sybr green reagent  (Thermo Fisher)


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    Structured Review

    Thermo Fisher sybr green reagent
    Real-time <t>RT-PCR</t> . Amplification of TNF- α , MIP-2, IL-6 and GAPDH cDNA in livers from mice that were pretreated with vehicle (i.e. normal saline), DMPP (1 mg/kg), or nicotine (1 mg/kg) prior to the onset of ischemia (90 min), followed by 3 h of reperfusion (IR). Results are expressed as fluorescence intensity of <t>SYBR</t> Green bound to DNA (Y-axis). The X-axis represents the number of cycles (i.e. 40 cycles). GAPDH was used as a housekeeping gene. NTC represents PCR products amplified in the absence of cDNA template. Graphs shown are representative of three separate experiments with similar results.
    Sybr Green Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybr green reagent/product/Thermo Fisher
    Average 99 stars, based on 316 article reviews
    Price from $9.99 to $1999.99
    sybr green reagent - by Bioz Stars, 2020-02
    99/100 stars

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    1) Product Images from "Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists"

    Article Title: Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists

    Journal: BMC Clinical Pathology

    doi: 10.1186/1472-6890-6-3

    Real-time RT-PCR . Amplification of TNF- α , MIP-2, IL-6 and GAPDH cDNA in livers from mice that were pretreated with vehicle (i.e. normal saline), DMPP (1 mg/kg), or nicotine (1 mg/kg) prior to the onset of ischemia (90 min), followed by 3 h of reperfusion (IR). Results are expressed as fluorescence intensity of SYBR Green bound to DNA (Y-axis). The X-axis represents the number of cycles (i.e. 40 cycles). GAPDH was used as a housekeeping gene. NTC represents PCR products amplified in the absence of cDNA template. Graphs shown are representative of three separate experiments with similar results.
    Figure Legend Snippet: Real-time RT-PCR . Amplification of TNF- α , MIP-2, IL-6 and GAPDH cDNA in livers from mice that were pretreated with vehicle (i.e. normal saline), DMPP (1 mg/kg), or nicotine (1 mg/kg) prior to the onset of ischemia (90 min), followed by 3 h of reperfusion (IR). Results are expressed as fluorescence intensity of SYBR Green bound to DNA (Y-axis). The X-axis represents the number of cycles (i.e. 40 cycles). GAPDH was used as a housekeeping gene. NTC represents PCR products amplified in the absence of cDNA template. Graphs shown are representative of three separate experiments with similar results.

    Techniques Used: Quantitative RT-PCR, Amplification, Mouse Assay, Fluorescence, SYBR Green Assay, Polymerase Chain Reaction

    Related Articles

    Amplification:

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    Article Snippet: The cDNA was then amplified by PCR using primers specific for full-length IRF-5 (5′-TTAGATAAGCTTGCTATGAACCACTCAGCCCCAG containing a HindIII site and 3′-TAAATATCTAGATGCTTGCATGCCAACTGGGTG containing an XbaI site), exon 2–3 (5′-CTTCAGTGGGTCAACGGG and 3′-TGTACGGCTGAGGTGGCAT), exon 7–8 (5′-ATCCGTCTGTGCCAGTGTAAC and 3′-GCTTCTTCTCTCGGGGTTTG), and β-actin (5′-CATGTTTGAGACCTTCAACACC and 3′-GCACAGCTTCTCTTTGATGTCAC). .. Quantitative PCR analysis for IFNB gene expression was performed using SYBR green reagent (Invitrogen) on a DNA engine Opticon 2 cycler (Bio-Rad) using the following primers: IFNB (5′-CAGCAATTTTCAGTGTCAGAAGC and 3′-CATCCTGTCCTTGAGGCAGT) and β-actin (5′-CCTGGCACCCAGCACAAT and 3′-GCCGATCCACACGGAGTA).

    Article Title: High glucose induces upregulation of scavenger receptors and promotes maturation of dendritic cells
    Article Snippet: LOX-1 was amplified using the sense primer 5′-GGGCTCATTTAACTGGGAAA-3′ and antisense primer 5′-GAAATTGCTTGCTGGATGAA-3′. .. Quantitative PCR using SYBR Green reagent was performed on the ABI 7500 Real-time PCR System (Applied Biosystems, United States).

    Article Title: Fatty Acid Synthase Modulates Homeostatic Responses to Myocardial Stress *
    Article Snippet: Quantitative PCR was performed with the ABI Prism7700 instrument using SYBR® Green reagent (Applied Biosystems). .. Assays were performed in duplicate and normalized to amplified mRNA for ribosomal protein L32.

    Article Title: Comparative evaluation of NOTCH signaling molecules in the endometrium of women with various gynecological diseases during the window of implantation
    Article Snippet: The identity of the amplified product was confirmed by the sequencing of the RT-PCR products. β-actin was considered as a reference gene ( ). .. The total reaction volume was 20 μl containing 250 ng cDNA, 5 pmol of each primer, and SYBR Green reagent (Applied Biosystems) with ROX dye as a passive control for signal intensity.

    Synthesized:

    Article Title: Bronchial epithelial injury in the context of alloimmunity promotes lymphocytic bronchiolitis through hyaluronan expression
    Article Snippet: RNA samples were treated with RNase-free DNase I (Ambion, Austin, TX) to clear DNA contamination. cDNA was synthesized using Invitrogen SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) or the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) following the manufacturer's instructions. .. Quantitative polymerase chain reaction was performed using ABI SDS 7500 and SYBR Green Reagent (Applied Biosystems).

    Article Title: Cleavage of MCM2 licensing protein fosters senescence in human keratinocytes
    Article Snippet: DNaseI treatment was done on column and cDNA was synthesized with the Superscript™ First Strand Synthesis System (Invitrogen, Carlsbad, CA). .. SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for GAPDH as an internal control.

