBR Green detection chemistry using the SensiMix SYBR Hi-ROX Kit (Bioline GmbH) and the StepOnePlus Cycler (Life Technologies). .. All reactions were carried out using clear MicroAmp® Fast 96-Well Reaction Plates (Life Technologies) that were sealed with adhesive films.

    Article Title: The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype
    Article Snippet: Samples were run in triplicate on a StepOnePlus Cycler (Thermo Fisher Scientific). .. For the nfkb−/− aging experiment, RNA was extracted from solubilized lung tissue using the RNeasy Mini Kit (74106, Qiagen), according to the manufacturer’s instructions. cDNA was generated using the Omniscript RT Kit (205110, Qiagen) as per the user manual. qRT-PCR was performed using 4 μl of cDNA as a template per reaction well using a Power SYBR Green (4367659, Invitrogen) PCR Master Mix and 100 nM of forward and reverse primers to form a final reaction volume of 10 μl.

    Article Title: A metabolite-triggered tuft cell-ILC2 circuit drives small intestinal remodeling
    Article Snippet: .. The resulting cDNA was used as template for quantitative PCR (qPCR) with the Power SYBR Green reagent on a StepOnePlus cycler (Applied Biosystems). .. Transcripts were normalized to Rps17 (40S ribosomal protein S17) expression and relative expression shown as 2−ΔCt .

    Article Title: Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s
    Article Snippet: .. The resulting cDNA was used as a template for quantitative PCR with the Power SYBR green reagent on a StepOnePlus cycler (Applied Biosystems). .. Transcripts were normalized to Rps17 (40S ribosomal protein S17) expression.

    Article Title: RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome
    Article Snippet: .. For quantitative real-time RT-PCR, cDNA was synthesized from 1 μg of total RNA with the Reverse-iT First-Strand Synthesis Kit (ABgene), oligo dT, and random hexamers in a 3:1 ratio. cDNA PCR amplification was carried out with the use of SYBR Green JumpStart Taq ReadyMix (Sigma) with ROX reference dye (Sigma) on a StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA), with gene-specific intron-crossing oligonucleotide pairs: 5′-CACAAACGGAGAACCACCAA-3′ and 5′-CTGAAATGCAACTCGGAGGTAAT-3′. .. For quantification, standard curves were obtained with the use of serially diluted cDNA amplified in the same real-time PCR run.

    RNA Expression:

    Article Title: Cancer-Predicting Gene Expression Changes in Colonic Mucosa of Western Diet Fed Mlh1+/- Mice
    Article Snippet: Paragraph title: RNA expression studies ... Thermal cycling and fluorescence data acquisition were performed with a StepOnePlus cycler (Life Technologies, Carlsbad, CA) and Cq values were called using the Data-assist v2.0 software (Life Technologies, Carlsbad, CA).

    Spectrophotometry:

    Article Title: Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)
    Article Snippet: DNA concentration in the extracts was quantified by spectrophotometry with A260/280 before adjustment to the concentration of 50 ng/μL in all assay. .. Real-time PCR was operated on StepOnePlus cycler (Applied Biosystems, Life technology) using 2X TaqMan® Universal PCR Master Mix.

    Produced:

    Article Title: Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime
    Article Snippet: .. Specific target amplification Pre-amplification of cDNA (produced from 25 to 65 ng of total RNA) was performed in the StepOnePlus cycler (Applied Biosystems) [at 95°C for 10 min activation step followed by 14 cycles: 95°C, (15 s), 60°C, (4 min)] in a total volume of 5 µl in the presence of all primers at a concentration of 50 nM. .. After pre-amplification, 20 µl Low EDTA TE Buffer [10 mM Tris pH8 (Ambion), 0.1 mM EDTA pH8 (Sigma)] was added to each sample.

    Activation Assay:

    Article Title: Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime
    Article Snippet: .. Specific target amplification Pre-amplification of cDNA (produced from 25 to 65 ng of total RNA) was performed in the StepOnePlus cycler (Applied Biosystems) [at 95°C for 10 min activation step followed by 14 cycles: 95°C, (15 s), 60°C, (4 min)] in a total volume of 5 µl in the presence of all primers at a concentration of 50 nM. .. After pre-amplification, 20 µl Low EDTA TE Buffer [10 mM Tris pH8 (Ambion), 0.1 mM EDTA pH8 (Sigma)] was added to each sample.

    FACS:

    Article Title: Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s
    Article Snippet: Quantitative RT-PCR ILCs and CD4+ cells were sorted from the lungs and eosinophils from the blood of naive mice, as described in the Flow cytometry and cell sorting section. .. The resulting cDNA was used as a template for quantitative PCR with the Power SYBR green reagent on a StepOnePlus cycler (Applied Biosystems).

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