taqman rna to ct 1 step kit  (Thermo Fisher)


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    Name:
    TaqMan RNA to CT 1 Step Kit
    Description:
    The Applied Biosystems TaqMan RNA to CT 1 Step Kit delivers consistent RNA target quantification for a wide variety of assays and is validated with Applied Biosystems comprehensive library of TaqMan Gene Expression assays The TaqMan RNA to CT 1 Step Kit is recommended for a variety of applications including general gene expression studies biomarker discovery and microarray validation • Accurate across a wide dynamic range for dependable performance • High sensitivity enabling low copy number detection and precise quantification • Validated with TaqMan Gene Expression Assays for easy experimental setup• Consistent performance with a wide variety of targets including AT rich GC rich and long sequences• Fewer pipetting steps means less hands on time and less chance for errorsEfficiency across a Broad Range of Input Concentrations Maximum flexibility in RNA template input quantity requires optimal real time PCR efficiency across a broad range of template quantities Figure 1 shows the excellent amplification efficiency across six orders of magnitude The result is comparable to that of the TaqMan RNA to CT 2 Step Kit and is a significant improvement in dynamic range and sensitivity over the TaqMan One Step RT PCR Master Mix Reagents High Sensitivity at Low Target Concentration The sensitivity of TaqMan RNA to CT 1 Step Kit was validated using a synthetic RNA template for which copy number is precisely known Significant sampling error occurs when measuring low quantities of target so statistical analysis is required for proper evaluation using multiple replicates Figure 2 shows the expected quantity of target and corresponding mean CT values Statistical analysis indicates high confidence of sample quantification based on a T test Table I consistent with as few as 10 copies of target
    Catalog Number:
    4392653
    Price:
    None
    Applications:
    Enzymes & Master Mixes for Real-Time PCR|One-Step qRT-PCR|PCR & Real-Time PCR|Real Time PCR (qPCR)|Real Time PCR-Based Gene Expression Profiling|Gene Expression Analysis & Genotyping
    Category:
    Kits and Assays
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    Structured Review

    Thermo Fisher taqman rna to ct 1 step kit
    Pathogenic Yersinia requires c-KIT activity for suppression of transcription factor and pro-inflammatory cytokine expression. (A-B) Analysis of signal transduction pathways in Y. pestis -infected THP1 cells in absence of c-KIT. THP1 cells untreated or pre-treated with 1μM OSI-930 for 18 h were infected with Y. pestis Ind195 at MOI 20 for 1h. <t>RNA</t> was isolated, converted to cDNA, and applied to a RT Profiler PCR Array for human signal transduction pathway expression analysis. Dot plots compare gene expression profiles between uninfected THP-1 cells and (A) Y. pestis Ind195-infected THP-1 cells or ( B ) OSI-930-pretreated, Y. pestis Ind195 infected THP-1 cells. Genes that do not exhibit significant expression changes between the control and experimental samples are concentrated between the two black lines. The black lines define the assay cut-off of 3-fold induction or 70% reduction of transcript levels. Genes of interest are highlighted in black. (C) Inhibition of c-KIT recovers EGR1, chemokine, and cell adhesion transcript levels in pathogenic Yersinia -infected THP1 cells. THP1 cells were pre-treated with 1μM OSI-930 for 18 h or were left untreated prior to infection with Y. pestis Ind195 at MOI 10 for 1 h. EGR1, VCAM1, CCL20, and IL-8 mRNA levels were determined by <t>Taqman</t> qPCR using total RNA isolated 24 h post-infection. Depicted RNA levels are relative to untreated THP1 control samples and were calculated using the 2 -ΔΔCt formula. A ‘*” denotes that relative RNA levels were significantly different (p
    The Applied Biosystems TaqMan RNA to CT 1 Step Kit delivers consistent RNA target quantification for a wide variety of assays and is validated with Applied Biosystems comprehensive library of TaqMan Gene Expression assays The TaqMan RNA to CT 1 Step Kit is recommended for a variety of applications including general gene expression studies biomarker discovery and microarray validation • Accurate across a wide dynamic range for dependable performance • High sensitivity enabling low copy number detection and precise quantification • Validated with TaqMan Gene Expression Assays for easy experimental setup• Consistent performance with a wide variety of targets including AT rich GC rich and long sequences• Fewer pipetting steps means less hands on time and less chance for errorsEfficiency across a Broad Range of Input Concentrations Maximum flexibility in RNA template input quantity requires optimal real time PCR efficiency across a broad range of template quantities Figure 1 shows the excellent amplification efficiency across six orders of magnitude The result is comparable to that of the TaqMan RNA to CT 2 Step Kit and is a significant improvement in dynamic range and sensitivity over the TaqMan One Step RT PCR Master Mix Reagents High Sensitivity at Low Target Concentration The sensitivity of TaqMan RNA to CT 1 Step Kit was validated using a synthetic RNA template for which copy number is precisely known Significant sampling error occurs when measuring low quantities of target so statistical analysis is required for proper evaluation using multiple replicates Figure 2 shows the expected quantity of target and corresponding mean CT values Statistical analysis indicates high confidence of sample quantification based on a T test Table I consistent with as few as 10 copies of target
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    Images

    1) Product Images from "c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response"

    Article Title: c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-13-249

    Pathogenic Yersinia requires c-KIT activity for suppression of transcription factor and pro-inflammatory cytokine expression. (A-B) Analysis of signal transduction pathways in Y. pestis -infected THP1 cells in absence of c-KIT. THP1 cells untreated or pre-treated with 1μM OSI-930 for 18 h were infected with Y. pestis Ind195 at MOI 20 for 1h. RNA was isolated, converted to cDNA, and applied to a RT Profiler PCR Array for human signal transduction pathway expression analysis. Dot plots compare gene expression profiles between uninfected THP-1 cells and (A) Y. pestis Ind195-infected THP-1 cells or ( B ) OSI-930-pretreated, Y. pestis Ind195 infected THP-1 cells. Genes that do not exhibit significant expression changes between the control and experimental samples are concentrated between the two black lines. The black lines define the assay cut-off of 3-fold induction or 70% reduction of transcript levels. Genes of interest are highlighted in black. (C) Inhibition of c-KIT recovers EGR1, chemokine, and cell adhesion transcript levels in pathogenic Yersinia -infected THP1 cells. THP1 cells were pre-treated with 1μM OSI-930 for 18 h or were left untreated prior to infection with Y. pestis Ind195 at MOI 10 for 1 h. EGR1, VCAM1, CCL20, and IL-8 mRNA levels were determined by Taqman qPCR using total RNA isolated 24 h post-infection. Depicted RNA levels are relative to untreated THP1 control samples and were calculated using the 2 -ΔΔCt formula. A ‘*” denotes that relative RNA levels were significantly different (p
    Figure Legend Snippet: Pathogenic Yersinia requires c-KIT activity for suppression of transcription factor and pro-inflammatory cytokine expression. (A-B) Analysis of signal transduction pathways in Y. pestis -infected THP1 cells in absence of c-KIT. THP1 cells untreated or pre-treated with 1μM OSI-930 for 18 h were infected with Y. pestis Ind195 at MOI 20 for 1h. RNA was isolated, converted to cDNA, and applied to a RT Profiler PCR Array for human signal transduction pathway expression analysis. Dot plots compare gene expression profiles between uninfected THP-1 cells and (A) Y. pestis Ind195-infected THP-1 cells or ( B ) OSI-930-pretreated, Y. pestis Ind195 infected THP-1 cells. Genes that do not exhibit significant expression changes between the control and experimental samples are concentrated between the two black lines. The black lines define the assay cut-off of 3-fold induction or 70% reduction of transcript levels. Genes of interest are highlighted in black. (C) Inhibition of c-KIT recovers EGR1, chemokine, and cell adhesion transcript levels in pathogenic Yersinia -infected THP1 cells. THP1 cells were pre-treated with 1μM OSI-930 for 18 h or were left untreated prior to infection with Y. pestis Ind195 at MOI 10 for 1 h. EGR1, VCAM1, CCL20, and IL-8 mRNA levels were determined by Taqman qPCR using total RNA isolated 24 h post-infection. Depicted RNA levels are relative to untreated THP1 control samples and were calculated using the 2 -ΔΔCt formula. A ‘*” denotes that relative RNA levels were significantly different (p

    Techniques Used: Activity Assay, Expressing, Transduction, Infection, Isolation, Polymerase Chain Reaction, Inhibition, Real-time Polymerase Chain Reaction

    2) Product Images from "Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay"

    Article Title: Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay

    Journal: Viruses

    doi: 10.3390/v10120714

    Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of capsid , NS1 , NS3 , and NS5 synthetic target RNA were amplified either individually (panels A – D , respectively) or as a mixture (panel E ) using multiplex LAMP-4GO assays containing LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10 5 copies of its target RNA) traces. The x -axis depicts the duration of endpoint signal measurement. ( F ) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. ( G ) Detection of Asian and African lineage ZIKV genomic RNA using TaqMan qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel F were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. ( H ) Detection limit of degenerate multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The x -axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.
    Figure Legend Snippet: Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of capsid , NS1 , NS3 , and NS5 synthetic target RNA were amplified either individually (panels A – D , respectively) or as a mixture (panel E ) using multiplex LAMP-4GO assays containing LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10 5 copies of its target RNA) traces. The x -axis depicts the duration of endpoint signal measurement. ( F ) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. ( G ) Detection of Asian and African lineage ZIKV genomic RNA using TaqMan qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel F were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. ( H ) Detection limit of degenerate multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The x -axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.