    Article Title: miR-181 promotes human NK cell development by regulating Notch signaling
    Article Snippet: .. For microRNA qRT-PCR, cDNA was synthesized with the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA), and primer/probe sets for miR-181a, miR-181b and RNU-24 were purchased from Applied Biosystems. qRT-PCR for Hes5 and NLK were performed with the SYBR Green reagent (Applied Biosystems). .. All primer/probe sets were purchased from Applied Biosystems except for NLK.

    Article Title: Gene Expression During Imidacloprid-Induced Hormesis in Green Peach Aphid
    Article Snippet: Later, cDNA was synthesized from 1 μg of total RNA using a QuantiTect® Reverse Transcription kit (Qiagen, Toronto, Ontario) and stored at −20° C until further analyses. .. Ace was used as an internal control for ANT , Hsp60 , and FPPS I genes. qRT PCR was performed on a StepOne™ RT PCR System (Applied Biosystems, Burlington, Ontario) in a 10 μL reaction following the manufacturer’s instructions using SYBR green reagent (Applied Biosystems, Burlington, Ontario).

    Quantitative RT-PCR:

    Article Title: Contrasting Transcriptional Responses of a Virulent and an Attenuated Strain of Mycobacterium tuberculosis Infecting Macrophages
    Article Snippet: .. Gene specific primers ( ) were used with SYBR Green reagent (Finnzymes) to assess expression of candidate genes. qRT-PCR conditions were as follows: 95°C for 10 minutes followed by 35 cycles of 94°C for 30 seconds, 57°C for 20 seconds, and 72°C for 30 seconds. qRT-PCR data is presented as fold difference of expression in H37Ra relative to H37Rv using the 2−ΔΔCt method as described previously with a 71 bp product (henceforth called rrnAP1) from the promoter region of rrnS (Rvnr01 ) in Mtb and dnaK used as normalizing genes. ..

    Article Title: EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors
    Article Snippet: Paragraph title: RNA isolation, cDNA synthesis and real-time RT-PCR ... SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for β-actin as described ( ).

    Article Title: The NIP7 protein is required for accurate pre-rRNA processing in human cells
    Article Snippet: Analysis of NIP7 mRNA levels NIP7 mRNA downregulation was confirmed by RT–qPCR using oligonucleotides complementary to NIP7 sequence (NIP7-shRNA-F and NIP7-shRNA-R). .. The mRNA levels were quantified using the SYBR Green® reagent on a 7500 Real Time PCR system (Applied Biosystems).

    Article Title: Fatty Acid Synthase Modulates Homeostatic Responses to Myocardial Stress *
    Article Snippet: Paragraph title: Quantitative RT-PCR ... Quantitative PCR was performed with the ABI Prism7700 instrument using SYBR® Green reagent (Applied Biosystems).

    Article Title: miR-181 promotes human NK cell development by regulating Notch signaling
    Article Snippet: .. For microRNA qRT-PCR, cDNA was synthesized with the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA), and primer/probe sets for miR-181a, miR-181b and RNU-24 were purchased from Applied Biosystems. qRT-PCR for Hes5 and NLK were performed with the SYBR Green reagent (Applied Biosystems). .. All primer/probe sets were purchased from Applied Biosystems except for NLK.

    Article Title: Gene Expression During Imidacloprid-Induced Hormesis in Green Peach Aphid
    Article Snippet: .. Ace was used as an internal control for ANT , Hsp60 , and FPPS I genes. qRT PCR was performed on a StepOne™ RT PCR System (Applied Biosystems, Burlington, Ontario) in a 10 μL reaction following the manufacturer’s instructions using SYBR green reagent (Applied Biosystems, Burlington, Ontario). .. The reaction mixture contained 2X SYBR green reagent master mix, 2 μL cDNA, 2.5 μL ultrapure water, and 0.25 μL each of forward and reverse primers (final concentration of 2.5 μM).

    Real-time Polymerase Chain Reaction:

    Article Title: Bronchial epithelial injury in the context of alloimmunity promotes lymphocytic bronchiolitis through hyaluronan expression
    Article Snippet: .. Quantitative polymerase chain reaction was performed using ABI SDS 7500 and SYBR Green Reagent (Applied Biosystems). .. Primers were produced by IDT (Coralville, IA), and their sequences are in .

    Article Title: Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists
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    Article Snippet: .. Quantitative PCR analysis for IFNB gene expression was performed using SYBR green reagent (Invitrogen) on a DNA engine Opticon 2 cycler (Bio-Rad) using the following primers: IFNB (5′-CAGCAATTTTCAGTGTCAGAAGC and 3′-CATCCTGTCCTTGAGGCAGT) and β-actin (5′-CCTGGCACCCAGCACAAT and 3′-GCCGATCCACACGGAGTA). .. The specificity of amplification was assessed for each sample by melting curve analysis, and the size of the amplicon was checked by electrophoresis.

    Article Title: High glucose induces upregulation of scavenger receptors and promotes maturation of dendritic cells
    Article Snippet: .. Quantitative PCR using SYBR Green reagent was performed on the ABI 7500 Real-time PCR System (Applied Biosystems, United States). .. Gene expression was analysed by the system software, and the copies of each mRNA molecule were determined by the standard curve method.

    Article Title: GADD45A Protein Plays an Essential Role in Active DNA Demethylation during Terminal Osteogenic Differentiation of Adipose-derived Mesenchymal Stem Cells *
    Article Snippet: .. The immunoprecipitated DNA was further reverse cross-linked, purified, and subjected to quantitative PCR analysis using SYBR Green reagent (Invitrogen). ..

    Article Title: Cleavage of MCM2 licensing protein fosters senescence in human keratinocytes
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    Article Title: The NIP7 protein is required for accurate pre-rRNA processing in human cells
    Article Snippet: .. The mRNA levels were quantified using the SYBR Green® reagent on a 7500 Real Time PCR system (Applied Biosystems). ..

