polymerase chain reaction pcr mixture  (Thermo Fisher)


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    Structured Review

    Thermo Fisher polymerase chain reaction pcr mixture
    The genotype characteristic of SNP rs231775 in CTLA-4 gene in AA and HIs. a Distribution of the genotype of SNP rs231775 in AA patients ( n = 67); b distribution of the genotype of SNP rs231775 in HIs ( n = 84); c agarose gel electrophoresis results for SNP rs231775 in CTLA-4 gene, lane 1 is the AG genotype, lane 2 is the GG genotype, lane 3 is a 50 bp <t>DNA</t> ladder, lane 4 is the AA genotype, and lane 5 is a <t>PCR</t> product without TseI digestion; d sequencing results of AA genotype; e sequencing results of AG genotype; f sequencing results of GG genotype; the red arrow indicates the single-nucleotide polymorphism sites
    Polymerase Chain Reaction Pcr Mixture, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Molecular alterations in the TCR signaling pathway in patients with aplastic anemia"

    Article Title: Molecular alterations in the TCR signaling pathway in patients with aplastic anemia

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-016-0261-6

    The genotype characteristic of SNP rs231775 in CTLA-4 gene in AA and HIs. a Distribution of the genotype of SNP rs231775 in AA patients ( n = 67); b distribution of the genotype of SNP rs231775 in HIs ( n = 84); c agarose gel electrophoresis results for SNP rs231775 in CTLA-4 gene, lane 1 is the AG genotype, lane 2 is the GG genotype, lane 3 is a 50 bp DNA ladder, lane 4 is the AA genotype, and lane 5 is a PCR product without TseI digestion; d sequencing results of AA genotype; e sequencing results of AG genotype; f sequencing results of GG genotype; the red arrow indicates the single-nucleotide polymorphism sites
    Figure Legend Snippet: The genotype characteristic of SNP rs231775 in CTLA-4 gene in AA and HIs. a Distribution of the genotype of SNP rs231775 in AA patients ( n = 67); b distribution of the genotype of SNP rs231775 in HIs ( n = 84); c agarose gel electrophoresis results for SNP rs231775 in CTLA-4 gene, lane 1 is the AG genotype, lane 2 is the GG genotype, lane 3 is a 50 bp DNA ladder, lane 4 is the AA genotype, and lane 5 is a PCR product without TseI digestion; d sequencing results of AA genotype; e sequencing results of AG genotype; f sequencing results of GG genotype; the red arrow indicates the single-nucleotide polymorphism sites

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing

    Related Articles

    Amplification:

    Article Title: The Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species
    Article Snippet: .. PCR amplification was conducted using 100 ng of gemonic DNA in a total volume of 20 μl with forward and reverse primers (400 nM each) using DreamTaq PCR Master Mix (Thermo Scientific). .. At the first round, fragments 1–3 were amplified by PCR with primer sets of CA5F and CA3R, CA1F and CA2R, CA4F and CA5R, respectively.

    Article Title: Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes *
    Article Snippet: .. For in vitro cleavage kinetics, ribozyme constructs were either amplified from their respective genomes or synthesized by mutual priming using primers listed below and the DreamTaq PCR master mix (Fermentas). .. The constructs were designed to include a 40-nucleotide leader sequence, the full ribozyme, and a short (2–8 nucleotide) tail.

    Article Title: Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate
    Article Snippet: .. T-RFLP Monitoring Amplification of a 900 bp fragment of the 16S rRNA gene for T-RFLP analysis from DNA extracts was achieved using DreamTaq PCR Master Mix (Thermo Fisher) and a Cy5-labeled 8F forward primer ( ) and 907R ( ) as described previously ( ). .. Up to 4 μL of PCR product were digested in a 10 μL reaction with 1 U of the restriction endonuclease HaeIII (Thermo Scientific) and 1 μL appropriate buffer.

    In Vitro:

    Article Title: Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes *
    Article Snippet: .. For in vitro cleavage kinetics, ribozyme constructs were either amplified from their respective genomes or synthesized by mutual priming using primers listed below and the DreamTaq PCR master mix (Fermentas). .. The constructs were designed to include a 40-nucleotide leader sequence, the full ribozyme, and a short (2–8 nucleotide) tail.

    Synthesized:

    Article Title: Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes *
    Article Snippet: .. For in vitro cleavage kinetics, ribozyme constructs were either amplified from their respective genomes or synthesized by mutual priming using primers listed below and the DreamTaq PCR master mix (Fermentas). .. The constructs were designed to include a 40-nucleotide leader sequence, the full ribozyme, and a short (2–8 nucleotide) tail.

