quantifast sybr green pcr kit  (Qiagen)

 
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    QuantiFast SYBR Green PCR Kit
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    For fast real time PCR and two step qRT PCR using SYBR Green Kit contents Qiagen QuantiFast SYBR Green PCR Kit 400 x 25L rxns SYBR Green I cDNA and DNA Sample Real time and two step RT PCR Reaction Q bond Technology For use in Gene Expression Analysis of cDNA Targets and Quantitative gDNA Analysis Delivers Fast and Specific Quantification of gDNA or cDNA Targets Includes 3 x 1 7mL 2x QuantiFast SYBR Green PCR Master Mix Contains ROX Dye 2 x 2mL RNase free Water Benefits Specific and sensitive detection of even low copy targets Optimized master mix for reliable results without optimization 5 minute enzyme activation and fast cycling conditions Accurate detection of a wide range of template amounts Universal protocol for all standard and fast cycl
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    QuantiFast SYBR Green PCR Kit
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    Qiagen quantifast sybr green pcr kit
    QuantiFast SYBR Green PCR Kit
    For fast real time PCR and two step qRT PCR using SYBR Green Kit contents Qiagen QuantiFast SYBR Green PCR Kit 400 x 25L rxns SYBR Green I cDNA and DNA Sample Real time and two step RT PCR Reaction Q bond Technology For use in Gene Expression Analysis of cDNA Targets and Quantitative gDNA Analysis Delivers Fast and Specific Quantification of gDNA or cDNA Targets Includes 3 x 1 7mL 2x QuantiFast SYBR Green PCR Master Mix Contains ROX Dye 2 x 2mL RNase free Water Benefits Specific and sensitive detection of even low copy targets Optimized master mix for reliable results without optimization 5 minute enzyme activation and fast cycling conditions Accurate detection of a wide range of template amounts Universal protocol for all standard and fast cycl
    https://www.bioz.com/result/quantifast sybr green pcr kit/product/Qiagen
    Average 99 stars, based on 1129 article reviews
    Price from $9.99 to $1999.99
    quantifast sybr green pcr kit - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues"

    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198795

    Allele-specific forward primers allow detection of single and double BRAF mutations. A. Schematic diagrams of primer design. Forward primers were chosen to specifically amplify BRAF V600E1 (c.1799T > A), V600E2 (c.1799_1800delTGinsAA) and V600K (c.1798_1799delGTinsAA) mutations. Detection of BRAF V600K mutation can be performed separately or together with BRAF V600E mutation detection. WT = wild-type. B and C. Detection of low abundant BRAF V600E1 (B) and V600K mutations (C) in diluted DNA reference standards, which allelic frequencies were measured by droplet digital PCR. DNA amplification is monitored at each cycle of PCR using SYBR Green reagent included in PCR premix. D and E. Detection of BRAF V600E2 mutation (D) and complex tandem mutation caused by nucleotide changes in codons 600 and 601 (BRAF V600E1 and K601E mutations) (E). Synthesized oligonucleotides ( S1 Table ) were diluted and used as PCR templates.
    Figure Legend Snippet: Allele-specific forward primers allow detection of single and double BRAF mutations. A. Schematic diagrams of primer design. Forward primers were chosen to specifically amplify BRAF V600E1 (c.1799T > A), V600E2 (c.1799_1800delTGinsAA) and V600K (c.1798_1799delGTinsAA) mutations. Detection of BRAF V600K mutation can be performed separately or together with BRAF V600E mutation detection. WT = wild-type. B and C. Detection of low abundant BRAF V600E1 (B) and V600K mutations (C) in diluted DNA reference standards, which allelic frequencies were measured by droplet digital PCR. DNA amplification is monitored at each cycle of PCR using SYBR Green reagent included in PCR premix. D and E. Detection of BRAF V600E2 mutation (D) and complex tandem mutation caused by nucleotide changes in codons 600 and 601 (BRAF V600E1 and K601E mutations) (E). Synthesized oligonucleotides ( S1 Table ) were diluted and used as PCR templates.