    Article Title: Fatty Acid Synthase Modulates Homeostatic Responses to Myocardial Stress *
    Article Snippet: .. Quantitative PCR was performed with the ABI Prism7700 instrument using SYBR® Green reagent (Applied Biosystems). ..

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    Article Title: Comparative evaluation of NOTCH signaling molecules in the endometrium of women with various gynecological diseases during the window of implantation
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    Article Title: Cell-to-cell variation in p53 dynamics leads to fractional killing
    Article Snippet: .. Quantitative PCR was performed with 8.4ng cDNA, 100nM primer and SYBR green reagent (Applied Biosystems). .. 72 hours after drug treatment 100µl of Cell Titer-Glo® reagent (Promega) was added to each well and luminescence was measured by a Victor2 plate reader (Perkin Elmer).

    Microarray:

    Article Title: Contrasting Transcriptional Responses of a Virulent and an Attenuated Strain of Mycobacterium tuberculosis Infecting Macrophages
    Article Snippet: Paragraph title: Quantitative RT-PCR analysis of selected candidate genes identified by microarray analysis ... Gene specific primers ( ) were used with SYBR Green reagent (Finnzymes) to assess expression of candidate genes. qRT-PCR conditions were as follows: 95°C for 10 minutes followed by 35 cycles of 94°C for 30 seconds, 57°C for 20 seconds, and 72°C for 30 seconds. qRT-PCR data is presented as fold difference of expression in H37Ra relative to H37Rv using the 2−ΔΔCt method as described previously with a 71 bp product (henceforth called rrnAP1) from the promoter region of rrnS (Rvnr01 ) in Mtb and dnaK used as normalizing genes.

    Incubation:

    Article Title: GADD45A Protein Plays an Essential Role in Active DNA Demethylation during Terminal Osteogenic Differentiation of Adipose-derived Mesenchymal Stem Cells *
    Article Snippet: Immune complexes were mixed with precoated protein A-Sepharose and incubated overnight via constant rotation at 4 °C. .. The immunoprecipitated DNA was further reverse cross-linked, purified, and subjected to quantitative PCR analysis using SYBR Green reagent (Invitrogen).

    Expressing:

    Article Title: Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists
    Article Snippet: Real-Time PCR, using SYBR Green reagent (Applied Biosystems) was performed using Stratagne Mx3000P. .. Each of the liver samples were tested for TNF-α, IL-6 and MIP2 expression levels.

    Article Title: Contrasting Transcriptional Responses of a Virulent and an Attenuated Strain of Mycobacterium tuberculosis Infecting Macrophages
    Article Snippet: .. Gene specific primers ( ) were used with SYBR Green reagent (Finnzymes) to assess expression of candidate genes. qRT-PCR conditions were as follows: 95°C for 10 minutes followed by 35 cycles of 94°C for 30 seconds, 57°C for 20 seconds, and 72°C for 30 seconds. qRT-PCR data is presented as fold difference of expression in H37Ra relative to H37Rv using the 2−ΔΔCt method as described previously with a 71 bp product (henceforth called rrnAP1) from the promoter region of rrnS (Rvnr01 ) in Mtb and dnaK used as normalizing genes. ..

    Article Title: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response *Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response * S⃞
    Article Snippet: .. Quantitative PCR analysis for IFNB gene expression was performed using SYBR green reagent (Invitrogen) on a DNA engine Opticon 2 cycler (Bio-Rad) using the following primers: IFNB (5′-CAGCAATTTTCAGTGTCAGAAGC and 3′-CATCCTGTCCTTGAGGCAGT) and β-actin (5′-CCTGGCACCCAGCACAAT and 3′-GCCGATCCACACGGAGTA). .. The specificity of amplification was assessed for each sample by melting curve analysis, and the size of the amplicon was checked by electrophoresis.

    Article Title: High glucose induces upregulation of scavenger receptors and promotes maturation of dendritic cells
    Article Snippet: Quantitative PCR using SYBR Green reagent was performed on the ABI 7500 Real-time PCR System (Applied Biosystems, United States). .. Gene expression was analysed by the system software, and the copies of each mRNA molecule were determined by the standard curve method.

    Article Title: EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors
    Article Snippet: Real-time RT-PCR was done with TaqMan® Gene Expression Assays (Applied Biosystems) for CDH1 (Hs00170423_m1), CDH2 (Hs00983062_m1), ZEB1 (Hs00232783_m1), ZEB2 (Hs00207691_m1), SNAI1 (Hs00195591_m1), SNAI2 (Hs00161804_m1), TWIST1 (Hs00361186_m1) and CDKN1A (Hs00355782_m1) using the ABI PRISM® 7000 Sequence Detection System (Applied Biosystems). .. SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for β-actin as described ( ).

    Article Title: Cleavage of MCM2 licensing protein fosters senescence in human keratinocytes
    Article Snippet: The MCM2 mRNA was determined by TaqMan® Gene Expression Assays (Applied Biosystems) using three independent sets of primers and the probes (Hs00170472_m1 for exons 2-3, Hs01091568_g1 for exons 3-4 and Hs01091564_m1 for exons 13-14), targeting different exon boundaries of the MCM2 transcript (MN_004526.2). .. SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for GAPDH as an internal control.

    Article Title: CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis
    Article Snippet: Paragraph title: Real-time gene expression analysis ... RNA from sorted cells was isolated using the ArrayPure Nano-Scale RNA Purification Kit (Epicentre, Singapore, Singapore). cDNA was generated using a cDNA synthesis kit (Roche, Basel, Switzerland), and quantitative real-time PCR (qPCR) analyses were performed using SYBR Green Reagent (Invitrogen, Carlsbad, CA, USA).