    Techniques Used: Multiplex Assay, Amplification, Fluorescence, Quantitative RT-PCR

    3) Product Images from "MicroRNA Controlled Adenovirus Mediates Anti-Cancer Efficacy without Affecting Endogenous MicroRNA Activity"

    Article Title: MicroRNA Controlled Adenovirus Mediates Anti-Cancer Efficacy without Affecting Endogenous MicroRNA Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016152

    The level and activity of mature mir122 in vivo is unaffected by Ad5mir122. Mice were injected with 2×10 10 vp of either Ad5mir122, Ad5WT, an E1A deleted Ad5luc vector or PBS. Quantification of mir122 mature RNA levels was performed using a Taqman microRNA assay specific for mir122. CT values were corrected against the levels of the microRNA, let7a, as a reference gene using the method published by Pfaffl M [25] . A) RT-QPCR for mir122 showing nine superimposed amplification curves (from three mice) in each treatment group, before correction against let7a. Samples were reverse transcribed using equal amounts of total RNA (5 ng) and RT-PCR was performed using equal amounts of cDNA. CT values shown here represent the average of the reactions from three mice plus or minus standard deviation. B) The total number of mRNA changes recorded by genome wide mRNA profiling of extracted murine hepatic RNA. Positive signals are those in which the median mRNA level changed by ≧2-fold from all mice in each group in comparison to median mRNA level in mice treated with PBS (n = 3 for all groups). The total number of genes altered is calculated using the average of the three independent replicates and therefore no error bars are shown. C) Western blot analysis of the mir122 regulated protein Aldolase A in mice treated as above. Liver protein extracts were subjected to a BCA protein assay and equal loading was confirmed by Ponceau stain (data not shown).
    Figure Legend Snippet: The level and activity of mature mir122 in vivo is unaffected by Ad5mir122. Mice were injected with 2×10 10 vp of either Ad5mir122, Ad5WT, an E1A deleted Ad5luc vector or PBS. Quantification of mir122 mature RNA levels was performed using a Taqman microRNA assay specific for mir122. CT values were corrected against the levels of the microRNA, let7a, as a reference gene using the method published by Pfaffl M [25] . A) RT-QPCR for mir122 showing nine superimposed amplification curves (from three mice) in each treatment group, before correction against let7a. Samples were reverse transcribed using equal amounts of total RNA (5 ng) and RT-PCR was performed using equal amounts of cDNA. CT values shown here represent the average of the reactions from three mice plus or minus standard deviation. B) The total number of mRNA changes recorded by genome wide mRNA profiling of extracted murine hepatic RNA. Positive signals are those in which the median mRNA level changed by ≧2-fold from all mice in each group in comparison to median mRNA level in mice treated with PBS (n = 3 for all groups). The total number of genes altered is calculated using the average of the three independent replicates and therefore no error bars are shown. C) Western blot analysis of the mir122 regulated protein Aldolase A in mice treated as above. Liver protein extracts were subjected to a BCA protein assay and equal loading was confirmed by Ponceau stain (data not shown).

    Techniques Used: Activity Assay, In Vivo, Mouse Assay, Injection, Plasmid Preparation, TaqMan microRNA Assay, Quantitative RT-PCR, Amplification, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Genome Wide, Western Blot, Bicinchoninic Acid Protein Assay, Staining

    4) Product Images from "Altered expression of MALAT1 lncRNA in nonalcoholic steatohepatitis Fibrosis Regulates CXCL5 in Hepatic Stellate Cells"

    Article Title: Altered expression of MALAT1 lncRNA in nonalcoholic steatohepatitis Fibrosis Regulates CXCL5 in Hepatic Stellate Cells

    Journal: Translational research : the journal of laboratory and clinical medicine

    doi: 10.1016/j.trsl.2017.09.001

    CXCL5 expression is increased in lobular inflammation and advanced fibrosis Total RNA was extracted from liver tissue as described in the Methods section. Relative quantification of CXCL5 was performed using TaqMan qPCR. Results are shown as inflammation and advanced fibrosis relative to normal histology, levels of which were set to a value of 1. Expression levels were normalized against GAPDH. All experiments were performed in triplicate. Data are expressed as mean ± standard deviation. *p > 0.05.
    Figure Legend Snippet: CXCL5 expression is increased in lobular inflammation and advanced fibrosis Total RNA was extracted from liver tissue as described in the Methods section. Relative quantification of CXCL5 was performed using TaqMan qPCR. Results are shown as inflammation and advanced fibrosis relative to normal histology, levels of which were set to a value of 1. Expression levels were normalized against GAPDH. All experiments were performed in triplicate. Data are expressed as mean ± standard deviation. *p > 0.05.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    Analysis of NEAT1 , MALAT1 , and HULC by RT-qPCR Total RNA was extracted from liver tissue as described in the Methods section. Relative quantification of lncRNAs was performed using TaqMan qPCR. Results are shown as inflammation and advanced fibrosis relative to normal histology, levels of which were set to a value of 1. Expression levels were normalized against GAPDH. All experiments were performed in triplicate. Data are expressed as mean ± standard deviation. *p > 0.05.
    Figure Legend Snippet: Analysis of NEAT1 , MALAT1 , and HULC by RT-qPCR Total RNA was extracted from liver tissue as described in the Methods section. Relative quantification of lncRNAs was performed using TaqMan qPCR. Results are shown as inflammation and advanced fibrosis relative to normal histology, levels of which were set to a value of 1. Expression levels were normalized against GAPDH. All experiments were performed in triplicate. Data are expressed as mean ± standard deviation. *p > 0.05.

    Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

    MALAT1 knockdown decreases CXCL5 transcript and protein levels HepG2 cells were transfected with MALAT1 siRNA for 48 hours and levels of A) MALAT1 , B) CXCL5 transcript and C) CXCL5 protein were quantified. Total RNA and protein were extracted and collected, respectively, as described in the Methods section. Relative quantification of transcripts was performed using TaqMan qPCR and normalized against GAPDH. Protein concentrations were assessed using commercially available enzyme-linked immunosorbent assays. All experiments were performed in triplicate. Data are expressed as mean ± standard deviation. *p > 0.05.
    Figure Legend Snippet: MALAT1 knockdown decreases CXCL5 transcript and protein levels HepG2 cells were transfected with MALAT1 siRNA for 48 hours and levels of A) MALAT1 , B) CXCL5 transcript and C) CXCL5 protein were quantified. Total RNA and protein were extracted and collected, respectively, as described in the Methods section. Relative quantification of transcripts was performed using TaqMan qPCR and normalized against GAPDH. Protein concentrations were assessed using commercially available enzyme-linked immunosorbent assays. All experiments were performed in triplicate. Data are expressed as mean ± standard deviation. *p > 0.05.

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Standard Deviation

    5) Product Images from "A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production"

    Article Title: A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production

    Journal: bioRxiv

    doi: 10.1101/2020.03.29.013342

    CDC SARS-CoV-2 N1, N2, and N3 TaqMan qRT-PCR assays performed using indicated copies of synthetic RNA and TaqPath™ commercial qRT-PCR mastermix. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.
    Figure Legend Snippet: CDC SARS-CoV-2 N1, N2, and N3 TaqMan qRT-PCR assays performed using indicated copies of synthetic RNA and TaqPath™ commercial qRT-PCR mastermix. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.