    Techniques Used: Mutagenesis, Digital PCR, Amplification, Polymerase Chain Reaction, SYBR Green Assay, Synthesized

    2) Product Images from "Analysis of a wild mouse promoter variant reveals a novel role for Fc?RIIb in the control of the germinal center and autoimmunity"

    Article Title: Analysis of a wild mouse promoter variant reveals a novel role for Fc?RIIb in the control of the germinal center and autoimmunity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20121752

    The reduced transcriptional activity of the haplotype I Fcgr2b promoter is caused by three single nucleotide substitutions leading to defective AP-1 binding. (A) The transcriptional activity of the WT, KI, NZB, and NZW promoter of Fcgr2b was determined in the Bal17 B cell line stimulated with LPS for 48 h. WT versus: KI, P = 0.015; NZW, P = 0.037; and NZB, P = 0.034. (B) The transcriptional activity of the WT promoter mutated at the indicated positions was determined as in A. WT versus: KI/NZB, P = 0.02; WT GG −1/+2 AA, P = 0.1; WT GG −1/+2 AA/T −161 C, P = 0.38; WT GG −1/+2 AA/G −79 C, P = 0.0025; WT GG −1/+2 AA/C −59 T, P = 0.88; WT GG −1/+2 AA/A −19 C, P = 0.5; and WT G −79 C, P = 0.3. (C) The transcriptional activity of the KI/NZB promoter mutated at the indicated positions was determined as in A. WT versus: KI/NZB, P = 0.001; KI/NZB AA −1/+2 GG/C −79 G, P = 0.07; KI/NZB C −79 G, P = 0.02; and KI/NZB AA −1/+2 GG, P = 0.94. KI/NZB versus: KI/NZB AA −1/+2 GG/C −79 G, P = 0.03; KI/NZB C −79 G, P = 0.06; and KI/NZB AA −1/+2 GG, P = 0.001. (B and C) The dashed lines represent the luciferase activity of the Fcgr2b KI promoter. (D) Bal17 cells were stimulated for 24 h with anti-Ig or LPS before ChIP with anti–c-Fos or –c-Jun or an isotype control. The region of the Fcgr2b promoter encompassing position −79 was amplified by PCR from input and coimmunoprecipitated DNA. (E) WT and FcγRIIb wild/H1 KI splenic CD19 + B cells were stimulated for 8 h with anti-Ig or LPS and processed as in D. The region of the Fcgr2b promoter encompassing position −79 was amplified by SYBR green quantitative PCR, and the ΔCT of isotype and c-Fos or c-Jun was plotted for each condition. In all panels, error bars represent SEM, and p-values were determined using the Mann–Whitney two-tailed test with a risk of 5%.
    Figure Legend Snippet: The reduced transcriptional activity of the haplotype I Fcgr2b promoter is caused by three single nucleotide substitutions leading to defective AP-1 binding. (A) The transcriptional activity of the WT, KI, NZB, and NZW promoter of Fcgr2b was determined in the Bal17 B cell line stimulated with LPS for 48 h. WT versus: KI, P = 0.015; NZW, P = 0.037; and NZB, P = 0.034. (B) The transcriptional activity of the WT promoter mutated at the indicated positions was determined as in A. WT versus: KI/NZB, P = 0.02; WT GG −1/+2 AA, P = 0.1; WT GG −1/+2 AA/T −161 C, P = 0.38; WT GG −1/+2 AA/G −79 C, P = 0.0025; WT GG −1/+2 AA/C −59 T, P = 0.88; WT GG −1/+2 AA/A −19 C, P = 0.5; and WT G −79 C, P = 0.3. (C) The transcriptional activity of the KI/NZB promoter mutated at the indicated positions was determined as in A. WT versus: KI/NZB, P = 0.001; KI/NZB AA −1/+2 GG/C −79 G, P = 0.07; KI/NZB C −79 G, P = 0.02; and KI/NZB AA −1/+2 GG, P = 0.94. KI/NZB versus: KI/NZB AA −1/+2 GG/C −79 G, P = 0.03; KI/NZB C −79 G, P = 0.06; and KI/NZB AA −1/+2 GG, P = 0.001. (B and C) The dashed lines represent the luciferase activity of the Fcgr2b KI promoter. (D) Bal17 cells were stimulated for 24 h with anti-Ig or LPS before ChIP with anti–c-Fos or –c-Jun or an isotype control. The region of the Fcgr2b promoter encompassing position −79 was amplified by PCR from input and coimmunoprecipitated DNA. (E) WT and FcγRIIb wild/H1 KI splenic CD19 + B cells were stimulated for 8 h with anti-Ig or LPS and processed as in D. The region of the Fcgr2b promoter encompassing position −79 was amplified by SYBR green quantitative PCR, and the ΔCT of isotype and c-Fos or c-Jun was plotted for each condition. In all panels, error bars represent SEM, and p-values were determined using the Mann–Whitney two-tailed test with a risk of 5%.

    Techniques Used: Activity Assay, Binding Assay, Luciferase, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, SYBR Green Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Two Tailed Test

    3) Product Images from "An ectopic CTCF binding element inhibits Tcrd rearrangement by limiting contact between Vδ and Dδ gene segments rearrangement by limiting contact between Vδ and Dδ gene segments"

    Article Title: An ectopic CTCF binding element inhibits Tcrd rearrangement by limiting contact between Vδ and Dδ gene segments rearrangement by limiting contact between Vδ and Dδ gene segments

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

    doi: 10.4049/jimmunol.1601124

    Tcrd rearrangement is perturbed in CBE KI mice ( A ) Locus map identifying Vδ and Vα gene segments analyzed. Trav12, Trav13 and Trav14 are V segment families whose members are distributed across the region indicated. ( B ) V-( Trdd1 )- Trdd2, Trdj1 rearrangements in genomic DNA of DN3 thymocytes sorted from WT and CBE KI mice, analyzed by SYBR-Green (left panel) or Taqman (right panel) qPCR. Data were normalized to Cd14 and represent the mean ± SEM of 3 WT and 3 CBE KI samples, with each sample representing a pool of 3 mice. Statistical significance was determined by unpaired Student’s t -test with Holm-Sidak correction for multiple comparisons. ( C ) RSS DSBs in genomic DNA of DN3 thymocytes sorted from WT and CBE KI mice, analyzed by LM-qPCR. Embryonic stem (ES) cells served as a negative control. LM-qPCR signals were normalized to those for standard Cd14 genomic PCR. Data represent the mean ± SEM, with each symbol representing an individual mouse. Statistical significance was determined by unpaired Student’s t -test with Holm-Sidak correction for multiple comparisons.
    Figure Legend Snippet: Tcrd rearrangement is perturbed in CBE KI mice ( A ) Locus map identifying Vδ and Vα gene segments analyzed. Trav12, Trav13 and Trav14 are V segment families whose members are distributed across the region indicated. ( B ) V-( Trdd1 )- Trdd2, Trdj1 rearrangements in genomic DNA of DN3 thymocytes sorted from WT and CBE KI mice, analyzed by SYBR-Green (left panel) or Taqman (right panel) qPCR. Data were normalized to Cd14 and represent the mean ± SEM of 3 WT and 3 CBE KI samples, with each sample representing a pool of 3 mice. Statistical significance was determined by unpaired Student’s t -test with Holm-Sidak correction for multiple comparisons. ( C ) RSS DSBs in genomic DNA of DN3 thymocytes sorted from WT and CBE KI mice, analyzed by LM-qPCR. Embryonic stem (ES) cells served as a negative control. LM-qPCR signals were normalized to those for standard Cd14 genomic PCR. Data represent the mean ± SEM, with each symbol representing an individual mouse. Statistical significance was determined by unpaired Student’s t -test with Holm-Sidak correction for multiple comparisons.

    Techniques Used: Mouse Assay, SYBR Green Assay, Real-time Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers
    Article Snippet: .. To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA). .. There was no difference in amplification efficiency (P -value = 0.69) for the five assays tested (let-7d, miR-20a, miR-21, miR-26a and miR-150) between the two master mixes and all the assays gave one peak in melting curve analysis and were comparable over eight log10 of template concentration (Figure ).

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    Article Snippet: .. Quantitative real-time PCR analysis was performed by following SYBR Green (QuantiFastTM SYBR Green PCR kit, QIAGEN) chemistry using the ABI PRISM 7500 Fast Realtime Platform (Applied Biosystems). .. Target genes were amplified by a two-step PCR reaction with an initial denaturation at 95 °C for 5min followed by 40 cycles of 95 °C for 30s and annealing/extension at 60 °C for 30s.

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines
    Article Snippet: .. The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche). .. U3 snoRNA and/or GAPDH mRNA was used as a normalisation control for RNA expression levels in all experiments.

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    Article Snippet: .. Quantitative PCR Quantitative PCR of biological samples was done in 10 μl total volume with 1 μl of cDNA diluted 8-10 times, 5 μl of 2x QuantiFast SYBR Green PCR master mix (Qiagen, Germany), 250 nM of each primer (Table ) or 2 μl microRNA LNATM primer sets (Exiqon, Denmark). .. Standard curves with 10-fold dilutions (made with a pool of equal amounts of cDNA from the 40 uterus samples) were made for all assays to calculate qPCR efficiency.

    Amplification:

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers
    Article Snippet: .. To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA). .. There was no difference in amplification efficiency (P -value = 0.69) for the five assays tested (let-7d, miR-20a, miR-21, miR-26a and miR-150) between the two master mixes and all the assays gave one peak in melting curve analysis and were comparable over eight log10 of template concentration (Figure ).

    Mutagenesis:

    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues
    Article Snippet: .. Briefly, we used reaction mixture comprising 7.5μl of QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen, Germany), the total of 400 nM of BRAF mutant-specific primers, and 12–48 ng of genomic DNA for a 15-μl reaction. .. We performed touchdown PCR with SYBR Green I fluorescence monitoring on a Rotor-Gene Q (Qiagen, Germany).

    Quantitative RT-PCR:

    Article Title: Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR
    Article Snippet: .. SYBR Green-based in real time for SARS-Cov-2 diagnosisThe SYBR Green RT-qPCR assay (one step) was performed, according to the manufacturer’s recommendations, with the QuantiFast SYBR® Green RT-PCR kit (QIAGEN, Hilden, Germany). .. Briefly, 8 μL of target RNA and 1 μL [10pM/μL] from each primer (E-Sarbeco F1 and R2) [ ] were added to the 12.5 μL 2x Master Mix QuantiFast SYBR Green RT-PCR, 0.25 μL of enzyme RT-Mix and Ultra-Pure Water (Thermo Fisher Scientific, Waltham, MA, USA) to complete 25 μL of reaction.

    Article Title: Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy
    Article Snippet: .. RT-qPCR design RT-qPCRs assays were carried out in a Light Cycler 480 II PCR platform (Roche Diagnostics, Penzberg, Germany), using QuantiFast SYBR Green RT-qPCR Kit (Qiagen™ Hilden Germany). ..

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines
    Article Snippet: .. The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche). .. U3 snoRNA and/or GAPDH mRNA was used as a normalisation control for RNA expression levels in all experiments.

    SYBR Green Assay:

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers
    Article Snippet: .. To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA). .. There was no difference in amplification efficiency (P -value = 0.69) for the five assays tested (let-7d, miR-20a, miR-21, miR-26a and miR-150) between the two master mixes and all the assays gave one peak in melting curve analysis and were comparable over eight log10 of template concentration (Figure ).

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    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues
    Article Snippet: .. Briefly, we used reaction mixture comprising 7.5μl of QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen, Germany), the total of 400 nM of BRAF mutant-specific primers, and 12–48 ng of genomic DNA for a 15-μl reaction. .. We performed touchdown PCR with SYBR Green I fluorescence monitoring on a Rotor-Gene Q (Qiagen, Germany).

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    Polymerase Chain Reaction:

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers
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    Article Title: Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy
    Article Snippet: .. RT-qPCR design RT-qPCRs assays were carried out in a Light Cycler 480 II PCR platform (Roche Diagnostics, Penzberg, Germany), using QuantiFast SYBR Green RT-qPCR Kit (Qiagen™ Hilden Germany). ..

    Article Title: Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription
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    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR
    Article Snippet: .. SYBR Green-based in real time for SARS-Cov-2 diagnosisThe SYBR Green RT-qPCR assay (one step) was performed, according to the manufacturer’s recommendations, with the QuantiFast SYBR® Green RT-PCR kit (QIAGEN, Hilden, Germany). .. Briefly, 8 μL of target RNA and 1 μL [10pM/μL] from each primer (E-Sarbeco F1 and R2) [ ] were added to the 12.5 μL 2x Master Mix QuantiFast SYBR Green RT-PCR, 0.25 μL of enzyme RT-Mix and Ultra-Pure Water (Thermo Fisher Scientific, Waltham, MA, USA) to complete 25 μL of reaction.

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines
    Article Snippet: .. The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche). .. U3 snoRNA and/or GAPDH mRNA was used as a normalisation control for RNA expression levels in all experiments.

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    Qiagen quantifast sybr green rt pcr kit
    Compensatory analysis of miR21 knock-down targeted to miR21 primary transcript by snoMEN vector. ( a ) Micrographs showing rescue experiment of apoptosis induction by co-transfecting with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (GFP+miR21)/Control plasmid that expresses GFP protein alone (GFP). Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( b ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after co-transfection with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (miR21)/Control plasmid that express GFP protein alone (GFP) and <t>qRT-PCR</t> was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21, Red). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta <t>SYBR</t> Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21, Green). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 3 independent experiments. ( c ) Micrographs show prevention of non-apoptosis induction by transfecting with pri-miR21-snoMEN mut, which has 3 base mismatches in each M box complementary sequence to pri-miR21. Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( d ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection with either pri-miR21-snoMEN mut or Control. Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments.
    Quantifast Sybr Green Rt Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantifast sybr green rt pcr kit/product/Qiagen
    Average 99 stars, based on 1169 article reviews
    Price from $9.99 to $1999.99
    quantifast sybr green rt pcr kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

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    Compensatory analysis of miR21 knock-down targeted to miR21 primary transcript by snoMEN vector. ( a ) Micrographs showing rescue experiment of apoptosis induction by co-transfecting with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (GFP+miR21)/Control plasmid that expresses GFP protein alone (GFP). Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( b ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after co-transfection with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (miR21)/Control plasmid that express GFP protein alone (GFP) and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21, Red). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21, Green). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 3 independent experiments. ( c ) Micrographs show prevention of non-apoptosis induction by transfecting with pri-miR21-snoMEN mut, which has 3 base mismatches in each M box complementary sequence to pri-miR21. Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( d ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection with either pri-miR21-snoMEN mut or Control. Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments.

    Journal: PLoS ONE

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines

    doi: 10.1371/journal.pone.0138668

    Figure Lengend Snippet: Compensatory analysis of miR21 knock-down targeted to miR21 primary transcript by snoMEN vector. ( a ) Micrographs showing rescue experiment of apoptosis induction by co-transfecting with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (GFP+miR21)/Control plasmid that expresses GFP protein alone (GFP). Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( b ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after co-transfection with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (miR21)/Control plasmid that express GFP protein alone (GFP) and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21, Red). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21, Green). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 3 independent experiments. ( c ) Micrographs show prevention of non-apoptosis induction by transfecting with pri-miR21-snoMEN mut, which has 3 base mismatches in each M box complementary sequence to pri-miR21. Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( d ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection with either pri-miR21-snoMEN mut or Control. Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments.

    Article Snippet: The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche).

    Techniques: Plasmid Preparation, Expressing, Transfection, Cotransfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation, Sequencing

    miR21 knock-down using si/shRNA targeted to endogenous miR21 primary transcript. ( a ) The regions on the pri-miR21 RNA targeted by the snoMEN vector used in this study are shown in a schematic diagram (sh/si21M1-M3). The same pri-miR21 sequences as targeted by the snoMEN vector were targeted by siRNA oligoribonucleotides and shRNA expression plasmids. ( b ) The graph shows the result of qRT-PCR/qPCR. Total RNA from HeLa cells was harvested 24 hours after transfection and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21, Red). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21, Green). U3 was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments. The right panel shows the result of Northern blot analysis for siRNA experiments. Detection of endogenous miR21 RNA levels following transfection of HeLa cells using either control siRNA (Scramble: lane1), miR21 siRNA (si21: lane2), miR21 M box siRNA-1 (si21M1: lane3), miR21 M box siRNA-2 (si21M2: lane4), miR21 M box siRNA-3 (si21M3: lane5). An equivalent amount of HeLa total RNA was loaded for each lane and the RNA separated by PAGE, electroblotted onto membrane and probed both with a miR21 probe and with a tRNA probe as a loading control. ( c ) The same series of experiments with siRNA transfection, as described in Fig 6b , except shRNA expression plasmids were transfected instead of siRNAs.

    Journal: PLoS ONE

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines

    doi: 10.1371/journal.pone.0138668

    Figure Lengend Snippet: miR21 knock-down using si/shRNA targeted to endogenous miR21 primary transcript. ( a ) The regions on the pri-miR21 RNA targeted by the snoMEN vector used in this study are shown in a schematic diagram (sh/si21M1-M3). The same pri-miR21 sequences as targeted by the snoMEN vector were targeted by siRNA oligoribonucleotides and shRNA expression plasmids. ( b ) The graph shows the result of qRT-PCR/qPCR. Total RNA from HeLa cells was harvested 24 hours after transfection and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21, Red). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21, Green). U3 was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments. The right panel shows the result of Northern blot analysis for siRNA experiments. Detection of endogenous miR21 RNA levels following transfection of HeLa cells using either control siRNA (Scramble: lane1), miR21 siRNA (si21: lane2), miR21 M box siRNA-1 (si21M1: lane3), miR21 M box siRNA-2 (si21M2: lane4), miR21 M box siRNA-3 (si21M3: lane5). An equivalent amount of HeLa total RNA was loaded for each lane and the RNA separated by PAGE, electroblotted onto membrane and probed both with a miR21 probe and with a tRNA probe as a loading control. ( c ) The same series of experiments with siRNA transfection, as described in Fig 6b , except shRNA expression plasmids were transfected instead of siRNAs.

    Article Snippet: The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche).

    Techniques: shRNA, Plasmid Preparation, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Transfection, SYBR Green Assay, Standard Deviation, Northern Blot, Polyacrylamide Gel Electrophoresis

    snoMEN vector targeted to miR21 primary transcript. ( a ) Structure for targeted endogenous miR21 primary transcript (mCherry–pri-miR21 snoMEN) and schematic diagram of miR21 maturation pathway. This construct has three snoMEN RNAs (blue pentagons) as previously described[ 1 ], except that here the M box sequences are complementary to specific sequences within the endogenous miR21 primary transcript. ( b ) Validation of snoMEN expression by Fluorescence In Situ Hybridisation (FISH) analysis. Each snoMEN RNA was detected by using a M box specific RNA probe labelled with Cy3 (Cy3). DNA is stained by DAPI (DAPI). Scale bar is 10 μm. ( c ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21). U3 was used as a loading control. Graph depicts mean and standard deviation from a minimum of 5 independent experiments. ( d ) HeLa cells transfected with pri-miR21-snoMEN/Control for 24 hours prior to total RNA extraction and northern blot analysis.

    Journal: PLoS ONE

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines

    doi: 10.1371/journal.pone.0138668

    Figure Lengend Snippet: snoMEN vector targeted to miR21 primary transcript. ( a ) Structure for targeted endogenous miR21 primary transcript (mCherry–pri-miR21 snoMEN) and schematic diagram of miR21 maturation pathway. This construct has three snoMEN RNAs (blue pentagons) as previously described[ 1 ], except that here the M box sequences are complementary to specific sequences within the endogenous miR21 primary transcript. ( b ) Validation of snoMEN expression by Fluorescence In Situ Hybridisation (FISH) analysis. Each snoMEN RNA was detected by using a M box specific RNA probe labelled with Cy3 (Cy3). DNA is stained by DAPI (DAPI). Scale bar is 10 μm. ( c ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21). U3 was used as a loading control. Graph depicts mean and standard deviation from a minimum of 5 independent experiments. ( d ) HeLa cells transfected with pri-miR21-snoMEN/Control for 24 hours prior to total RNA extraction and northern blot analysis.

    Article Snippet: The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche).

    Techniques: Plasmid Preparation, Construct, Expressing, Fluorescence, In Situ, Hybridization, Fluorescence In Situ Hybridization, Staining, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation, RNA Extraction, Northern Blot

    Ago2 is important for snoMEN RNA interference. ( a ) HeLa cells were transfected with siRNA oligonucleotides for Ago1, Ago2, and Upf1. Whole cell lysates were prepared 48 hours after transfections and analysed by western blot using the indicated antibodies. ( b ) HeLa cells were transfected with siRNA oligonucleotides prior to pri-miR21-snoMEN transfection for 24h. Total RNA from HeLa cells was harvested and qPCR was performed, followed by cDNA synthesis, using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ). ( c ) HeLa cells transfected with pri-miR21-snoMEN for 24 hours prior to protein extraction. The same amount of proteins/RNAs from each subcellular fraction was loaded on each lane and analysed by western blot/Northern blot using the indicated antibodies (Ago1, Ago2, Upf1) / RI-labelled probes (snoMEN, tRNA). The fractionation markers, i.e. Tubulin (Cytoplasm), Lamin A/C (Nucleoplasm), Fibrillarin (Nucleoli), were tested to evaluate fractionation quality. ( d ) Images show localisation pattern of DNA (DAPI, Blue), a transfection FP-marker of pri-miR21-snoMEN (mCherry, Red) and the indicated proteins, i.e. Ago1, Ago2 and Upf1 (FITC, Green). Scale bar is 10 μm. Arrows and arrowheads show transfected and non-transfected cells, respectively. Specificity of each antibody was also verified by siRNA transfections ( Fig K in S1 File ). ( e ) HeLa cells were transfected with pri-miR21-snoMEN for 24 hours prior to fractionation. Cell lysates of each subcellular fraction were prepared and immunoprecipitated with either normal mouse IgG, or anti-Ago2 antibodies. Total protein was isolated from precipitates and resolved by SDS-PAGE and then analysed by western blotting using an Ago2 antibody to validate the efficiency of purification. In parallel, total RNA was also isolated from each precipitate and qRT-PCR was performed using the specific primers indicated.

    Journal: PLoS ONE

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines

    doi: 10.1371/journal.pone.0138668

    Figure Lengend Snippet: Ago2 is important for snoMEN RNA interference. ( a ) HeLa cells were transfected with siRNA oligonucleotides for Ago1, Ago2, and Upf1. Whole cell lysates were prepared 48 hours after transfections and analysed by western blot using the indicated antibodies. ( b ) HeLa cells were transfected with siRNA oligonucleotides prior to pri-miR21-snoMEN transfection for 24h. Total RNA from HeLa cells was harvested and qPCR was performed, followed by cDNA synthesis, using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ). ( c ) HeLa cells transfected with pri-miR21-snoMEN for 24 hours prior to protein extraction. The same amount of proteins/RNAs from each subcellular fraction was loaded on each lane and analysed by western blot/Northern blot using the indicated antibodies (Ago1, Ago2, Upf1) / RI-labelled probes (snoMEN, tRNA). The fractionation markers, i.e. Tubulin (Cytoplasm), Lamin A/C (Nucleoplasm), Fibrillarin (Nucleoli), were tested to evaluate fractionation quality. ( d ) Images show localisation pattern of DNA (DAPI, Blue), a transfection FP-marker of pri-miR21-snoMEN (mCherry, Red) and the indicated proteins, i.e. Ago1, Ago2 and Upf1 (FITC, Green). Scale bar is 10 μm. Arrows and arrowheads show transfected and non-transfected cells, respectively. Specificity of each antibody was also verified by siRNA transfections ( Fig K in S1 File ). ( e ) HeLa cells were transfected with pri-miR21-snoMEN for 24 hours prior to fractionation. Cell lysates of each subcellular fraction were prepared and immunoprecipitated with either normal mouse IgG, or anti-Ago2 antibodies. Total protein was isolated from precipitates and resolved by SDS-PAGE and then analysed by western blotting using an Ago2 antibody to validate the efficiency of purification. In parallel, total RNA was also isolated from each precipitate and qRT-PCR was performed using the specific primers indicated.

    Article Snippet: The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche).

    Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay, Protein Extraction, Northern Blot, Fractionation, Marker, Immunoprecipitation, Isolation, SDS Page, Purification, Quantitative RT-PCR

    Primer distribution in a 96-well PCR plate. All the wells contain a QuantiFast SYBR Green RT-qPCR Master Mix. Additionally, the wells contain the primers for DENV ( blue ), WNV ( violet ), Rickettsia spp. ( red ) and Leptospira spp. ( green ). Numbers represent the sample that must be added to each set of four wells. The plate includes a set of four wells for negative control [ light-colored wells with a “(−)” sign] and another for the Universal Positive Control ( dark-colored wells with “UPC”)

    Journal: SpringerPlus

    Article Title: Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy

    doi: 10.1186/s40064-016-2318-y

    Figure Lengend Snippet: Primer distribution in a 96-well PCR plate. All the wells contain a QuantiFast SYBR Green RT-qPCR Master Mix. Additionally, the wells contain the primers for DENV ( blue ), WNV ( violet ), Rickettsia spp. ( red ) and Leptospira spp. ( green ). Numbers represent the sample that must be added to each set of four wells. The plate includes a set of four wells for negative control [ light-colored wells with a “(−)” sign] and another for the Universal Positive Control ( dark-colored wells with “UPC”)

    Article Snippet: RT-qPCR design RT-qPCRs assays were carried out in a Light Cycler 480 II PCR platform (Roche Diagnostics, Penzberg, Germany), using QuantiFast SYBR Green RT-qPCR Kit (Qiagen™ Hilden Germany).

    Techniques: Polymerase Chain Reaction, SYBR Green Assay, Quantitative RT-PCR, Negative Control, Positive Control

    Comparison of Ct values obtained by TaqMan, SYBR Green, and one-step/two-step RT-PCR assays. a Ct values of 63 clinical samples detected with TaqMan and SYBR Green RT-qPCR. b , c Agarose gels of amplified fragments by one-step RT-PCR and two-step PCR assays, respectively, of 14 representatives’ clinical samples. The 600-bp marker in the ladder (LD) is indicated in the picture. The arrow points to the fragment of 113 base pairs (bp) amplified with SARS-CoV-2-specific E gene primers

    Journal: Brazilian Journal of Microbiology

    Article Title: Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR

    doi: 10.1007/s42770-020-00347-5

    Figure Lengend Snippet: Comparison of Ct values obtained by TaqMan, SYBR Green, and one-step/two-step RT-PCR assays. a Ct values of 63 clinical samples detected with TaqMan and SYBR Green RT-qPCR. b , c Agarose gels of amplified fragments by one-step RT-PCR and two-step PCR assays, respectively, of 14 representatives’ clinical samples. The 600-bp marker in the ladder (LD) is indicated in the picture. The arrow points to the fragment of 113 base pairs (bp) amplified with SARS-CoV-2-specific E gene primers

    Article Snippet: SYBR Green-based in real time for SARS-Cov-2 diagnosisThe SYBR Green RT-qPCR assay (one step) was performed, according to the manufacturer’s recommendations, with the QuantiFast SYBR® Green RT-PCR kit (QIAGEN, Hilden, Germany).

    Techniques: SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Polymerase Chain Reaction, Marker

    Allele-specific forward primers allow detection of single and double BRAF mutations. A. Schematic diagrams of primer design. Forward primers were chosen to specifically amplify BRAF V600E1 (c.1799T > A), V600E2 (c.1799_1800delTGinsAA) and V600K (c.1798_1799delGTinsAA) mutations. Detection of BRAF V600K mutation can be performed separately or together with BRAF V600E mutation detection. WT = wild-type. B and C. Detection of low abundant BRAF V600E1 (B) and V600K mutations (C) in diluted DNA reference standards, which allelic frequencies were measured by droplet digital PCR. DNA amplification is monitored at each cycle of PCR using SYBR Green reagent included in PCR premix. D and E. Detection of BRAF V600E2 mutation (D) and complex tandem mutation caused by nucleotide changes in codons 600 and 601 (BRAF V600E1 and K601E mutations) (E). Synthesized oligonucleotides ( S1 Table ) were diluted and used as PCR templates.

    Journal: PLoS ONE

    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

    doi: 10.1371/journal.pone.0198795

    Figure Lengend Snippet: Allele-specific forward primers allow detection of single and double BRAF mutations. A. Schematic diagrams of primer design. Forward primers were chosen to specifically amplify BRAF V600E1 (c.1799T > A), V600E2 (c.1799_1800delTGinsAA) and V600K (c.1798_1799delGTinsAA) mutations. Detection of BRAF V600K mutation can be performed separately or together with BRAF V600E mutation detection. WT = wild-type. B and C. Detection of low abundant BRAF V600E1 (B) and V600K mutations (C) in diluted DNA reference standards, which allelic frequencies were measured by droplet digital PCR. DNA amplification is monitored at each cycle of PCR using SYBR Green reagent included in PCR premix. D and E. Detection of BRAF V600E2 mutation (D) and complex tandem mutation caused by nucleotide changes in codons 600 and 601 (BRAF V600E1 and K601E mutations) (E). Synthesized oligonucleotides ( S1 Table ) were diluted and used as PCR templates.

    Article Snippet: Briefly, we used reaction mixture comprising 7.5μl of QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen, Germany), the total of 400 nM of BRAF mutant-specific primers, and 12–48 ng of genomic DNA for a 15-μl reaction.

    Techniques: Mutagenesis, Digital PCR, Amplification, Polymerase Chain Reaction, SYBR Green Assay, Synthesized