    Article Title: Gene Expression During Imidacloprid-Induced Hormesis in Green Peach Aphid
    Article Snippet: Paragraph title: Gene expression analyses ... Ace was used as an internal control for ANT , Hsp60 , and FPPS I genes. qRT PCR was performed on a StepOne™ RT PCR System (Applied Biosystems, Burlington, Ontario) in a 10 μL reaction following the manufacturer’s instructions using SYBR green reagent (Applied Biosystems, Burlington, Ontario).

    Immunoprecipitation:

    Article Title: GADD45A Protein Plays an Essential Role in Active DNA Demethylation during Terminal Osteogenic Differentiation of Adipose-derived Mesenchymal Stem Cells *
    Article Snippet: .. The immunoprecipitated DNA was further reverse cross-linked, purified, and subjected to quantitative PCR analysis using SYBR Green reagent (Invitrogen). ..

    Step One RT-PCR:

    Article Title: Gene Expression During Imidacloprid-Induced Hormesis in Green Peach Aphid
    Article Snippet: .. Ace was used as an internal control for ANT , Hsp60 , and FPPS I genes. qRT PCR was performed on a StepOne™ RT PCR System (Applied Biosystems, Burlington, Ontario) in a 10 μL reaction following the manufacturer’s instructions using SYBR green reagent (Applied Biosystems, Burlington, Ontario). .. The reaction mixture contained 2X SYBR green reagent master mix, 2 μL cDNA, 2.5 μL ultrapure water, and 0.25 μL each of forward and reverse primers (final concentration of 2.5 μM).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Bronchial epithelial injury in the context of alloimmunity promotes lymphocytic bronchiolitis through hyaluronan expression
    Article Snippet: Paragraph title: RT-PCR. ... Quantitative polymerase chain reaction was performed using ABI SDS 7500 and SYBR Green Reagent (Applied Biosystems).

    Article Title: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response *Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response * S⃞
    Article Snippet: RT, PCR, Quantitative PCR, and Sequencing —Total RNA was isolated using the RNeasy miniprep kit (Qiagen). .. Quantitative PCR analysis for IFNB gene expression was performed using SYBR green reagent (Invitrogen) on a DNA engine Opticon 2 cycler (Bio-Rad) using the following primers: IFNB (5′-CAGCAATTTTCAGTGTCAGAAGC and 3′-CATCCTGTCCTTGAGGCAGT) and β-actin (5′-CCTGGCACCCAGCACAAT and 3′-GCCGATCCACACGGAGTA).

    Article Title: Comparative evaluation of NOTCH signaling molecules in the endometrium of women with various gynecological diseases during the window of implantation
    Article Snippet: The identity of the amplified product was confirmed by the sequencing of the RT-PCR products. β-actin was considered as a reference gene ( ). .. The total reaction volume was 20 μl containing 250 ng cDNA, 5 pmol of each primer, and SYBR Green reagent (Applied Biosystems) with ROX dye as a passive control for signal intensity.

    Generated:

    Article Title: CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis
    Article Snippet: .. RNA from sorted cells was isolated using the ArrayPure Nano-Scale RNA Purification Kit (Epicentre, Singapore, Singapore). cDNA was generated using a cDNA synthesis kit (Roche, Basel, Switzerland), and quantitative real-time PCR (qPCR) analyses were performed using SYBR Green Reagent (Invitrogen, Carlsbad, CA, USA). .. Primer sequences are available upon request.

    Sequencing:

    Article Title: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response *Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response * S⃞
    Article Snippet: Sequencing was conducted using T7 primers by Macrogen (Seoul, South Korea). .. Quantitative PCR analysis for IFNB gene expression was performed using SYBR green reagent (Invitrogen) on a DNA engine Opticon 2 cycler (Bio-Rad) using the following primers: IFNB (5′-CAGCAATTTTCAGTGTCAGAAGC and 3′-CATCCTGTCCTTGAGGCAGT) and β-actin (5′-CCTGGCACCCAGCACAAT and 3′-GCCGATCCACACGGAGTA).

    Article Title: EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors
    Article Snippet: Real-time RT-PCR was done with TaqMan® Gene Expression Assays (Applied Biosystems) for CDH1 (Hs00170423_m1), CDH2 (Hs00983062_m1), ZEB1 (Hs00232783_m1), ZEB2 (Hs00207691_m1), SNAI1 (Hs00195591_m1), SNAI2 (Hs00161804_m1), TWIST1 (Hs00361186_m1) and CDKN1A (Hs00355782_m1) using the ABI PRISM® 7000 Sequence Detection System (Applied Biosystems). .. SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for β-actin as described ( ).

    Article Title: Cleavage of MCM2 licensing protein fosters senescence in human keratinocytes
    Article Snippet: Real-time PCR was performed using the ABI PRISM® 7000 Sequence Detection System (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. .. SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for GAPDH as an internal control.

    Article Title: The NIP7 protein is required for accurate pre-rRNA processing in human cells
    Article Snippet: Analysis of NIP7 mRNA levels NIP7 mRNA downregulation was confirmed by RT–qPCR using oligonucleotides complementary to NIP7 sequence (NIP7-shRNA-F and NIP7-shRNA-R). .. The mRNA levels were quantified using the SYBR Green® reagent on a 7500 Real Time PCR system (Applied Biosystems).

    Article Title: Comparative evaluation of NOTCH signaling molecules in the endometrium of women with various gynecological diseases during the window of implantation
    Article Snippet: The identity of the amplified product was confirmed by the sequencing of the RT-PCR products. β-actin was considered as a reference gene ( ). .. The total reaction volume was 20 μl containing 250 ng cDNA, 5 pmol of each primer, and SYBR Green reagent (Applied Biosystems) with ROX dye as a passive control for signal intensity.