    Construct:

    Article Title: Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes *
    Article Snippet: .. For in vitro cleavage kinetics, ribozyme constructs were either amplified from their respective genomes or synthesized by mutual priming using primers listed below and the DreamTaq PCR master mix (Fermentas). .. The constructs were designed to include a 40-nucleotide leader sequence, the full ribozyme, and a short (2–8 nucleotide) tail.

    Terminal Restriction Fragment Length Polymorphism:

    Article Title: Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate
    Article Snippet: .. T-RFLP Monitoring Amplification of a 900 bp fragment of the 16S rRNA gene for T-RFLP analysis from DNA extracts was achieved using DreamTaq PCR Master Mix (Thermo Fisher) and a Cy5-labeled 8F forward primer ( ) and 907R ( ) as described previously ( ). .. Up to 4 μL of PCR product were digested in a 10 μL reaction with 1 U of the restriction endonuclease HaeIII (Thermo Scientific) and 1 μL appropriate buffer.

    Polymerase Chain Reaction:

    Article Title: Using an in-vitro biofilm model to assess the virulence potential of Bacterial Vaginosis or non-Bacterial Vaginosis Gardnerella vaginalis isolates
    Article Snippet: .. Genomic DNA was extracted as described before and the thermocycling program (Mini-MJ, Bio-Rad, Hercules, CA, USA) was performed using the DreamTaq PCR Master Mix 2x (Finnzymes, Espoo, Finland) and consisted on the following steps: 94 °C for 2 minutes followed by 40 cycles of 94 °C for 30 seconds, 58 °C for 30 seconds, 72 °C for 60 seconds and finally 72 °C for 5 minutes. ..

    Article Title: The Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species
    Article Snippet: .. PCR amplification was conducted using 100 ng of gemonic DNA in a total volume of 20 μl with forward and reverse primers (400 nM each) using DreamTaq PCR Master Mix (Thermo Scientific). .. At the first round, fragments 1–3 were amplified by PCR with primer sets of CA5F and CA3R, CA1F and CA2R, CA4F and CA5R, respectively.

    Article Title: Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes *
    Article Snippet: .. For in vitro cleavage kinetics, ribozyme constructs were either amplified from their respective genomes or synthesized by mutual priming using primers listed below and the DreamTaq PCR master mix (Fermentas). .. The constructs were designed to include a 40-nucleotide leader sequence, the full ribozyme, and a short (2–8 nucleotide) tail.

    Article Title: Phenotype-Independent Isolation of Interspecies Saccharomyces Hybrids by Dual-Dye Fluorescent Staining and Fluorescence-Activated Cell Sorting
    Article Snippet: .. Identification of Interspecies Hybrids by PCR The presence of genetic material from S. cerevisiae and from S. eubayanus and the mating type of potential hybrids was verified by PCR using DreamTaq PCR Mastermix (Life Technologies), as described previously ( ). .. To ensure that single cells isolates were tested, sorted dual-stained cells were grown in YPD and a second FACS step was used to sort a single cell from each culture.

    Article Title: Single-Cell Resolution of Uncultured Magnetotactic Bacteria via Fluorescence-Coupled Electron Microscopy
    Article Snippet: .. Briefly, each 50-μl PCR mixture contained 1 μl of template, 25 μl of DreamTaq PCR master mix (MBI Fermentas), 2 μl of each primer (10 μM), and 20 μl of Milli-Q water. ..

    Article Title: Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate
    Article Snippet: .. T-RFLP Monitoring Amplification of a 900 bp fragment of the 16S rRNA gene for T-RFLP analysis from DNA extracts was achieved using DreamTaq PCR Master Mix (Thermo Fisher) and a Cy5-labeled 8F forward primer ( ) and 907R ( ) as described previously ( ). .. Up to 4 μL of PCR product were digested in a 10 μL reaction with 1 U of the restriction endonuclease HaeIII (Thermo Scientific) and 1 μL appropriate buffer.

    Article Title: Molecular prevalence and haemato-biochemical profile of canine monocytic ehrlichiosis in dogs in and around Hisar, Haryana, India
    Article Snippet: .. The reaction (10 μl) contained 1.0 μl of template DNA in DreamTaq PCR master mix (Thermo Fisher Scientific) containing 5.0 μl DreamTaq polymerase, optimized DreamTaq buffer, MgCl2 and dNTPs; 0.3 μl of each primer and DMSO and 3.1 μl NFW. .. The thermocycling conditions consisted of initial denaturation at 94 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 45 s and extension at 72 °C for 30 s. This was followed by a final extension at 72 °C for 6 min. All the PCR products and a molecular weight marker (100 base-pair ladder) were electrophoresed through 1.5% agarose gels stained with ethidium bromide in Tris-acetate-EDTA (TAE) buffer for 35 min at 80 V and the DNA fragments were visualized under UV fluorescence using a gel documentation system (GeNei Uvitec, UK).