    Techniques Used: Quantitative RT-PCR, Amplification, Negative Control

    CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed in 1X RTX buffer using indicated copies of synthetic RNA and RTX or RTX Exo- and Taq DNA polymerases. Panels A, B, and C depict TaqMan assays containing RTX and Taq DNA polymerases. Panels D, E, and F depict TaqMan assays containing RTX Exo- and Taq DNA polymerases. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.
    Figure Legend Snippet: CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed in 1X RTX buffer using indicated copies of synthetic RNA and RTX or RTX Exo- and Taq DNA polymerases. Panels A, B, and C depict TaqMan assays containing RTX and Taq DNA polymerases. Panels D, E, and F depict TaqMan assays containing RTX Exo- and Taq DNA polymerases. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.

    Techniques Used: Quantitative RT-PCR, Amplification, Negative Control

    CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed in 1X ThermoPol buffer using indicated copies of synthetic RNA and RTX or RTX Exo- and Taq DNA polymerases. Panels A, B, and C depict TaqMan assays containing both RTX and Taq DNA polymerases. Panels D, E, and F depict TaqMan assays containing only Taq DNA polymerase. Panels G, H, and I depict TaqMan assays containing RTX Exo- and Taq DNA polymerases. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.
    Figure Legend Snippet: CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed in 1X ThermoPol buffer using indicated copies of synthetic RNA and RTX or RTX Exo- and Taq DNA polymerases. Panels A, B, and C depict TaqMan assays containing both RTX and Taq DNA polymerases. Panels D, E, and F depict TaqMan assays containing only Taq DNA polymerase. Panels G, H, and I depict TaqMan assays containing RTX Exo- and Taq DNA polymerases. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.

    Techniques Used: Quantitative RT-PCR, Amplification, Negative Control

    6) Product Images from "Detection of EWS/FLI-1 fusion in non-Ewing soft tissue tumors"

    Article Title: Detection of EWS/FLI-1 fusion in non-Ewing soft tissue tumors

    Journal: Journal of Medicine and Life

    doi:

    Quantitative analysis on Green fluorescent channel for RNA samples (normal tissue RNA / tumor tissue RNA). a. Cases no. 3, 4; b. Cases no. 5-9; c. Cases no. 10-14; d. Cases no. 15-19; e. Cases no. 20-22 RT-qPCR at RotorGene 6000 termocycler with TaqMan RNA-to-Ct 1 Step Kit and Hs03024497_ft - TaqMan Gene Expression Assay
    Figure Legend Snippet: Quantitative analysis on Green fluorescent channel for RNA samples (normal tissue RNA / tumor tissue RNA). a. Cases no. 3, 4; b. Cases no. 5-9; c. Cases no. 10-14; d. Cases no. 15-19; e. Cases no. 20-22 RT-qPCR at RotorGene 6000 termocycler with TaqMan RNA-to-Ct 1 Step Kit and Hs03024497_ft - TaqMan Gene Expression Assay

    Techniques Used: Quantitative RT-PCR, Expressing

    Quantitative analysis on Green fluorescence channel for reference genes and NTC RT-qPCR at RotorGene 6000 termocycler with TaqMan RNA-to-Ct 1 Step Kit and Hs03928990_g1- TaqMan Gene Expression Assay. The cycling kit threshold (Ct) is featured pointed
    Figure Legend Snippet: Quantitative analysis on Green fluorescence channel for reference genes and NTC RT-qPCR at RotorGene 6000 termocycler with TaqMan RNA-to-Ct 1 Step Kit and Hs03928990_g1- TaqMan Gene Expression Assay. The cycling kit threshold (Ct) is featured pointed

    Techniques Used: Fluorescence, Quantitative RT-PCR, Expressing

    7) Product Images from "Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay"

    Article Title: Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay

    Journal: Viruses

    doi: 10.3390/v10120714

    Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of capsid , NS1 , NS3 , and NS5 synthetic target RNA were amplified either individually (panels A – D , respectively) or as a mixture (panel E ) using multiplex LAMP-4GO assays containing LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10 5 copies of its target RNA) traces. The x -axis depicts the duration of endpoint signal measurement. ( F ) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. ( G ) Detection of Asian and African lineage ZIKV genomic RNA using TaqMan qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel F were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. ( H ) Detection limit of degenerate multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The x -axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.
    Figure Legend Snippet: Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of capsid , NS1 , NS3 , and NS5 synthetic target RNA were amplified either individually (panels A – D , respectively) or as a mixture (panel E ) using multiplex LAMP-4GO assays containing LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10 5 copies of its target RNA) traces. The x -axis depicts the duration of endpoint signal measurement. ( F ) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. ( G ) Detection of Asian and African lineage ZIKV genomic RNA using TaqMan qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel F were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. ( H ) Detection limit of degenerate multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The x -axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.

    Techniques Used: Multiplex Assay, Amplification, Fluorescence, Quantitative RT-PCR

    8) Product Images from "Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing 1Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing 1 2"

    Article Title: Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing 1Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing 1 2

    Journal: Neoplasia (New York, N.Y.)

    doi:

    TMED6-COG8 ( TC ) read-through in RCC. (A) DNA: Schematic representation of the genomic structure of TMED6 and COG8 on the DNA (-) strand. RNA: Three isoforms were expressed with TCv1 being the prominent one. The TaqMan assay used to detect TC levels in
    Figure Legend Snippet: TMED6-COG8 ( TC ) read-through in RCC. (A) DNA: Schematic representation of the genomic structure of TMED6 and COG8 on the DNA (-) strand. RNA: Three isoforms were expressed with TCv1 being the prominent one. The TaqMan assay used to detect TC levels in

    Techniques Used: TaqMan Assay

    9) Product Images from "MicroRNA-375 Suppresses Extracellular Matrix Degradation and Invadopodial Activity in Head and Neck Squamous Cell Carcinoma"

    Article Title: MicroRNA-375 Suppresses Extracellular Matrix Degradation and Invadopodial Activity in Head and Neck Squamous Cell Carcinoma

    Journal: Archives of pathology & laboratory medicine

    doi: 10.5858/arpa.2014-0471-OA

    Elevated expression of microRNA-375 in HNSCC cells significantly reduces kallikrein 6, kallikrein 10, and MMP-9 messenger RNA (mRNA) expression. The mRNA expression levels in the transductant lines were measured by TaqMan quantitative real-time polymerase
    Figure Legend Snippet: Elevated expression of microRNA-375 in HNSCC cells significantly reduces kallikrein 6, kallikrein 10, and MMP-9 messenger RNA (mRNA) expression. The mRNA expression levels in the transductant lines were measured by TaqMan quantitative real-time polymerase

    Techniques Used: Expressing

    10) Product Images from "ER stress in rodent islets of Langerhans is concomitant with obesity and ?-cell compensation but not with ?-cell dysfunction and diabetes"

    Article Title: ER stress in rodent islets of Langerhans is concomitant with obesity and ?-cell compensation but not with ?-cell dysfunction and diabetes

    Journal: Nutrition & Diabetes

    doi: 10.1038/nutd.2013.35

    Obesity-induced ER stress in the islets of Langerhans is not exacerbated by diet-induced diabetes. ( a i) Body weight in grams, ( a ii) plasma insulin after an overnight fast and ( a iii) %GHb in chow-fed fZDF rats and HF-fZDF rats. ( b ) Differential gene expression in the islets of fZDF rats and HF-fZDF rats assessed using TLDA and expressed as a fold change and normalised to 18S ribosomal RNA. Data are shown as mean±s.e.m. (fZDF, n =3; HF-fZDF, n =3). ( c ) Relative expression of selected transcripts from the islets of HF-fZDF versus chow-fed fZDF rats by single-gene Taqman RT-qPCR. Results are expressed as relative expression levels and normalised to ribosomal protein P2 (RPP2). Values are mean±s.e.m. determined from control chow-fed fZDF ( n =6) and HF-fZDF rats ( n =6). Statistical significance was determined using an unpaired two-tailed Student t -test. No significant changes were found.
    Figure Legend Snippet: Obesity-induced ER stress in the islets of Langerhans is not exacerbated by diet-induced diabetes. ( a i) Body weight in grams, ( a ii) plasma insulin after an overnight fast and ( a iii) %GHb in chow-fed fZDF rats and HF-fZDF rats. ( b ) Differential gene expression in the islets of fZDF rats and HF-fZDF rats assessed using TLDA and expressed as a fold change and normalised to 18S ribosomal RNA. Data are shown as mean±s.e.m. (fZDF, n =3; HF-fZDF, n =3). ( c ) Relative expression of selected transcripts from the islets of HF-fZDF versus chow-fed fZDF rats by single-gene Taqman RT-qPCR. Results are expressed as relative expression levels and normalised to ribosomal protein P2 (RPP2). Values are mean±s.e.m. determined from control chow-fed fZDF ( n =6) and HF-fZDF rats ( n =6). Statistical significance was determined using an unpaired two-tailed Student t -test. No significant changes were found.