    Sonication:

    Article Title: GADD45A Protein Plays an Essential Role in Active DNA Demethylation during Terminal Osteogenic Differentiation of Adipose-derived Mesenchymal Stem Cells *
    Article Snippet: The chromatin aliquots were precleared with protein A-Sepharose coated with sonicated salmon sperm DNA and bovine serum albumin. .. The immunoprecipitated DNA was further reverse cross-linked, purified, and subjected to quantitative PCR analysis using SYBR Green reagent (Invitrogen).

    Nucleic Acid Electrophoresis:

    Article Title: Gene Expression During Imidacloprid-Induced Hormesis in Green Peach Aphid
    Article Snippet: Quality (A260/280 > 2.0) and quantity of total RNA was assessed with a Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, Delaware) and gel electrophoresis (rRNA band intensity: 28s = 2X 18s). .. Ace was used as an internal control for ANT , Hsp60 , and FPPS I genes. qRT PCR was performed on a StepOne™ RT PCR System (Applied Biosystems, Burlington, Ontario) in a 10 μL reaction following the manufacturer’s instructions using SYBR green reagent (Applied Biosystems, Burlington, Ontario).

    Isolation:

    Article Title: Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists
    Article Snippet: Analysis of mRNA by Real-Time PCR Total RNA was isolated from the livers using Ultraspec™-II RNA isolation system (Biotecx, Houston, TX). .. Real-Time PCR, using SYBR Green reagent (Applied Biosystems) was performed using Stratagne Mx3000P.

    Article Title: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response *Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response * S⃞
    Article Snippet: RT, PCR, Quantitative PCR, and Sequencing —Total RNA was isolated using the RNeasy miniprep kit (Qiagen). .. Quantitative PCR analysis for IFNB gene expression was performed using SYBR green reagent (Invitrogen) on a DNA engine Opticon 2 cycler (Bio-Rad) using the following primers: IFNB (5′-CAGCAATTTTCAGTGTCAGAAGC and 3′-CATCCTGTCCTTGAGGCAGT) and β-actin (5′-CCTGGCACCCAGCACAAT and 3′-GCCGATCCACACGGAGTA).

    Article Title: High glucose induces upregulation of scavenger receptors and promotes maturation of dendritic cells
    Article Snippet: Total RNA was isolated and treated with DNase using a Trizol Reagent kit according to the manufacturer’s instructions. .. Quantitative PCR using SYBR Green reagent was performed on the ABI 7500 Real-time PCR System (Applied Biosystems, United States).

    Article Title: EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors
    Article Snippet: Paragraph title: RNA isolation, cDNA synthesis and real-time RT-PCR ... SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for β-actin as described ( ).

    Article Title: Cleavage of MCM2 licensing protein fosters senescence in human keratinocytes
    Article Snippet: Briefly, total RNA was isolated from cells with the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). .. SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for GAPDH as an internal control.

    Article Title: CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis
    Article Snippet: .. RNA from sorted cells was isolated using the ArrayPure Nano-Scale RNA Purification Kit (Epicentre, Singapore, Singapore). cDNA was generated using a cDNA synthesis kit (Roche, Basel, Switzerland), and quantitative real-time PCR (qPCR) analyses were performed using SYBR Green Reagent (Invitrogen, Carlsbad, CA, USA). .. Primer sequences are available upon request.

    Size-exclusion Chromatography:

    Article Title: Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists
    Article Snippet: Real-Time PCR, using SYBR Green reagent (Applied Biosystems) was performed using Stratagne Mx3000P. .. PCR parameters consisted of 40 cycles using a 3-step cycle process that began with melting at 95°C for 20 sec, annealing at 55°C for 75 sec, followed by extension at 72°C for 60 sec.

    Article Title: Comparative evaluation of NOTCH signaling molecules in the endometrium of women with various gynecological diseases during the window of implantation
    Article Snippet: The RT-PCR conditions for 40 cycles were as follows: 95 ° C for 30 sec, 60 ° C for 1 min, and 72 ° C for 1 min. To separate PCR products, agarose gel (1.2%) electrophoresis and visualizing via an ultraviolet transillumination were carried out. .. The total reaction volume was 20 μl containing 250 ng cDNA, 5 pmol of each primer, and SYBR Green reagent (Applied Biosystems) with ROX dye as a passive control for signal intensity.

    Purification:

    Article Title: GADD45A Protein Plays an Essential Role in Active DNA Demethylation during Terminal Osteogenic Differentiation of Adipose-derived Mesenchymal Stem Cells *
    Article Snippet: .. The immunoprecipitated DNA was further reverse cross-linked, purified, and subjected to quantitative PCR analysis using SYBR Green reagent (Invitrogen). ..

    Article Title: CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis
    Article Snippet: .. RNA from sorted cells was isolated using the ArrayPure Nano-Scale RNA Purification Kit (Epicentre, Singapore, Singapore). cDNA was generated using a cDNA synthesis kit (Roche, Basel, Switzerland), and quantitative real-time PCR (qPCR) analyses were performed using SYBR Green Reagent (Invitrogen, Carlsbad, CA, USA). .. Primer sequences are available upon request.

    Polymerase Chain Reaction:

    Article Title: Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists
    Article Snippet: Real-Time PCR, using SYBR Green reagent (Applied Biosystems) was performed using Stratagne Mx3000P. .. PCR parameters consisted of 40 cycles using a 3-step cycle process that began with melting at 95°C for 20 sec, annealing at 55°C for 75 sec, followed by extension at 72°C for 60 sec.