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    Thermo Fisher genes has1 3
    Factor analysis for correlation of gene expression levels. <t>HAS1-3</t> , HYAL1-2 , CEMIP , CD44 , VCAN , and TSG6 in non-failing left wall and septum (n = 9) ( a ) and HCM myectomies (n = 5) ( b ). ( a ) In left wall and septum there are two clusters where HAS1 , CEMIP , CD44 , VCAN , and TSG6 form one cluster, and HAS2 , HAS3, and HYAL2 form another. ( b ) In basal septal myectomies from HCM patients the expression levels of CEMIP , CD44, and VCAN formed a new correlation cluster with HAS3 . HAS2 , HYAL1 , and TSG6 formed another cluster. The levels of HAS1 and HYAL2 no longer correlated with any of the other genes investigated. Factor analysis was performed with the principal components method to analyze the correlation matrix and two factors were extracted.
    Genes Has1 3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp abcb1 hs00184500 m1
    Quantification of mRNA expression of CSC-LC property-related genes in sorted cell fractions by real-time PCR. In SAS, CD44v3 + /CD24 − cells show significantly higher mRNA expression of transporter-related genes <t>(ABCB1</t> and ABCG2), ALDH1A1, anti-apoptotic genes (BCL2) and self-replication genes (Oct-4 and Nanog) than the other fractions. The experiments were repeated at least three times for each cell line, and almost identical results were obtained ( * P
    Gene Exp Abcb1 Hs00184500 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp abcb1 hs00184500 m1/product/Thermo Fisher
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    89
    Thermo Fisher qrt pcr reaction mixture
    Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by <t>qRT-PCR</t> with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P
    Qrt Pcr Reaction Mixture, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman universal master mix ii
    Dysregulation of <t>miR-125b</t> and miR-22 expression in RNA extracted from prostate tissue samples by LCM. (A) Frozen prostate samples were dissected via LCM into benign glandular epithelial versus tumor. Total RNA was extracted using the PicoPure RNA extraction method, as described in Materials and Methods. qRT-PCR was performed with specific primers against miR-125b and miR-22 using the <t>TaqMan</t> ® Universal Master Mix II. C T values were normalized to RNU48 and reported as fold differences compared to benign tissue using the ΔΔCT method of Livak et al. [ 23 ]. Results are the average from frozen tissue samples (n = 4) repeated in 3 technical replicates. (B) From 10 successive slides of a single FFPE prostate sample, miR-125b was quantitated by qRT-PCR as described in panel A for RNA extracted from stroma, benign glandular tissue, BPH, PIN and tumor of stage G4.
    Taqman Universal Master Mix Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Factor analysis for correlation of gene expression levels. HAS1-3 , HYAL1-2 , CEMIP , CD44 , VCAN , and TSG6 in non-failing left wall and septum (n = 9) ( a ) and HCM myectomies (n = 5) ( b ). ( a ) In left wall and septum there are two clusters where HAS1 , CEMIP , CD44 , VCAN , and TSG6 form one cluster, and HAS2 , HAS3, and HYAL2 form another. ( b ) In basal septal myectomies from HCM patients the expression levels of CEMIP , CD44, and VCAN formed a new correlation cluster with HAS3 . HAS2 , HYAL1 , and TSG6 formed another cluster. The levels of HAS1 and HYAL2 no longer correlated with any of the other genes investigated. Factor analysis was performed with the principal components method to analyze the correlation matrix and two factors were extracted.

    Journal: Cells

    Article Title: Low Molecular Mass Myocardial Hyaluronan in Human Hypertrophic Cardiomyopathy

    doi: 10.3390/cells8020097

    Figure Lengend Snippet: Factor analysis for correlation of gene expression levels. HAS1-3 , HYAL1-2 , CEMIP , CD44 , VCAN , and TSG6 in non-failing left wall and septum (n = 9) ( a ) and HCM myectomies (n = 5) ( b ). ( a ) In left wall and septum there are two clusters where HAS1 , CEMIP , CD44 , VCAN , and TSG6 form one cluster, and HAS2 , HAS3, and HYAL2 form another. ( b ) In basal septal myectomies from HCM patients the expression levels of CEMIP , CD44, and VCAN formed a new correlation cluster with HAS3 . HAS2 , HYAL1 , and TSG6 formed another cluster. The levels of HAS1 and HYAL2 no longer correlated with any of the other genes investigated. Factor analysis was performed with the principal components method to analyze the correlation matrix and two factors were extracted.