    Techniques Used: Expressing, TLDA Assay, Quantitative RT-PCR, Two Tailed Test

    Obesity leads to increased ER stress in ZO rats. ( a i) Body weight in grams, ( a ii) plasma insulin after an overnight fast and ( a iii) % GHb in age-matched Zucker rats (obese) and their lean littermates (lean). ( b ) Differential gene expression between Zucker rats (obese) and their lean littermates (lean) assessed using TLDA is expressed as a fold change and normalised to 18S ribosomal RNA. Data are shown as mean±s.e.m. (Zucker obese, n =5; lean control, n =4). ( c ) Relative expression of selected transcripts from the islets of Zucker rats (obese) and their lean littermates (lean) assessed by single-gene Taqman RT-qPCR. Results are expressed as relative expression levels and normalised to the housekeeping gene ribosomal protein P2 (RPP2). Values are mean±s.e.m. determined from control lean ( n =4) and obese rats ( n =5). Statistical significance was determined using an unpaired two-tailed Student t -test. * P
    Figure Legend Snippet: Obesity leads to increased ER stress in ZO rats. ( a i) Body weight in grams, ( a ii) plasma insulin after an overnight fast and ( a iii) % GHb in age-matched Zucker rats (obese) and their lean littermates (lean). ( b ) Differential gene expression between Zucker rats (obese) and their lean littermates (lean) assessed using TLDA is expressed as a fold change and normalised to 18S ribosomal RNA. Data are shown as mean±s.e.m. (Zucker obese, n =5; lean control, n =4). ( c ) Relative expression of selected transcripts from the islets of Zucker rats (obese) and their lean littermates (lean) assessed by single-gene Taqman RT-qPCR. Results are expressed as relative expression levels and normalised to the housekeeping gene ribosomal protein P2 (RPP2). Values are mean±s.e.m. determined from control lean ( n =4) and obese rats ( n =5). Statistical significance was determined using an unpaired two-tailed Student t -test. * P

    Techniques Used: Expressing, TLDA Assay, Quantitative RT-PCR, Two Tailed Test

    Obesity leads to increased ER stress in female ZDF rats. ( a i) Body weight in grams, ( a ii) plasma insulin after an overnight fast and ( a iii) %GHb in fZDF rats (obese) and their lean littermates (lean). ( b ) Differential gene expression in the islets of fZDF rats (obese) and their lean littermates (lean) assessed using TLDA is expressed as a fold change and is normalised to 18S ribosomal RNA. Data are shown as mean±s.e.m. ( n =3). ( c ) Relative expression of selected transcripts from the islets of fZDF rats (obese) and their lean littermates (lean) by single-gene Taqman RT-qPCR. Results are expressed as relative expression levels and normalised to ribosomal protein P2 (RPP2). Values are mean±s.e.m. determined from control lean ( n =3) and obese rats ( n =3). Statistical significance was determined using an unpaired two-tailed Student t -test. * P
    Figure Legend Snippet: Obesity leads to increased ER stress in female ZDF rats. ( a i) Body weight in grams, ( a ii) plasma insulin after an overnight fast and ( a iii) %GHb in fZDF rats (obese) and their lean littermates (lean). ( b ) Differential gene expression in the islets of fZDF rats (obese) and their lean littermates (lean) assessed using TLDA is expressed as a fold change and is normalised to 18S ribosomal RNA. Data are shown as mean±s.e.m. ( n =3). ( c ) Relative expression of selected transcripts from the islets of fZDF rats (obese) and their lean littermates (lean) by single-gene Taqman RT-qPCR. Results are expressed as relative expression levels and normalised to ribosomal protein P2 (RPP2). Values are mean±s.e.m. determined from control lean ( n =3) and obese rats ( n =3). Statistical significance was determined using an unpaired two-tailed Student t -test. * P

    Techniques Used: Expressing, TLDA Assay, Quantitative RT-PCR, Two Tailed Test

    11) Product Images from "The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs"

    Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbagrm.2013.09.007

    The ACE2 gene promoter is bipartite in humans and mice. (A) Diagram of the human ACE2 promoter with two conserved regions separated by a repetitive Alu element giving rise to two transcript forms encoding the same protein. (B) Design of DPT and PPT assays involves a common reverse primer, unique forward primers, and unique Taqman probes. (C) The DPT and PPT assays were run with different concentration of pCRII-TOPO plasmids containing cloned DPT and PPT amplicons as samples. A water sample was run as a no-template control. The cycle-threshold values (Ct) were recorded. (D) Comparisons of the concentrations of DPT and PPT in mouse tissues. Concentrations are shown as the C t values obtained for RNA samples diluted to 20 ng/μl. Samples giving no amplification were assigned a Ct value of 40 which is the highest cycle number in the assays. The lung, kidney, heart, and brain were harvested from 3 female mice and the pancreas from 5 male mice. *: p
    Figure Legend Snippet: The ACE2 gene promoter is bipartite in humans and mice. (A) Diagram of the human ACE2 promoter with two conserved regions separated by a repetitive Alu element giving rise to two transcript forms encoding the same protein. (B) Design of DPT and PPT assays involves a common reverse primer, unique forward primers, and unique Taqman probes. (C) The DPT and PPT assays were run with different concentration of pCRII-TOPO plasmids containing cloned DPT and PPT amplicons as samples. A water sample was run as a no-template control. The cycle-threshold values (Ct) were recorded. (D) Comparisons of the concentrations of DPT and PPT in mouse tissues. Concentrations are shown as the C t values obtained for RNA samples diluted to 20 ng/μl. Samples giving no amplification were assigned a Ct value of 40 which is the highest cycle number in the assays. The lung, kidney, heart, and brain were harvested from 3 female mice and the pancreas from 5 male mice. *: p

    Techniques Used: Mouse Assay, Concentration Assay, Clone Assay, Amplification

    12) Product Images from "Diet-induced adipose tissue expansion is mitigated in mice with a targeted inactivation of mesoderm specific transcript (Mest)"

    Article Title: Diet-induced adipose tissue expansion is mitigated in mice with a targeted inactivation of mesoderm specific transcript (Mest)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0179879

    Dietary obesity in mice with Adipoq -cre mediated inactivation of Mest . (A) Mest mRNA expression in inguinal (iWAT) and epididymal (eWAT) white adipose tissue of wildtype (WT; n = 9), WT. Adipoq -cre (WT-cre; n = 5), paternal floxed Mest (pFL; n = 11) and pFL. Adipoq -cre (ApKO; n = 17) mice after being fed a HFD from 8–24 weeks of age. Mest mRNA expression measured by TaqMan QRT-PCR is represented as the mean ± SEM of arbitrary units (AU) normalized to TATA-box binding protein ( Tbp ). Significance in Mest RNA expression between groups was determined via one-way ANOVA using Tukey’s multiple comparisons test. (B) Data shows the longitudinal measurements of bodyweight (BWT) for WT (n = 9), WT-cre (n = 5), pFL (n = 11) and ApKO (n = 17) mice fed a HFD from 8 to 24 weeks of age as indicated by the arrow along the X-axis. (C) Longitudinal measurements of fat mass (g) and (D) fat-free mass (g) measured by DEXA at the times indicated on the X-axis are shown for WT (n = 9), WT-cre (n = 5), pFL (n = 11) and ApKO (n = 17) mice fed a HFD from 8 to 24 weeks of age as indicated by the arrow along the X-axis. (B-D) All data in the longitudinal studies are presented as the mean ± SEM. Significance at each time point of the longitudinal phenotypic analyses was determined by 2-way ANOVA and Tukey’s multiple comparisons test. Time points annotated with ‘a’ and ‘b’ indicates significant differences between ‘ApKO vs pFL’ and ‘ApKO vs WT’.
    Figure Legend Snippet: Dietary obesity in mice with Adipoq -cre mediated inactivation of Mest . (A) Mest mRNA expression in inguinal (iWAT) and epididymal (eWAT) white adipose tissue of wildtype (WT; n = 9), WT. Adipoq -cre (WT-cre; n = 5), paternal floxed Mest (pFL; n = 11) and pFL. Adipoq -cre (ApKO; n = 17) mice after being fed a HFD from 8–24 weeks of age. Mest mRNA expression measured by TaqMan QRT-PCR is represented as the mean ± SEM of arbitrary units (AU) normalized to TATA-box binding protein ( Tbp ). Significance in Mest RNA expression between groups was determined via one-way ANOVA using Tukey’s multiple comparisons test. (B) Data shows the longitudinal measurements of bodyweight (BWT) for WT (n = 9), WT-cre (n = 5), pFL (n = 11) and ApKO (n = 17) mice fed a HFD from 8 to 24 weeks of age as indicated by the arrow along the X-axis. (C) Longitudinal measurements of fat mass (g) and (D) fat-free mass (g) measured by DEXA at the times indicated on the X-axis are shown for WT (n = 9), WT-cre (n = 5), pFL (n = 11) and ApKO (n = 17) mice fed a HFD from 8 to 24 weeks of age as indicated by the arrow along the X-axis. (B-D) All data in the longitudinal studies are presented as the mean ± SEM. Significance at each time point of the longitudinal phenotypic analyses was determined by 2-way ANOVA and Tukey’s multiple comparisons test. Time points annotated with ‘a’ and ‘b’ indicates significant differences between ‘ApKO vs pFL’ and ‘ApKO vs WT’.

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Binding Assay, RNA Expression

    WAT gene expression in mice with inactivated Mest . (A) Data shows expression of Mest , (B) Sfrp5 , (C) Bmp3 , (D) Nkd1 , (E) Serpine1 , and (F) Pparg in epididymal (eWAT) and inguinal (iWAT) white adipose tissue of wildtype (WT; n = 17) mice with a paternal inactivation of Mest (pKO; n = 10) and subgroups of WT mice with low (LM; n = 6) and high (HM; n = 6) WAT Mest expression after being fed a HFD from 8–16 weeks of age. Gene expression was determined via TaqMan qRT-PCR in total RNA from eWAT and iWAT and represented as arbitrary units (AU) normalized to cyclophilin b ( Ppib ). Gene expression is presented as the mean ± SEM and significance between groups was determined using two-tailed unpaired parametric t-tests. Datasets annotated with the same letter indicate no significant differences between groups.
    Figure Legend Snippet: WAT gene expression in mice with inactivated Mest . (A) Data shows expression of Mest , (B) Sfrp5 , (C) Bmp3 , (D) Nkd1 , (E) Serpine1 , and (F) Pparg in epididymal (eWAT) and inguinal (iWAT) white adipose tissue of wildtype (WT; n = 17) mice with a paternal inactivation of Mest (pKO; n = 10) and subgroups of WT mice with low (LM; n = 6) and high (HM; n = 6) WAT Mest expression after being fed a HFD from 8–16 weeks of age. Gene expression was determined via TaqMan qRT-PCR in total RNA from eWAT and iWAT and represented as arbitrary units (AU) normalized to cyclophilin b ( Ppib ). Gene expression is presented as the mean ± SEM and significance between groups was determined using two-tailed unpaired parametric t-tests. Datasets annotated with the same letter indicate no significant differences between groups.

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Two Tailed Test

    Dietary obesity in mice with inactivated Mest . (A) Longitudinal measurement of bodyweight (BWT) in 17 wildtype (WT) mice and 10 mice with inactivation of Mest on the paternal allele (pKO). Mice were fed high fat diet (HFD) from 8 to 16 weeks of age as indicated by the arrow along the X-axis. (B) Longitudinal measurements show the ratio of fat mass (FM) and lean mass (LM) measured by NMR, indicated as adiposity index (FM/LM), in 17 WT and 10 pKO mice fed a HFD from 8 to 16 weeks as shown by the arrow along the X-axis. (C) Data shows the cumulative change of fat mass (Δ Fat Mass) and fat-free mass (Δ FFM; inset graph) of 17 WT and 10 Mest pKO (pKO) mice after initiation of a HFD at 8 weeks of age until 16 weeks of age as indicated on the X-axis. (D) Data represents tertiles of mice from the 17 WT mice in the study with the lowest (WT-LM; n = 6) and highest (WT-HM; n = 6) epididymal white adipose tissue (eWAT) Mest expression. Gene expression was measured by TaqMan QRT-PCR and is represented as arbitrary units (AU) normalized to cyclophilin b ( Ppib ). (E) Longitudinal analyses of BWT and (F) adiposity index in mice within WT-LM and WT-HM tertiles for eWAT Mest and pKO mice are shown. Data in all figures are presented as the mean ± SEM. Significance at each time point of the longitudinal phenotypic analyses between WT and pKO (A- C) was determined by 2-way ANOVA using the Holm-Sidak multiple comparisons test. Time points annotated with 1, 2, 3 or 4 asterisks indicate significant differences of P≤0.05, P≤0.01, P≤0.001 and P≤0.0001 respectively. Significance in eWAT Mest RNA expression between the WT-LM and WT-HM groups (D) was measured by a two-tailed unpaired parametric t-test and is indicated on the bar graph. Longitudinal data for WT-LM, WT-HM and pKO (E and F) was performed using 2-way ANOVA using Tukey’s multiple comparisons test. Annotation with ‘a’ and ‘b’ indicates significant differences between WT-HM vs pKO and WT-HM vs WT-LM mice respectively.
    Figure Legend Snippet: Dietary obesity in mice with inactivated Mest . (A) Longitudinal measurement of bodyweight (BWT) in 17 wildtype (WT) mice and 10 mice with inactivation of Mest on the paternal allele (pKO). Mice were fed high fat diet (HFD) from 8 to 16 weeks of age as indicated by the arrow along the X-axis. (B) Longitudinal measurements show the ratio of fat mass (FM) and lean mass (LM) measured by NMR, indicated as adiposity index (FM/LM), in 17 WT and 10 pKO mice fed a HFD from 8 to 16 weeks as shown by the arrow along the X-axis. (C) Data shows the cumulative change of fat mass (Δ Fat Mass) and fat-free mass (Δ FFM; inset graph) of 17 WT and 10 Mest pKO (pKO) mice after initiation of a HFD at 8 weeks of age until 16 weeks of age as indicated on the X-axis. (D) Data represents tertiles of mice from the 17 WT mice in the study with the lowest (WT-LM; n = 6) and highest (WT-HM; n = 6) epididymal white adipose tissue (eWAT) Mest expression. Gene expression was measured by TaqMan QRT-PCR and is represented as arbitrary units (AU) normalized to cyclophilin b ( Ppib ). (E) Longitudinal analyses of BWT and (F) adiposity index in mice within WT-LM and WT-HM tertiles for eWAT Mest and pKO mice are shown. Data in all figures are presented as the mean ± SEM. Significance at each time point of the longitudinal phenotypic analyses between WT and pKO (A- C) was determined by 2-way ANOVA using the Holm-Sidak multiple comparisons test. Time points annotated with 1, 2, 3 or 4 asterisks indicate significant differences of P≤0.05, P≤0.01, P≤0.001 and P≤0.0001 respectively. Significance in eWAT Mest RNA expression between the WT-LM and WT-HM groups (D) was measured by a two-tailed unpaired parametric t-test and is indicated on the bar graph. Longitudinal data for WT-LM, WT-HM and pKO (E and F) was performed using 2-way ANOVA using Tukey’s multiple comparisons test. Annotation with ‘a’ and ‘b’ indicates significant differences between WT-HM vs pKO and WT-HM vs WT-LM mice respectively.

    Techniques Used: Mouse Assay, Nuclear Magnetic Resonance, Expressing, Quantitative RT-PCR, RNA Expression, Two Tailed Test

    Inflammatory genes in WAT of mice with inactivated Mest . A, B, C and D show expression of gene markers for inflammation and macrophage infiltration in WAT of WT and Mest pKO (pKO) mice. Gene expression was determined via TaqMan qRT-PCR in total RNA from eWAT and iWAT of wildtype (WT; n = 17), mice with a paternal inactivation of Mest (pKO; n = 10) and subgroups of WT mice with low (LM; n = 6) and high (HM; n = 6) WAT Mest expression after being fed a HFD from 8–16 weeks of age. Gene expression is represented as arbitrary units (AU) normalized to Tbp and presented as the mean ± SEM. Statistical analysis between genotypes within each WAT depot was performed using two-tailed unpaired parametric t-tests and datasets annotated with the same letter indicate no significant differences between groups.
    Figure Legend Snippet: Inflammatory genes in WAT of mice with inactivated Mest . A, B, C and D show expression of gene markers for inflammation and macrophage infiltration in WAT of WT and Mest pKO (pKO) mice. Gene expression was determined via TaqMan qRT-PCR in total RNA from eWAT and iWAT of wildtype (WT; n = 17), mice with a paternal inactivation of Mest (pKO; n = 10) and subgroups of WT mice with low (LM; n = 6) and high (HM; n = 6) WAT Mest expression after being fed a HFD from 8–16 weeks of age. Gene expression is represented as arbitrary units (AU) normalized to Tbp and presented as the mean ± SEM. Statistical analysis between genotypes within each WAT depot was performed using two-tailed unpaired parametric t-tests and datasets annotated with the same letter indicate no significant differences between groups.

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Two Tailed Test

    13) Product Images from "Detection of EWS/FLI-1 fusion in non-Ewing soft tissue tumors"

    Article Title: Detection of EWS/FLI-1 fusion in non-Ewing soft tissue tumors

    Journal: Journal of Medicine and Life

    doi:

    Quantitative analysis on Green fluorescent channel for RNA samples (normal tissue RNA / tumor tissue RNA). a. Cases no. 3, 4; b. Cases no. 5-9; c. Cases no. 10-14; d. Cases no. 15-19; e. Cases no. 20-22 RT-qPCR at RotorGene 6000 termocycler with TaqMan RNA-to-Ct 1 Step Kit and Hs03024497_ft - TaqMan Gene Expression Assay
    Figure Legend Snippet: Quantitative analysis on Green fluorescent channel for RNA samples (normal tissue RNA / tumor tissue RNA). a. Cases no. 3, 4; b. Cases no. 5-9; c. Cases no. 10-14; d. Cases no. 15-19; e. Cases no. 20-22 RT-qPCR at RotorGene 6000 termocycler with TaqMan RNA-to-Ct 1 Step Kit and Hs03024497_ft - TaqMan Gene Expression Assay

    Techniques Used: Quantitative RT-PCR, Expressing

    Quantitative analysis on Green fluorescence channel for reference genes and NTC RT-qPCR at RotorGene 6000 termocycler with TaqMan RNA-to-Ct 1 Step Kit and Hs03928990_g1- TaqMan Gene Expression Assay. The cycling kit threshold (Ct) is featured pointed
    Figure Legend Snippet: Quantitative analysis on Green fluorescence channel for reference genes and NTC RT-qPCR at RotorGene 6000 termocycler with TaqMan RNA-to-Ct 1 Step Kit and Hs03928990_g1- TaqMan Gene Expression Assay. The cycling kit threshold (Ct) is featured pointed

    Techniques Used: Fluorescence, Quantitative RT-PCR, Expressing

    14) Product Images from "Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning"

    Article Title: Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning

    Journal: bioRxiv

    doi: 10.1101/2020.08.17.253559

    Quantitative measurement of TNFSF11 RNA and RANKL protein. TNFSF11 transcription was assessed by two qPCRs upstream (A) and downstream (B) of the gRNAs target sites. hTERT MSC and the TNFSF11 knockout clones g2d, g23b and g23d were analyzed (n=3, representative runs are displayed). Upstream reaction was positive for hTERT MSCs and g23b, while downstream reaction was positive only for hTERT MSCs. (C) Schematic presentation of the major splice variant of TNFSF11 . Blue boxes represent exons, red bars indicate the target site of the CRISPR/Cas9 gRNAs and black arrows symbolize the TaqMan probes with the respective fluorophore. (D) Protein expression was measured using emulsion coupling. RANKL-positive hTERT MSCs applied as positive control and 3 different RANKL KO hTERT MSCs were analyzed. The results are in the absolute counts of detected RANKL proteins. The values were normalized against control reaction without interaction (ABC) (red = positive control, cyan = samples); boxes represent the interquartile range (IQR; first to third quartiles); whiskers are 1.5× IQR; horizontal mid-line, median; dot, mean. Statistical significance is indicated: Kolmogorov-Smirnov-test (n = 3) *P ¡ 0.05; zero level means no RANKL protein detected.
    Figure Legend Snippet: Quantitative measurement of TNFSF11 RNA and RANKL protein. TNFSF11 transcription was assessed by two qPCRs upstream (A) and downstream (B) of the gRNAs target sites. hTERT MSC and the TNFSF11 knockout clones g2d, g23b and g23d were analyzed (n=3, representative runs are displayed). Upstream reaction was positive for hTERT MSCs and g23b, while downstream reaction was positive only for hTERT MSCs. (C) Schematic presentation of the major splice variant of TNFSF11 . Blue boxes represent exons, red bars indicate the target site of the CRISPR/Cas9 gRNAs and black arrows symbolize the TaqMan probes with the respective fluorophore. (D) Protein expression was measured using emulsion coupling. RANKL-positive hTERT MSCs applied as positive control and 3 different RANKL KO hTERT MSCs were analyzed. The results are in the absolute counts of detected RANKL proteins. The values were normalized against control reaction without interaction (ABC) (red = positive control, cyan = samples); boxes represent the interquartile range (IQR; first to third quartiles); whiskers are 1.5× IQR; horizontal mid-line, median; dot, mean. Statistical significance is indicated: Kolmogorov-Smirnov-test (n = 3) *P ¡ 0.05; zero level means no RANKL protein detected.

    Techniques Used: Knock-Out, Clone Assay, Variant Assay, CRISPR, Expressing, Positive Control

    15) Product Images from "Parasite specific 7SL-derived small RNA is an effective target for diagnosis of active trypanosomiasis infection"

    Article Title: Parasite specific 7SL-derived small RNA is an effective target for diagnosis of active trypanosomiasis infection

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007189

    7SL-sRNA sequence enables differentiation between animal-infective trypanosome species. A) The 26-bp 7SL-sRNA sequence identified in T . congolense serum was aligned to that from other trypanosomatids, revealing species-specific differences that were flanked by conserved guanosine repeats. The polymorphisms were utilised to design species-specific Taqman RT-qPCR assays. B) Schematic representation of the Taqman RT-qPCR assay, a 2 step based assay that makes use of step-loop primers as previously described in [ 56 ]. C) Assays generated for the detection of T . b . brucei , T . vivax and T . congolense were applied to RNA samples extracted from plasma from infected animals, adjudged to exhibit similar parasitaemia scores. In each case, only the species for which the assay was designed was detected by RT-qPCR and were statistically significant (alpha = 0.05) as determined by Wilcoxon Signed Rank test, P value (two tailed)
    Figure Legend Snippet: 7SL-sRNA sequence enables differentiation between animal-infective trypanosome species. A) The 26-bp 7SL-sRNA sequence identified in T . congolense serum was aligned to that from other trypanosomatids, revealing species-specific differences that were flanked by conserved guanosine repeats. The polymorphisms were utilised to design species-specific Taqman RT-qPCR assays. B) Schematic representation of the Taqman RT-qPCR assay, a 2 step based assay that makes use of step-loop primers as previously described in [ 56 ]. C) Assays generated for the detection of T . b . brucei , T . vivax and T . congolense were applied to RNA samples extracted from plasma from infected animals, adjudged to exhibit similar parasitaemia scores. In each case, only the species for which the assay was designed was detected by RT-qPCR and were statistically significant (alpha = 0.05) as determined by Wilcoxon Signed Rank test, P value (two tailed)

    Techniques Used: Sequencing, Quantitative RT-PCR, Generated, Infection, Two Tailed Test

    16) Product Images from "Functional characterization of BC039389-GATM and KLK4-KRSP1 chimeric read-through transcripts which are up-regulated in renal cell cancer"

    Article Title: Functional characterization of BC039389-GATM and KLK4-KRSP1 chimeric read-through transcripts which are up-regulated in renal cell cancer

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1446-z

    Expression levels of the read-throughs’ parent genes in tissues. ( A ) Expression of the BC039389 and GATM parent genes alone (wt, wild-type levels) or with read-throughs (tot, total levels) in 32 frozen tumor and matched normal samples. One sample had not suffient RNA left for measuring wild-type and BC039389 tot levels. The location of the TaqMan assays is indicated in Figure 2 A. ( B ) Expression of the KLK4 and KRSP1 parent genes (wild-type and total transcript levels) in 13 samples expressing KKv1 read-through (left panel) or 19 samples not expressing KKv1 (right panel). To be consistent in showing normal tissues, we artificially calculated KKv1 expression for 11 of 13 normal samples using the cut-off of Ct40, as KKv1 was expressed only in 2 normal tissues of the KKv1 -expressing cohort. The location of the TaqMan assays is indicated in Figure 3 A. Significance was calculated using student’s t -test in paired samples (*p
    Figure Legend Snippet: Expression levels of the read-throughs’ parent genes in tissues. ( A ) Expression of the BC039389 and GATM parent genes alone (wt, wild-type levels) or with read-throughs (tot, total levels) in 32 frozen tumor and matched normal samples. One sample had not suffient RNA left for measuring wild-type and BC039389 tot levels. The location of the TaqMan assays is indicated in Figure 2 A. ( B ) Expression of the KLK4 and KRSP1 parent genes (wild-type and total transcript levels) in 13 samples expressing KKv1 read-through (left panel) or 19 samples not expressing KKv1 (right panel). To be consistent in showing normal tissues, we artificially calculated KKv1 expression for 11 of 13 normal samples using the cut-off of Ct40, as KKv1 was expressed only in 2 normal tissues of the KKv1 -expressing cohort. The location of the TaqMan assays is indicated in Figure 3 A. Significance was calculated using student’s t -test in paired samples (*p

    Techniques Used: Expressing

    Description of read-through BC039389-GATM. ( A ) DNA: Genomic structure of BC039389 and GATM on the DNA (−) strand. RNA: Two isoforms are expressed from the genomic locus both utilizing the same alternative ATG in GATM exon 3 for putative protein translation. TaqMan assay positions and the positions targeted by siRNAs are indicated. ( B ) Differential expression of BC039389-GATM isoform 1 ( BGv1 ) in tumor vs. normal kidney tissue (**p
    Figure Legend Snippet: Description of read-through BC039389-GATM. ( A ) DNA: Genomic structure of BC039389 and GATM on the DNA (−) strand. RNA: Two isoforms are expressed from the genomic locus both utilizing the same alternative ATG in GATM exon 3 for putative protein translation. TaqMan assay positions and the positions targeted by siRNAs are indicated. ( B ) Differential expression of BC039389-GATM isoform 1 ( BGv1 ) in tumor vs. normal kidney tissue (**p

    Techniques Used: TaqMan Assay, Expressing

    Description of read-through KLK4-KRSP1. ( A ) DNA: Genomic structure of KLK4 and KRSP1 on the DNA (−) strand. RNA: At least five isoforms are expressed from the genomic locus. A pink line represents the splice site in KLK4 exon 2 which leads to a frameshift and subsequent STOP codon in the RNA and the use of an alternative (alt) ATG START codon in putative KKv1 and KKv2 proteins. Green and brown lines indicate the splice sites in KLK4 exon 5 (5 m = exon 5 middle, 5 m2 = exon 5 middle with different splice site) utilized in KKv2 and KKv3 , respectively. TaqMan assay positions and the positions targeted by siRNAs are indicated. ( B ) Expression levels of KLK4-KRSP1 isoform 1 ( KKv1 ) in 46 frozen tumor/normal sample pairs using TaqMan assay. ( C ) KKv2 expression in tumor/normal tissue pairs was screend for 46 KKv1 -positive samples.
    Figure Legend Snippet: Description of read-through KLK4-KRSP1. ( A ) DNA: Genomic structure of KLK4 and KRSP1 on the DNA (−) strand. RNA: At least five isoforms are expressed from the genomic locus. A pink line represents the splice site in KLK4 exon 2 which leads to a frameshift and subsequent STOP codon in the RNA and the use of an alternative (alt) ATG START codon in putative KKv1 and KKv2 proteins. Green and brown lines indicate the splice sites in KLK4 exon 5 (5 m = exon 5 middle, 5 m2 = exon 5 middle with different splice site) utilized in KKv2 and KKv3 , respectively. TaqMan assay positions and the positions targeted by siRNAs are indicated. ( B ) Expression levels of KLK4-KRSP1 isoform 1 ( KKv1 ) in 46 frozen tumor/normal sample pairs using TaqMan assay. ( C ) KKv2 expression in tumor/normal tissue pairs was screend for 46 KKv1 -positive samples.

    Techniques Used: TaqMan Assay, Expressing

    A panel of read-throughs is expressed in RCC. ( A ) The fraction of different classes of RNA chimeras called by FusionSeq from the RNASeq data in seven frozen RCC samples. The clinical characteristics of the samples are given elsewhere [ 30 ]. ccRCC, clear cell RCC; chrRCC, chromophobe RCC; tRCC, Xp11 translocation RCC. ( B ) 27 read-throughs which were chosen for further validation by reverse transcription (RT)-PCR. Plotted are the individual RESPER scores per sample and the mean RESPER with range if the read-through was found in more than 1 sample. ( C ) Quantitative evaluation of 12 read-throughs by TaqMan assays in frozen tissue of RCC and matched adjacent normal kidney, plotted as Tukey boxplots. No read-through has significant differential expression between tumor and normal tissue as calculated by student’s t -test with correction for multiple testing by Bonferroni method. The blue outlier dot in TMED6-COG8 is the tRCC whose detailed analysis is featured elsewhere [ 30 ].
    Figure Legend Snippet: A panel of read-throughs is expressed in RCC. ( A ) The fraction of different classes of RNA chimeras called by FusionSeq from the RNASeq data in seven frozen RCC samples. The clinical characteristics of the samples are given elsewhere [ 30 ]. ccRCC, clear cell RCC; chrRCC, chromophobe RCC; tRCC, Xp11 translocation RCC. ( B ) 27 read-throughs which were chosen for further validation by reverse transcription (RT)-PCR. Plotted are the individual RESPER scores per sample and the mean RESPER with range if the read-through was found in more than 1 sample. ( C ) Quantitative evaluation of 12 read-throughs by TaqMan assays in frozen tissue of RCC and matched adjacent normal kidney, plotted as Tukey boxplots. No read-through has significant differential expression between tumor and normal tissue as calculated by student’s t -test with correction for multiple testing by Bonferroni method. The blue outlier dot in TMED6-COG8 is the tRCC whose detailed analysis is featured elsewhere [ 30 ].

    Techniques Used: Translocation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

    17) Product Images from "Increased MicroRNA-34b and -34c Predominantly Expressed in Stromal Tissues Is Associated with Poor Prognosis in Human Colon Cancer"

    Article Title: Increased MicroRNA-34b and -34c Predominantly Expressed in Stromal Tissues Is Associated with Poor Prognosis in Human Colon Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124899

    miR-34 family tends to be expressed predominantly in stromal tissue. RNA samples from cancer epithelium, cancer stroma, normal adjacent epithelium and normal adjacent stroma were extracted separately using laser microdissection technique from 5 paired samples in the American cohort. Dot plots represent miR-34a/b/c threshold cycle values of tumor tissue from TaqMan qRT-PCR normalized to U48. Horizontal bars indicate median expression value. Un-paired t test was used.
    Figure Legend Snippet: miR-34 family tends to be expressed predominantly in stromal tissue. RNA samples from cancer epithelium, cancer stroma, normal adjacent epithelium and normal adjacent stroma were extracted separately using laser microdissection technique from 5 paired samples in the American cohort. Dot plots represent miR-34a/b/c threshold cycle values of tumor tissue from TaqMan qRT-PCR normalized to U48. Horizontal bars indicate median expression value. Un-paired t test was used.

    Techniques Used: Laser Capture Microdissection, Quantitative RT-PCR, Expressing

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response
    Article Snippet: .. Total RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA) following the manufacturer’s instructions. mRNA expression levels were determined by real-time quantitative PCR (qPCR) with TaqMan Gene Expression Assays and the TaqMan RNA-to-CT ™ 1-Step Kit (Applied Biosystems, Foster City, CA) using a 7300 real-time cycler (Applied Biosystems). .. NF-κB-driven luciferase activity was quantified using the Cell Titer-Glo assay.

    Article Title: Detection of EWS/FLI-1 fusion in non-Ewing soft tissue tumors
    Article Snippet: .. Detection of EWS/FLI-1 fusion EWS/FLI-1 fusion was screened by quantitative real-time PCR (qPCR) with Rotor Gene 6000 thermocycler (Corbett Life Science, Qiagen Sciences, Maryland, USA) using the TaqMan RNA-to-Ct 1 Step Kit (Applied Biosystems - Life Technologies, Carlsbad, CA, USA), with Hs03024497_ft primer specific for fusion. .. PCR data normalization was performed by using 18S rRNA gene reference and Hs03928990_g1 primer.

    Article Title: Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay
    Article Snippet: .. Reactions containing Evoscript RNA Probes Master were analyzed using the LightCycler96 qPCR machine while TaqMan RNA-to-Ct 1-Step assays were performed on the StepOnePlus real-time PCR machine (Thermo Scientific). .. Heat-inactivated virions of Asian ZIKV strain PRVABC59 (BEI Resources, Manassas, VA, USA) were used as templates for LAMP-based assays and for one-step qRT-PCR.

    Isolation:

    Article Title: c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response
    Article Snippet: .. Total RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA) following the manufacturer’s instructions. mRNA expression levels were determined by real-time quantitative PCR (qPCR) with TaqMan Gene Expression Assays and the TaqMan RNA-to-CT ™ 1-Step Kit (Applied Biosystems, Foster City, CA) using a 7300 real-time cycler (Applied Biosystems). .. NF-κB-driven luciferase activity was quantified using the Cell Titer-Glo assay.

    Expressing:

    Article Title: c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response
    Article Snippet: .. Total RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA) following the manufacturer’s instructions. mRNA expression levels were determined by real-time quantitative PCR (qPCR) with TaqMan Gene Expression Assays and the TaqMan RNA-to-CT ™ 1-Step Kit (Applied Biosystems, Foster City, CA) using a 7300 real-time cycler (Applied Biosystems). .. NF-κB-driven luciferase activity was quantified using the Cell Titer-Glo assay.

    Quantitative RT-PCR:

    Article Title: A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production
    Article Snippet: .. TaqMan qRT-PCR reaction set up shown in was used for assembling CDC SARS-CoV-2 N1, N2, and N3 assays using either RTX buffer or ThermoPol buffer. ..

    Article Title: Regulation of Hypoxia-Inducible Factor 1α during Hypoxia by DAP5-Induced Translation of PHD2
    Article Snippet: .. For RT-qPCR analysis, an RNA-to-CT one-step kit (Invitrogen) was used according to the manufacturer's protocol on a 7900 HT TaqMan machine; samples were measured in triplicate, and the average value was used for further calculation. ..

    Article Title: Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay
    Article Snippet: .. The previously reported ZIKV NS2b-specific TaqMan qRT-PCR assay ( ) [ ] was performed using either the Evoscript RNA Probes Master (Roche, Basel, Switzerland) or the TaqMan RNA-to-Ct 1-Step Kit (Thermo Scientific, Waltham MA, USA). ..

    Article Title: Altered expression of MALAT1 lncRNA in nonalcoholic steatohepatitis Fibrosis Regulates CXCL5 in Hepatic Stellate Cells
    Article Snippet: .. We converted RNA to cDNA using the TaqMan RNA-to-Ct 1-Step kit (Life Technologies) according to the manufacturer’s protocol, followed by RT-qPCR analysis in conjunction with the QuantStudio 6 Flex (Life Technologies). .. Cycle threshold value was generated using QuantStudio Real-Time PCR Software 1.0 (Life Technologies).

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    Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of capsid , NS1 , NS3 , and NS5 synthetic target <t>RNA</t> were amplified either individually (panels A – D , respectively) or as a mixture (panel E ) using multiplex LAMP-4GO assays containing LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10 5 copies of its target RNA) traces. The x -axis depicts the duration of endpoint signal measurement. ( F ) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. ( G ) Detection of Asian and African lineage ZIKV genomic RNA using <t>TaqMan</t> qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel F were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. ( H ) Detection limit of degenerate multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The x -axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.
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    Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of capsid , NS1 , NS3 , and NS5 synthetic target RNA were amplified either individually (panels A – D , respectively) or as a mixture (panel E ) using multiplex LAMP-4GO assays containing LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10 5 copies of its target RNA) traces. The x -axis depicts the duration of endpoint signal measurement. ( F ) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. ( G ) Detection of Asian and African lineage ZIKV genomic RNA using TaqMan qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel F were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. ( H ) Detection limit of degenerate multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The x -axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.

    Journal: Viruses

    Article Title: Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay

    doi: 10.3390/v10120714

    Figure Lengend Snippet: Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of capsid , NS1 , NS3 , and NS5 synthetic target RNA were amplified either individually (panels A – D , respectively) or as a mixture (panel E ) using multiplex LAMP-4GO assays containing LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10 5 copies of its target RNA) traces. The x -axis depicts the duration of endpoint signal measurement. ( F ) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. ( G ) Detection of Asian and African lineage ZIKV genomic RNA using TaqMan qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel F were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. ( H ) Detection limit of degenerate multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The x -axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.

    Article Snippet: The previously reported ZIKV NS2b-specific TaqMan qRT-PCR assay ( ) [ ] was performed using either the Evoscript RNA Probes Master (Roche, Basel, Switzerland) or the TaqMan RNA-to-Ct 1-Step Kit (Thermo Scientific, Waltham MA, USA).

    Techniques: Multiplex Assay, Amplification, Fluorescence, Quantitative RT-PCR

    CDC SARS-CoV-2 N1, N2, and N3 TaqMan qRT-PCR assays performed using indicated copies of synthetic RNA and TaqPath™ commercial qRT-PCR mastermix. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.

    Journal: bioRxiv

    Article Title: A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production

    doi: 10.1101/2020.03.29.013342

    Figure Lengend Snippet: CDC SARS-CoV-2 N1, N2, and N3 TaqMan qRT-PCR assays performed using indicated copies of synthetic RNA and TaqPath™ commercial qRT-PCR mastermix. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.

    Article Snippet: TaqMan qRT-PCR reaction set up shown in was used for assembling CDC SARS-CoV-2 N1, N2, and N3 assays using either RTX buffer or ThermoPol buffer.

    Techniques: Quantitative RT-PCR, Amplification, Negative Control

    CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed in 1X RTX buffer using indicated copies of synthetic RNA and RTX or RTX Exo- and Taq DNA polymerases. Panels A, B, and C depict TaqMan assays containing RTX and Taq DNA polymerases. Panels D, E, and F depict TaqMan assays containing RTX Exo- and Taq DNA polymerases. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.

    Journal: bioRxiv

    Article Title: A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production

    doi: 10.1101/2020.03.29.013342

    Figure Lengend Snippet: CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed in 1X RTX buffer using indicated copies of synthetic RNA and RTX or RTX Exo- and Taq DNA polymerases. Panels A, B, and C depict TaqMan assays containing RTX and Taq DNA polymerases. Panels D, E, and F depict TaqMan assays containing RTX Exo- and Taq DNA polymerases. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.

    Article Snippet: TaqMan qRT-PCR reaction set up shown in was used for assembling CDC SARS-CoV-2 N1, N2, and N3 assays using either RTX buffer or ThermoPol buffer.

    Techniques: Quantitative RT-PCR, Amplification, Negative Control

    CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed in 1X ThermoPol buffer using indicated copies of synthetic RNA and RTX or RTX Exo- and Taq DNA polymerases. Panels A, B, and C depict TaqMan assays containing both RTX and Taq DNA polymerases. Panels D, E, and F depict TaqMan assays containing only Taq DNA polymerase. Panels G, H, and I depict TaqMan assays containing RTX Exo- and Taq DNA polymerases. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.

    Journal: bioRxiv

    Article Title: A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production

    doi: 10.1101/2020.03.29.013342

    Figure Lengend Snippet: CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed in 1X ThermoPol buffer using indicated copies of synthetic RNA and RTX or RTX Exo- and Taq DNA polymerases. Panels A, B, and C depict TaqMan assays containing both RTX and Taq DNA polymerases. Panels D, E, and F depict TaqMan assays containing only Taq DNA polymerase. Panels G, H, and I depict TaqMan assays containing RTX Exo- and Taq DNA polymerases. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.

    Article Snippet: TaqMan qRT-PCR reaction set up shown in was used for assembling CDC SARS-CoV-2 N1, N2, and N3 assays using either RTX buffer or ThermoPol buffer.

    Techniques: Quantitative RT-PCR, Amplification, Negative Control

    Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of capsid , NS1 , NS3 , and NS5 synthetic target RNA were amplified either individually (panels A – D , respectively) or as a mixture (panel E ) using multiplex LAMP-4GO assays containing LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10 5 copies of its target RNA) traces. The x -axis depicts the duration of endpoint signal measurement. ( F ) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. ( G ) Detection of Asian and African lineage ZIKV genomic RNA using TaqMan qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel F were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. ( H ) Detection limit of degenerate multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The x -axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.

    Journal: Viruses

    Article Title: Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay

    doi: 10.3390/v10120714

    Figure Lengend Snippet: Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of capsid , NS1 , NS3 , and NS5 synthetic target RNA were amplified either individually (panels A – D , respectively) or as a mixture (panel E ) using multiplex LAMP-4GO assays containing LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10 5 copies of its target RNA) traces. The x -axis depicts the duration of endpoint signal measurement. ( F ) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. ( G ) Detection of Asian and African lineage ZIKV genomic RNA using TaqMan qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel F were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. ( H ) Detection limit of degenerate multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The x -axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.

    Article Snippet: Reactions containing Evoscript RNA Probes Master were analyzed using the LightCycler96 qPCR machine while TaqMan RNA-to-Ct 1-Step assays were performed on the StepOnePlus real-time PCR machine (Thermo Scientific).

    Techniques: Multiplex Assay, Amplification, Fluorescence, Quantitative RT-PCR