    Article Title: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response *Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response * S⃞
    Article Snippet: The cDNA was then amplified by PCR using primers specific for full-length IRF-5 (5′-TTAGATAAGCTTGCTATGAACCACTCAGCCCCAG containing a HindIII site and 3′-TAAATATCTAGATGCTTGCATGCCAACTGGGTG containing an XbaI site), exon 2–3 (5′-CTTCAGTGGGTCAACGGG and 3′-TGTACGGCTGAGGTGGCAT), exon 7–8 (5′-ATCCGTCTGTGCCAGTGTAAC and 3′-GCTTCTTCTCTCGGGGTTTG), and β-actin (5′-CATGTTTGAGACCTTCAACACC and 3′-GCACAGCTTCTCTTTGATGTCAC). .. Quantitative PCR analysis for IFNB gene expression was performed using SYBR green reagent (Invitrogen) on a DNA engine Opticon 2 cycler (Bio-Rad) using the following primers: IFNB (5′-CAGCAATTTTCAGTGTCAGAAGC and 3′-CATCCTGTCCTTGAGGCAGT) and β-actin (5′-CCTGGCACCCAGCACAAT and 3′-GCCGATCCACACGGAGTA).

    Article Title: Cleavage of MCM2 licensing protein fosters senescence in human keratinocytes
    Article Snippet: SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for GAPDH as an internal control. .. All PCR reactions were performed in triplicate.

    Article Title: Comparative evaluation of NOTCH signaling molecules in the endometrium of women with various gynecological diseases during the window of implantation
    Article Snippet: The RT-PCR conditions for 40 cycles were as follows: 95 ° C for 30 sec, 60 ° C for 1 min, and 72 ° C for 1 min. To separate PCR products, agarose gel (1.2%) electrophoresis and visualizing via an ultraviolet transillumination were carried out. .. The total reaction volume was 20 μl containing 250 ng cDNA, 5 pmol of each primer, and SYBR Green reagent (Applied Biosystems) with ROX dye as a passive control for signal intensity.

    IA:

    Article Title: Bronchial epithelial injury in the context of alloimmunity promotes lymphocytic bronchiolitis through hyaluronan expression
    Article Snippet: Quantitative polymerase chain reaction was performed using ABI SDS 7500 and SYBR Green Reagent (Applied Biosystems). .. Primers were produced by IDT (Coralville, IA), and their sequences are in .

    Chromatin Immunoprecipitation:

    Article Title: GADD45A Protein Plays an Essential Role in Active DNA Demethylation during Terminal Osteogenic Differentiation of Adipose-derived Mesenchymal Stem Cells *
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation (ChIP) Assay ... The immunoprecipitated DNA was further reverse cross-linked, purified, and subjected to quantitative PCR analysis using SYBR Green reagent (Invitrogen).

    Software:

    Article Title: High glucose induces upregulation of scavenger receptors and promotes maturation of dendritic cells
    Article Snippet: Quantitative PCR using SYBR Green reagent was performed on the ABI 7500 Real-time PCR System (Applied Biosystems, United States). .. Gene expression was analysed by the system software, and the copies of each mRNA molecule were determined by the standard curve method.

    Article Title: Cleavage of MCM2 licensing protein fosters senescence in human keratinocytes
    Article Snippet: SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for GAPDH as an internal control. .. Data were analyzed using ABI PRISM® 7000 sequence detection system software (Applied Biosystems).

    SYBR Green Assay:

    Article Title: Bronchial epithelial injury in the context of alloimmunity promotes lymphocytic bronchiolitis through hyaluronan expression
    Article Snippet: .. Quantitative polymerase chain reaction was performed using ABI SDS 7500 and SYBR Green Reagent (Applied Biosystems). .. Primers were produced by IDT (Coralville, IA), and their sequences are in .

    Article Title: Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists
    Article Snippet: .. Real-Time PCR, using SYBR Green reagent (Applied Biosystems) was performed using Stratagne Mx3000P. .. PCR parameters consisted of 40 cycles using a 3-step cycle process that began with melting at 95°C for 20 sec, annealing at 55°C for 75 sec, followed by extension at 72°C for 60 sec.

    Article Title: Contrasting Transcriptional Responses of a Virulent and an Attenuated Strain of Mycobacterium tuberculosis Infecting Macrophages
    Article Snippet: .. Gene specific primers ( ) were used with SYBR Green reagent (Finnzymes) to assess expression of candidate genes. qRT-PCR conditions were as follows: 95°C for 10 minutes followed by 35 cycles of 94°C for 30 seconds, 57°C for 20 seconds, and 72°C for 30 seconds. qRT-PCR data is presented as fold difference of expression in H37Ra relative to H37Rv using the 2−ΔΔCt method as described previously with a 71 bp product (henceforth called rrnAP1) from the promoter region of rrnS (Rvnr01 ) in Mtb and dnaK used as normalizing genes. ..

    Article Title: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response *Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response * S⃞
    Article Snippet: .. Quantitative PCR analysis for IFNB gene expression was performed using SYBR green reagent (Invitrogen) on a DNA engine Opticon 2 cycler (Bio-Rad) using the following primers: IFNB (5′-CAGCAATTTTCAGTGTCAGAAGC and 3′-CATCCTGTCCTTGAGGCAGT) and β-actin (5′-CCTGGCACCCAGCACAAT and 3′-GCCGATCCACACGGAGTA). .. The specificity of amplification was assessed for each sample by melting curve analysis, and the size of the amplicon was checked by electrophoresis.

    Article Title: High glucose induces upregulation of scavenger receptors and promotes maturation of dendritic cells
    Article Snippet: .. Quantitative PCR using SYBR Green reagent was performed on the ABI 7500 Real-time PCR System (Applied Biosystems, United States). .. Gene expression was analysed by the system software, and the copies of each mRNA molecule were determined by the standard curve method.

    Article Title: GADD45A Protein Plays an Essential Role in Active DNA Demethylation during Terminal Osteogenic Differentiation of Adipose-derived Mesenchymal Stem Cells *
    Article Snippet: .. The immunoprecipitated DNA was further reverse cross-linked, purified, and subjected to quantitative PCR analysis using SYBR Green reagent (Invitrogen). ..

    Article Title: EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors
    Article Snippet: .. SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for β-actin as described ( ). ..

    Article Title: Cleavage of MCM2 licensing protein fosters senescence in human keratinocytes
    Article Snippet: .. SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for GAPDH as an internal control. .. All PCR reactions were performed in triplicate.

    Article Title: The NIP7 protein is required for accurate pre-rRNA processing in human cells
    Article Snippet: .. The mRNA levels were quantified using the SYBR Green® reagent on a 7500 Real Time PCR system (Applied Biosystems). ..

    Article Title: Fatty Acid Synthase Modulates Homeostatic Responses to Myocardial Stress *
    Article Snippet: .. Quantitative PCR was performed with the ABI Prism7700 instrument using SYBR® Green reagent (Applied Biosystems). ..

    Article Title: CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis
    Article Snippet: .. RNA from sorted cells was isolated using the ArrayPure Nano-Scale RNA Purification Kit (Epicentre, Singapore, Singapore). cDNA was generated using a cDNA synthesis kit (Roche, Basel, Switzerland), and quantitative real-time PCR (qPCR) analyses were performed using SYBR Green Reagent (Invitrogen, Carlsbad, CA, USA). .. Primer sequences are available upon request.

    Article Title: miR-181 promotes human NK cell development by regulating Notch signaling
    Article Snippet: .. For microRNA qRT-PCR, cDNA was synthesized with the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA), and primer/probe sets for miR-181a, miR-181b and RNU-24 were purchased from Applied Biosystems. qRT-PCR for Hes5 and NLK were performed with the SYBR Green reagent (Applied Biosystems). .. All primer/probe sets were purchased from Applied Biosystems except for NLK.

    Article Title: Comparative evaluation of NOTCH signaling molecules in the endometrium of women with various gynecological diseases during the window of implantation
    Article Snippet: .. The total reaction volume was 20 μl containing 250 ng cDNA, 5 pmol of each primer, and SYBR Green reagent (Applied Biosystems) with ROX dye as a passive control for signal intensity. .. Amplification was carried out at 95 ° C for 30 sec, 60 ° C for 30 sec, and 72 ° C for 30 sec. Melting curves of PCR reactions were used to check the formation of nonspecific products and primer-dimer.

    Article Title: Cell-to-cell variation in p53 dynamics leads to fractional killing
    Article Snippet: .. Quantitative PCR was performed with 8.4ng cDNA, 100nM primer and SYBR green reagent (Applied Biosystems). .. 72 hours after drug treatment 100µl of Cell Titer-Glo® reagent (Promega) was added to each well and luminescence was measured by a Victor2 plate reader (Perkin Elmer).

    Article Title: Gene Expression During Imidacloprid-Induced Hormesis in Green Peach Aphid
    Article Snippet: .. Ace was used as an internal control for ANT , Hsp60 , and FPPS I genes. qRT PCR was performed on a StepOne™ RT PCR System (Applied Biosystems, Burlington, Ontario) in a 10 μL reaction following the manufacturer’s instructions using SYBR green reagent (Applied Biosystems, Burlington, Ontario). .. The reaction mixture contained 2X SYBR green reagent master mix, 2 μL cDNA, 2.5 μL ultrapure water, and 0.25 μL each of forward and reverse primers (final concentration of 2.5 μM).

    RNA Extraction:

    Article Title: EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors
    Article Snippet: RNA extraction and cDNA synthesis were performed as described previously ( ). .. SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for β-actin as described ( ).

    Article Title: Cleavage of MCM2 licensing protein fosters senescence in human keratinocytes
    Article Snippet: Paragraph title: RNA extraction, cDNA preparation and real-time PCR ... SYBR green reagent (Applied Biosystems) was used to quantitate mRNA for GAPDH as an internal control.

    Agarose Gel Electrophoresis:

    Article Title: Comparative evaluation of NOTCH signaling molecules in the endometrium of women with various gynecological diseases during the window of implantation
    Article Snippet: The RT-PCR conditions for 40 cycles were as follows: 95 ° C for 30 sec, 60 ° C for 1 min, and 72 ° C for 1 min. To separate PCR products, agarose gel (1.2%) electrophoresis and visualizing via an ultraviolet transillumination were carried out. .. The total reaction volume was 20 μl containing 250 ng cDNA, 5 pmol of each primer, and SYBR Green reagent (Applied Biosystems) with ROX dye as a passive control for signal intensity.

    Electrophoresis:

    Article Title: Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response *Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response * S⃞
    Article Snippet: Quantitative PCR analysis for IFNB gene expression was performed using SYBR green reagent (Invitrogen) on a DNA engine Opticon 2 cycler (Bio-Rad) using the following primers: IFNB (5′-CAGCAATTTTCAGTGTCAGAAGC and 3′-CATCCTGTCCTTGAGGCAGT) and β-actin (5′-CCTGGCACCCAGCACAAT and 3′-GCCGATCCACACGGAGTA). .. The specificity of amplification was assessed for each sample by melting curve analysis, and the size of the amplicon was checked by electrophoresis.

    Article Title: Comparative evaluation of NOTCH signaling molecules in the endometrium of women with various gynecological diseases during the window of implantation
    Article Snippet: The RT-PCR conditions for 40 cycles were as follows: 95 ° C for 30 sec, 60 ° C for 1 min, and 72 ° C for 1 min. To separate PCR products, agarose gel (1.2%) electrophoresis and visualizing via an ultraviolet transillumination were carried out. .. The total reaction volume was 20 μl containing 250 ng cDNA, 5 pmol of each primer, and SYBR Green reagent (Applied Biosystems) with ROX dye as a passive control for signal intensity.

    Produced:

    Article Title: Bronchial epithelial injury in the context of alloimmunity promotes lymphocytic bronchiolitis through hyaluronan expression
    Article Snippet: Quantitative polymerase chain reaction was performed using ABI SDS 7500 and SYBR Green Reagent (Applied Biosystems). .. Primers were produced by IDT (Coralville, IA), and their sequences are in .

    Concentration Assay:

    Article Title: Cell-to-cell variation in p53 dynamics leads to fractional killing
    Article Snippet: Quantitative PCR was performed with 8.4ng cDNA, 100nM primer and SYBR green reagent (Applied Biosystems). .. Quantitative PCR was performed with 8.4ng cDNA, 100nM primer and SYBR green reagent (Applied Biosystems).

    Article Title: Gene Expression During Imidacloprid-Induced Hormesis in Green Peach Aphid
    Article Snippet: Ace was used as an internal control for ANT , Hsp60 , and FPPS I genes. qRT PCR was performed on a StepOne™ RT PCR System (Applied Biosystems, Burlington, Ontario) in a 10 μL reaction following the manufacturer’s instructions using SYBR green reagent (Applied Biosystems, Burlington, Ontario). .. The reaction mixture contained 2X SYBR green reagent master mix, 2 μL cDNA, 2.5 μL ultrapure water, and 0.25 μL each of forward and reverse primers (final concentration of 2.5 μM).

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  • 90
    Thermo Fisher turbo dna free kit
    Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated <t>DNA</t> damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p
    Turbo Dna Free Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher trizol reagent
    MHV-ExoN(-) evolved WT-like genomic <t>RNA</t> accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using <t>TRIzol</t> at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P
    Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 44742 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript rt pcr reagents
    Improved <t>PCR</t> detection following MTE. BDBV (A), influenza B strain 38 (B), MARV (C), EBOV (D), and P . falciparum (E) nucleic acid were serially diluted. One dilution series was amplified by MTE prior to being run in the <t>SYBR</t> Green assay. The amount of pathogen-specific nucleic acid was determined by real-time PCR using primers internal to the MTE amplification primers. Fig B is shown as a dilution factor because influenza B strain 38 did not titer. Samples were run in triplicate, and the error bars represent the standard deviation of the mean. A two-way ANOVA with Bonferroni determined all dilutions for all viruses tested were significantly different with the exception of the highest influenza B virus concentration (~10 3 PFU/rxn).
    Superscript Rt Pcr Reagents, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated DNA damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p

    Journal: Molecular Cell

    Article Title: P-TEFb Activation by RBM7 Shapes a Pro-survival Transcriptional Response to Genotoxic Stress

    doi: 10.1016/j.molcel.2019.01.033

    Figure Lengend Snippet: Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated DNA damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p

    Article Snippet: RNA samples were DNase-treated with the Turbo DNA-Free kit (Thermo Fisher Scientific), reverse transcribed with SuperScript III reverse transcriptase (Thermo Fisher Sceintific) and random hexamers (Thermo Fisher Scientific), and amplified using FastStart Universal SYBR Green QPCR Master (Rox) (Sigma), RNA-specific primer pair, and Stratagene Mx3005 qPCR machine.

    Techniques: Labeling, Expressing, Indirect Immunoperoxidase Assay, Quantitative RT-PCR

    MHV-ExoN(-) evolved WT-like genomic RNA accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using TRIzol at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P

    Journal: mBio

    Article Title: Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations

    doi: 10.1128/mBio.01503-17

    Figure Lengend Snippet: MHV-ExoN(-) evolved WT-like genomic RNA accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using TRIzol at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P

    Article Snippet: For each passage, supernatants were harvested at 24 h. RNA was extracted from 100 μl of supernatant using 900 μl of TRIzol reagent and PureLink RNA minikit columns (Thermo Scientific, Waltham, MA), and 150 μl of supernatant was used to infect fresh cells in a 24-well plate (total MOI estimated at 1 PFU/cell).

    Techniques: Infection, SYBR Green Assay, Plaque Assay, MANN-WHITNEY

    Improved PCR detection following MTE. BDBV (A), influenza B strain 38 (B), MARV (C), EBOV (D), and P . falciparum (E) nucleic acid were serially diluted. One dilution series was amplified by MTE prior to being run in the SYBR Green assay. The amount of pathogen-specific nucleic acid was determined by real-time PCR using primers internal to the MTE amplification primers. Fig B is shown as a dilution factor because influenza B strain 38 did not titer. Samples were run in triplicate, and the error bars represent the standard deviation of the mean. A two-way ANOVA with Bonferroni determined all dilutions for all viruses tested were significantly different with the exception of the highest influenza B virus concentration (~10 3 PFU/rxn).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A highly multiplexed broad pathogen detection assay for infectious disease diagnostics

    doi: 10.1371/journal.pntd.0006889

    Figure Lengend Snippet: Improved PCR detection following MTE. BDBV (A), influenza B strain 38 (B), MARV (C), EBOV (D), and P . falciparum (E) nucleic acid were serially diluted. One dilution series was amplified by MTE prior to being run in the SYBR Green assay. The amount of pathogen-specific nucleic acid was determined by real-time PCR using primers internal to the MTE amplification primers. Fig B is shown as a dilution factor because influenza B strain 38 did not titer. Samples were run in triplicate, and the error bars represent the standard deviation of the mean. A two-way ANOVA with Bonferroni determined all dilutions for all viruses tested were significantly different with the exception of the highest influenza B virus concentration (~10 3 PFU/rxn).

    Article Snippet: The levels of enrichment were measured by real-time RT-PCR using Superscript RT-PCR reagents (Thermo Fisher Scientific) and SYBR Green.

    Techniques: Polymerase Chain Reaction, Amplification, SYBR Green Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Concentration Assay