    Article Snippet: The real-time quantitative PCR was performed on a 7900 HT Fast Real-Time PCR system (Thermo Fisher Scientific, MA, USA) using 1 µg cDNA, TaqMan® Gene Expression Assays, and 1 µL Gene Assay Mix for the genes HAS1-3 , HYAL 1 and 2 , CEMIP , CD44 , VCAN , and TSG6 (Thermo Fisher Scientific, MA, USA).

    Techniques: Expressing

    Quantification of mRNA expression of CSC-LC property-related genes in sorted cell fractions by real-time PCR. In SAS, CD44v3 + /CD24 − cells show significantly higher mRNA expression of transporter-related genes (ABCB1 and ABCG2), ALDH1A1, anti-apoptotic genes (BCL2) and self-replication genes (Oct-4 and Nanog) than the other fractions. The experiments were repeated at least three times for each cell line, and almost identical results were obtained ( * P

    Journal: International Journal of Oncology

    Article Title: CD44v3+/CD24− cells possess cancer stem cell-like properties in human oral squamous cell carcinoma

    doi: 10.3892/ijo.2015.3261

    Figure Lengend Snippet: Quantification of mRNA expression of CSC-LC property-related genes in sorted cell fractions by real-time PCR. In SAS, CD44v3 + /CD24 − cells show significantly higher mRNA expression of transporter-related genes (ABCB1 and ABCG2), ALDH1A1, anti-apoptotic genes (BCL2) and self-replication genes (Oct-4 and Nanog) than the other fractions. The experiments were repeated at least three times for each cell line, and almost identical results were obtained ( * P

    Article Snippet: Gene expression assays and primer and probe mixes were used for ABCB1, ABCG2, ALDH1A1, BCL2, CFLAR, Oct4, Nanog, HIF1α, and β-actin [assay IDs (Hs 00184500_m1, Hs01053790_m1, Hs00946916_m1, Hs00608023_m1, Hs00153439_m1, Hs03666771, Hs04260366, Hs00153153_m1, and Hs99999903_m1, respectively; Applied Biosystems)], and thermal conditions were as follows: initial incubation at 95°C for 10 min, then 40 cycles alternating in turn with 95°C for 10 sec, 60°C for 20 sec, and 72°C for 15 sec, and then maintained at 72°C for 10 min.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Over Expression, Expressing, Plasmid Preparation, Construct, Quantitative RT-PCR, High Performance Liquid Chromatography

    Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR

    PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR

    Dysregulation of miR-125b and miR-22 expression in RNA extracted from prostate tissue samples by LCM. (A) Frozen prostate samples were dissected via LCM into benign glandular epithelial versus tumor. Total RNA was extracted using the PicoPure RNA extraction method, as described in Materials and Methods. qRT-PCR was performed with specific primers against miR-125b and miR-22 using the TaqMan ® Universal Master Mix II. C T values were normalized to RNU48 and reported as fold differences compared to benign tissue using the ΔΔCT method of Livak et al. [ 23 ]. Results are the average from frozen tissue samples (n = 4) repeated in 3 technical replicates. (B) From 10 successive slides of a single FFPE prostate sample, miR-125b was quantitated by qRT-PCR as described in panel A for RNA extracted from stroma, benign glandular tissue, BPH, PIN and tumor of stage G4.

    Journal: PLoS ONE

    Article Title: Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis

    doi: 10.1371/journal.pone.0142373

    Figure Lengend Snippet: Dysregulation of miR-125b and miR-22 expression in RNA extracted from prostate tissue samples by LCM. (A) Frozen prostate samples were dissected via LCM into benign glandular epithelial versus tumor. Total RNA was extracted using the PicoPure RNA extraction method, as described in Materials and Methods. qRT-PCR was performed with specific primers against miR-125b and miR-22 using the TaqMan ® Universal Master Mix II. C T values were normalized to RNU48 and reported as fold differences compared to benign tissue using the ΔΔCT method of Livak et al. [ 23 ]. Results are the average from frozen tissue samples (n = 4) repeated in 3 technical replicates. (B) From 10 successive slides of a single FFPE prostate sample, miR-125b was quantitated by qRT-PCR as described in panel A for RNA extracted from stroma, benign glandular tissue, BPH, PIN and tumor of stage G4.

    Article Snippet: Each individual assay was performed in a 20 μl reaction volume with 1.33 μl of cDNA, 1.0 μl specific miR assay, 10 μl TaqMan™ Universal PCR Master Mix II with no AmpErase UNG and 7.67 μl of nuclease free water.

    Techniques: Expressing, Laser Capture Microdissection, RNA Extraction, Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded