luna universal one step rt qpcr kit  (New England Biolabs)


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    Name:
    Luna Universal One Step RT qPCR Kit
    Description:
    Luna Universal One Step RT qPCR Kit 2 500 rxns
    Catalog Number:
    E3005E
    Price:
    2074
    Category:
    RT PCR Kits
    Size:
    2 500 rxns
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    New England Biolabs luna universal one step rt qpcr kit
    Luna Universal One Step RT qPCR Kit
    Luna Universal One Step RT qPCR Kit 2 500 rxns
    https://www.bioz.com/result/luna universal one step rt qpcr kit/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
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    luna universal one step rt qpcr kit - by Bioz Stars, 2021-09
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    1) Product Images from "Characterization and functional interrogation of SARS-CoV-2 RNA interactome"

    Article Title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome

    Journal: bioRxiv

    doi: 10.1101/2021.03.23.436611

    Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p
    Figure Legend Snippet: Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p

    Techniques Used: Activity Assay, Infection, Quantitative RT-PCR

    Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.
    Figure Legend Snippet: Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.

    Techniques Used: Functional Assay, Transfection, Infection, Quantitative RT-PCR, Flow Cytometry

    2) Product Images from "LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts"

    Article Title: LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts

    Journal: Journal of Orthopaedic Surgery and Research

    doi: 10.1186/s13018-019-1430-4

    Plasma lncRNA TUG1 was upregulated in osteoporosis patients than in healthy participants. RT-qPCR results showed that plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients than in healthy participants (* p
    Figure Legend Snippet: Plasma lncRNA TUG1 was upregulated in osteoporosis patients than in healthy participants. RT-qPCR results showed that plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients than in healthy participants (* p

    Techniques Used: Quantitative RT-PCR

    3) Product Images from "SARS-CoV-2 and SARS-CoV differ in their cell tropism and drug sensitivity profiles"

    Article Title: SARS-CoV-2 and SARS-CoV differ in their cell tropism and drug sensitivity profiles

    Journal: bioRxiv

    doi: 10.1101/2020.04.03.024257

    N A) Western blots indicating cellular ACE2 and TMPRSS2 protein levels. B) CPE formation in SARS-CoV and SARS-CoV-2 (MOI 0.01)-infected ACE2-negative 293 cells and 293 cells stably expressing ACE2 cells (293/ACE2) 48h post infection. C) Immunostaining for double-stranded RNA in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. D) Quantification of virus genomes by qPCR in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. E) Cytopathogenic effect (CPE) formation in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected Caco2 cells in the presence of antibodies directed against ACE2 or DPP4 (MERS-CoV receptor) 48h post infection.
    Figure Legend Snippet: N A) Western blots indicating cellular ACE2 and TMPRSS2 protein levels. B) CPE formation in SARS-CoV and SARS-CoV-2 (MOI 0.01)-infected ACE2-negative 293 cells and 293 cells stably expressing ACE2 cells (293/ACE2) 48h post infection. C) Immunostaining for double-stranded RNA in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. D) Quantification of virus genomes by qPCR in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. E) Cytopathogenic effect (CPE) formation in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected Caco2 cells in the presence of antibodies directed against ACE2 or DPP4 (MERS-CoV receptor) 48h post infection.

    Techniques Used: Western Blot, Infection, Stable Transfection, Expressing, Immunostaining, Real-time Polymerase Chain Reaction

    SARS-CoV-2 and SARS-CoV susceptibility of colorectal cancer cell lines. A) Cytopathogenic effect (CPE) formation 48h post infection in MOI 0.01-infected cells. B) Representative images showing MOI 0.01-infected cells immunostained for double-stranded RNA 48h post infection. C) Quantification of virus genomes by qPCR at different time points post infection (p.i.).
    Figure Legend Snippet: SARS-CoV-2 and SARS-CoV susceptibility of colorectal cancer cell lines. A) Cytopathogenic effect (CPE) formation 48h post infection in MOI 0.01-infected cells. B) Representative images showing MOI 0.01-infected cells immunostained for double-stranded RNA 48h post infection. C) Quantification of virus genomes by qPCR at different time points post infection (p.i.).

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    4) Product Images from "Dual-level autoregulation of the E. coli DeaD RNA helicase via mRNA stability and Rho-dependent transcription termination"

    Article Title: Dual-level autoregulation of the E. coli DeaD RNA helicase via mRNA stability and Rho-dependent transcription termination

    Journal: bioRxiv

    doi: 10.1101/2020.04.19.049098

    Autoregulation of deaD is mediated via mRNA stability and transcription termination. (A) Northern-blot analysis. WT or DM strains were treated with rifampicin and aliquots of the cell cultures were harvested at different times after rifampicin addition, as indicated at the top. Total RNA was prepared from these cultures and analyzed by northern blot using a probe for deaD mRNA. (B) Half-life analysis. The levels of deaD mRNA from (A) were normalized and plotted as a function of time after rifampicin addition on a semi-logarithmic scale. Each data point represents an average value from three experiments. The calculated half-life of the deaD mRNA in each strain is indicated. (C) Inactivation of RNase E increases deaD mRNA levels. WT and DM strains, as well as their rne-3071 derivatives, were transferred to 42°C for 30 min after growth to midlog phase at 30°C, followed by harvesting of cultures, RNA preparation and northern blot analysis. (D) Effects of Rho inactivation on deaD mRNA. Total RNA was isolated from WT or DM strains containing either wild-type rho or a rho ts allele. The strains were grown to mid-log phase at 30°C and then transferred to 42°C for 30 min before harvesting. RNA was isolated from the strains and analyzed by northern blotting. (E) Effect of BCM addition on deaD mRNA. Cultures of WT or DM strains were grown at 37°C and were either untreated or treated with 20 or 40 μg/ml of BCM for 20 min before harvesting. RNA was isolated from the strains and analyzed by northern blotting. (F) RNA was isolated from WT, rho ts or DM strains after growth at 42°C for 30 min and the levels of RNA transcription from 538 to 638 bps downstream of the guaC coding region were quantified by qRT-PCR. The convergently oriented guaC and hofC coding regions are shown in blue and yellow, respectively. The arrow depicts transcription from the guaC promoter with dotted lines indicating readthrough antisense transcription into the hofC coding region. The region analyzed by qRT-PCR is indicated by a red line.
    Figure Legend Snippet: Autoregulation of deaD is mediated via mRNA stability and transcription termination. (A) Northern-blot analysis. WT or DM strains were treated with rifampicin and aliquots of the cell cultures were harvested at different times after rifampicin addition, as indicated at the top. Total RNA was prepared from these cultures and analyzed by northern blot using a probe for deaD mRNA. (B) Half-life analysis. The levels of deaD mRNA from (A) were normalized and plotted as a function of time after rifampicin addition on a semi-logarithmic scale. Each data point represents an average value from three experiments. The calculated half-life of the deaD mRNA in each strain is indicated. (C) Inactivation of RNase E increases deaD mRNA levels. WT and DM strains, as well as their rne-3071 derivatives, were transferred to 42°C for 30 min after growth to midlog phase at 30°C, followed by harvesting of cultures, RNA preparation and northern blot analysis. (D) Effects of Rho inactivation on deaD mRNA. Total RNA was isolated from WT or DM strains containing either wild-type rho or a rho ts allele. The strains were grown to mid-log phase at 30°C and then transferred to 42°C for 30 min before harvesting. RNA was isolated from the strains and analyzed by northern blotting. (E) Effect of BCM addition on deaD mRNA. Cultures of WT or DM strains were grown at 37°C and were either untreated or treated with 20 or 40 μg/ml of BCM for 20 min before harvesting. RNA was isolated from the strains and analyzed by northern blotting. (F) RNA was isolated from WT, rho ts or DM strains after growth at 42°C for 30 min and the levels of RNA transcription from 538 to 638 bps downstream of the guaC coding region were quantified by qRT-PCR. The convergently oriented guaC and hofC coding regions are shown in blue and yellow, respectively. The arrow depicts transcription from the guaC promoter with dotted lines indicating readthrough antisense transcription into the hofC coding region. The region analyzed by qRT-PCR is indicated by a red line.

    Techniques Used: Northern Blot, Isolation, Quantitative RT-PCR

    (A) Deletion mapping of regions important for autoregulation. A wild-type single-copy deaD-lacZ fusion or variants containing the indicated deletions within the deaD 5’ UTR were incorporated into DlacZ derivatives of the WT or DdeaD strain and the β-galactosidase activity of the resulting strains was measured. (B) RNA was isolated from WT or rho ts strains containing a WT deaD-lacZ fusion or a fusion containing a 720 nt deletion in the deaD 5’ UTR. The RNA samples were analyzed by qRT-PCR, with amplification performed either over the junction region of the deaD-lacZ transcript (100 nts of deaD and 24 nts of lacZ sequence) or a 123 nt region near the 5’ end of the transcript. n. d., not determined.
    Figure Legend Snippet: (A) Deletion mapping of regions important for autoregulation. A wild-type single-copy deaD-lacZ fusion or variants containing the indicated deletions within the deaD 5’ UTR were incorporated into DlacZ derivatives of the WT or DdeaD strain and the β-galactosidase activity of the resulting strains was measured. (B) RNA was isolated from WT or rho ts strains containing a WT deaD-lacZ fusion or a fusion containing a 720 nt deletion in the deaD 5’ UTR. The RNA samples were analyzed by qRT-PCR, with amplification performed either over the junction region of the deaD-lacZ transcript (100 nts of deaD and 24 nts of lacZ sequence) or a 123 nt region near the 5’ end of the transcript. n. d., not determined.

    Techniques Used: Activity Assay, Isolation, Quantitative RT-PCR, Amplification, Sequencing

    5) Product Images from "A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing"

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

    Journal: bioRxiv

    doi: 10.1101/2020.06.23.166397

    A sensitive, robust RT-LAMP assay compatible with crude patient samples. A) Schematic illustrating loop-mediated amplification (LAMP) of SARS-CoV-2 RNA and the regions targeted in this study (Orf1ab, E and N genes; depicted above). Each target region is recognized by a defined set of primers (B3, LB, BIP, LF, FIP, F3). The RNA template (red) is reverse transcribed and displaced after first-strand synthesis; the outer primer binding sites are added in the subsequent amplification step. The resulting dumbbell DNA structure acts as template for further rounds of amplification, ultimately leading to high molecular weight amplicons. B) Readout of a real-time fluorescence RT-LAMP reaction using 500 copies of synthetic SARS-CoV-2 (red) or water as non-targeting control (NTC, black) as input. ‘Time to threshold’ indicates the time at which the fluorescence value reaches threshold level (equivalent to Cq value in RT-qPCR assays), ‘end-point RFU’ indicates the fluorescence value (FAM filter set, absorption/emission at 494 nm/518 nm) after 35 minutes reaction time (used throughout this study unless indicated otherwise); RFU: relative fluorescence units. C) Performance of the three top primer sets for RT-LAMP-based SARS-CoV-2 detection. End-point relative fluorescence units (RFUs) of RT-LAMP reactions (in duplicates) using the indicated primer sets and serially diluted synthetic SARS-CoV-2 RNA standard as input. Water was used as no-target control (NTC). D) Cartoon indicating the workflow for SARS-CoV-2 detection by either RT-LAMP or 1-step RT-qPCR from patient samples (nasopharyngeal swab or gargle) with prior RNA isolation. E) Comparison of RT-LAMP and RT-qPCR performance. Plotted are RT-LAMP end-point fluorescence values after 35 minutes versus the respective RT-qPCR Cq values. RNA was derived from gargle (green) or nasopharyngeal swabs (black); two no-target controls were included (black cross). Reactions in which no amplification was recorded are labelled as qPCR negative. F) Predictive agreement between RT-LAMP and 1-step RT-qPCR assays. Shown are percentages of positive (detected in RT-LAMP and RT-qPCR, black bars) and negative (not detected in either RT-LAMP or RT-qPCR, purple bars) predictive agreement for sample groups (defined by RT-qPCR-derived Cq values) between RT-LAMP (using E- and/or N-gene primers) and 1-step RT-qPCR. G) Performance of different crude sample preparation methods in RT-LAMP. Shown are end-point relative fluorescence units (RFUs) for RT-LAMP reactions targeting human RNAseP on sample inputs derived from defined numbers of HEK293 cells mixed 1:1 with indicated 2x buffers (extracted RNsA served as a positive control). H) Cartoon indicating the workflow for RT-LAMP using QuickExtract crude lysate as sample input. I) Comparison of QuickExtract crude sample input versus extracted RNA as input using 1-step RT-qPCR. Covid-19 patient nasopharyngeal swabs or gargle samples (color coded according to the indicated collection medium) were either processed with the QuickExtract workflow (crude sample input) or RNA was extracted using an automated King Fisher RNA bead purification protocol. Reactions in which no amplification was recorded are labelled as qPCR negative. J) Performance of RT-LAMP with QuickExtract treated crude Covid-19 patient sample input (same samples as in I). Depicted is the correlation of Cq values from RT-qPCR performed on QuickExtract treated samples versus corresponding end-point relative fluorescence units (RFUs) from RT-LAMP reactions.
    Figure Legend Snippet: A sensitive, robust RT-LAMP assay compatible with crude patient samples. A) Schematic illustrating loop-mediated amplification (LAMP) of SARS-CoV-2 RNA and the regions targeted in this study (Orf1ab, E and N genes; depicted above). Each target region is recognized by a defined set of primers (B3, LB, BIP, LF, FIP, F3). The RNA template (red) is reverse transcribed and displaced after first-strand synthesis; the outer primer binding sites are added in the subsequent amplification step. The resulting dumbbell DNA structure acts as template for further rounds of amplification, ultimately leading to high molecular weight amplicons. B) Readout of a real-time fluorescence RT-LAMP reaction using 500 copies of synthetic SARS-CoV-2 (red) or water as non-targeting control (NTC, black) as input. ‘Time to threshold’ indicates the time at which the fluorescence value reaches threshold level (equivalent to Cq value in RT-qPCR assays), ‘end-point RFU’ indicates the fluorescence value (FAM filter set, absorption/emission at 494 nm/518 nm) after 35 minutes reaction time (used throughout this study unless indicated otherwise); RFU: relative fluorescence units. C) Performance of the three top primer sets for RT-LAMP-based SARS-CoV-2 detection. End-point relative fluorescence units (RFUs) of RT-LAMP reactions (in duplicates) using the indicated primer sets and serially diluted synthetic SARS-CoV-2 RNA standard as input. Water was used as no-target control (NTC). D) Cartoon indicating the workflow for SARS-CoV-2 detection by either RT-LAMP or 1-step RT-qPCR from patient samples (nasopharyngeal swab or gargle) with prior RNA isolation. E) Comparison of RT-LAMP and RT-qPCR performance. Plotted are RT-LAMP end-point fluorescence values after 35 minutes versus the respective RT-qPCR Cq values. RNA was derived from gargle (green) or nasopharyngeal swabs (black); two no-target controls were included (black cross). Reactions in which no amplification was recorded are labelled as qPCR negative. F) Predictive agreement between RT-LAMP and 1-step RT-qPCR assays. Shown are percentages of positive (detected in RT-LAMP and RT-qPCR, black bars) and negative (not detected in either RT-LAMP or RT-qPCR, purple bars) predictive agreement for sample groups (defined by RT-qPCR-derived Cq values) between RT-LAMP (using E- and/or N-gene primers) and 1-step RT-qPCR. G) Performance of different crude sample preparation methods in RT-LAMP. Shown are end-point relative fluorescence units (RFUs) for RT-LAMP reactions targeting human RNAseP on sample inputs derived from defined numbers of HEK293 cells mixed 1:1 with indicated 2x buffers (extracted RNsA served as a positive control). H) Cartoon indicating the workflow for RT-LAMP using QuickExtract crude lysate as sample input. I) Comparison of QuickExtract crude sample input versus extracted RNA as input using 1-step RT-qPCR. Covid-19 patient nasopharyngeal swabs or gargle samples (color coded according to the indicated collection medium) were either processed with the QuickExtract workflow (crude sample input) or RNA was extracted using an automated King Fisher RNA bead purification protocol. Reactions in which no amplification was recorded are labelled as qPCR negative. J) Performance of RT-LAMP with QuickExtract treated crude Covid-19 patient sample input (same samples as in I). Depicted is the correlation of Cq values from RT-qPCR performed on QuickExtract treated samples versus corresponding end-point relative fluorescence units (RFUs) from RT-LAMP reactions.

    Techniques Used: RT Lamp Assay, Amplification, Binding Assay, Molecular Weight, Fluorescence, Quantitative RT-PCR, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction, Sample Prep, Positive Control, Purification

    6) Product Images from "A streamlined, cost-effective, and specific method to deplete transcripts for RNA-seq"

    Article Title: A streamlined, cost-effective, and specific method to deplete transcripts for RNA-seq

    Journal: bioRxiv

    doi: 10.1101/2020.05.21.109033

    RNaseH-mediated rRNA and non-specific mRNA depletion. A) Nondenaturing 1.2% agarose gel depicting the following lanes from left to right: 1) ssRNA ladder; 2) total RNA input; 3) input with mock incubations; 4) RNaseH treatment and DNaseI treatment without oligos; 5) DNaseI and oligos only; and 6-8) increasing oligo:RNA mass ratio (1:1, 2:1, and 4:1; total RNA fixed at 1 µg) with RNaseH and DNaseI treatment. B) Fold depletion of 18S rRNA, ACTB, and GAPDH transcripts normalized to input total RNA by RT-qPCR performed on the RNA samples in panel A. Total RNA samples incubated with RNaseH and DNaseI without oligos (grey bar) or increasing oligo:RNA ratio (red bars) with RNaseH and DNaseI treatment. NEB E.coli RNaseH treatment for 1 hour at 37°C and NEB E.coli DNaseI treatment for 30 minutes at 37°C used for samples in this experiment.
    Figure Legend Snippet: RNaseH-mediated rRNA and non-specific mRNA depletion. A) Nondenaturing 1.2% agarose gel depicting the following lanes from left to right: 1) ssRNA ladder; 2) total RNA input; 3) input with mock incubations; 4) RNaseH treatment and DNaseI treatment without oligos; 5) DNaseI and oligos only; and 6-8) increasing oligo:RNA mass ratio (1:1, 2:1, and 4:1; total RNA fixed at 1 µg) with RNaseH and DNaseI treatment. B) Fold depletion of 18S rRNA, ACTB, and GAPDH transcripts normalized to input total RNA by RT-qPCR performed on the RNA samples in panel A. Total RNA samples incubated with RNaseH and DNaseI without oligos (grey bar) or increasing oligo:RNA ratio (red bars) with RNaseH and DNaseI treatment. NEB E.coli RNaseH treatment for 1 hour at 37°C and NEB E.coli DNaseI treatment for 30 minutes at 37°C used for samples in this experiment.

    Techniques Used: Agarose Gel Electrophoresis, Quantitative RT-PCR, Incubation

    7) Product Images from "Attenuation of a very virulent Marek's disease herpesvirus (MDV) by codon pair bias deoptimization"

    Article Title: Attenuation of a very virulent Marek's disease herpesvirus (MDV) by codon pair bias deoptimization

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006857

    Quantification of RNA expression and protein production from the recoded UL30 genes. HEK 293T cells were transfected with dual expression plasmids pVITRO2-TagBFP-UL30-EGFP that carried differently recoded UL30 genes fused in frame with EGFP gene. 24 h post transfection RNA expression (A) from the recoded genes was quantified by qPCR, and protein production by flow cytometry (B). The UL30 RNA levels were normalized against the TagBFP levels. We used EGFP fluorescence as a reporter to quantify protein production of the fusion UL30-EGFP genes. The EGFP fluorescence was normalized against the TagBFP fluorescence. P-values were calculated using Kruskal-Wallis H test, * indicates P
    Figure Legend Snippet: Quantification of RNA expression and protein production from the recoded UL30 genes. HEK 293T cells were transfected with dual expression plasmids pVITRO2-TagBFP-UL30-EGFP that carried differently recoded UL30 genes fused in frame with EGFP gene. 24 h post transfection RNA expression (A) from the recoded genes was quantified by qPCR, and protein production by flow cytometry (B). The UL30 RNA levels were normalized against the TagBFP levels. We used EGFP fluorescence as a reporter to quantify protein production of the fusion UL30-EGFP genes. The EGFP fluorescence was normalized against the TagBFP fluorescence. P-values were calculated using Kruskal-Wallis H test, * indicates P

    Techniques Used: RNA Expression, Transfection, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

    Characterization of recoded MDV UL30 mutants. (A) Effect of recoding on UL30 expression from the virus background. CEC were transfected with the parental or mutant BAC clones that carried differently recoded UL30 genes. 24 h post transfection RNA levels of UL29, UL30 and UL42 genes were quantified by qPCR. P-values were calculated using Kruskal-Wallis H test, * indicates P
    Figure Legend Snippet: Characterization of recoded MDV UL30 mutants. (A) Effect of recoding on UL30 expression from the virus background. CEC were transfected with the parental or mutant BAC clones that carried differently recoded UL30 genes. 24 h post transfection RNA levels of UL29, UL30 and UL42 genes were quantified by qPCR. P-values were calculated using Kruskal-Wallis H test, * indicates P

    Techniques Used: Expressing, Capillary Electrochromatography, Transfection, Mutagenesis, BAC Assay, Clone Assay, Real-time Polymerase Chain Reaction

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    Article Snippet: .. Quantitative real-time PCR (RT-qPCR) experiments were performed in triplicate. mRNA expression levels were normalized to the level of GAPDH. ..

    Article Title: Modulatory Effect of 4-(methylthio)butyl Isothiocyanate Isolated From Eruca Sativa Thell. on DMBA Induced Overexpression of Hypoxia and Glycolytic Pathway in Sprague-Dawley Female Rats
    Article Snippet: .. Using Luna One-Step RT-qPCR (New England BioLabs), the gene expression of hexokinase, phosphoglucose isomerase, aldolase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate isomerase, phosphoglycerate mutase, enolase, pyruvate kinase, HIF-1α, HIF-1β, prolyl hydroxylase (PHD), mTOR, phosphatase and tensin homolog (PTEN), Heat shock protein-90α (HSP-90α), and succinate dehydrogenase was recorded. ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Function of cofactor Akirin2 in the regulation of gene expression in model human Caucasian neutrophil-like HL60 cells
    Article Snippet: .. Total RNA was used to characterize the mRNA levels of selected genes by qRT-PCR using gene-specific oligonucleotide forward (F) and reverse (R) primers (ELANE , F: 5′-CGTGGCGAATGTAAACGTCC-3′, R: 5′-CCCGTTGAGCTGGAGAATCA-3′; LHX2 , F: 5′-GACCACTTCGGCATGAAGGA-3′, R: 5′-TGCCCACGCCATTGTAGTAG-3′) using the Luna Universal One-Step qRT-PCR Kit (New England Biolabs, Ipswich, MA, U.S.A.) and the CFX96 Touch RT-PCR Detection System (Bio-Rad, Hercules, CA, U.S.A.). ..

    Amplification:

    Article Title: Zika virus NS2A inhibits interferon signaling by degradation of STAT1 and STAT2
    Article Snippet: .. Luna Universal One-Step RT-qPCR kit (New England Biolabs) was used to reverse transcribed and amplified mRNA. ..

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  • 99
    New England Biolabs luna universal one step rt qpcr kit
    Screening of compounds with antiviral activity targeting <t>SARS-CoV-2</t> host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by <t>RT-qPCR</t> (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p
    Luna Universal One Step Rt Qpcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luna universal one step rt qpcr kit/product/New England Biolabs
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    luna universal one step rt qpcr kit - by Bioz Stars, 2021-09
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    99
    New England Biolabs luna universal qpcr master mix
    Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) <t>PCR</t> analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) <t>RT-qPCR</t> analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p
    Luna Universal Qpcr Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luna universal qpcr master mix/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    luna universal qpcr master mix - by Bioz Stars, 2021-09
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    95
    New England Biolabs luna universal one step reverse transcription quantitative pcr rt qpcr kit
    B3GalNT1 KO UT7/Epo cells lack B3GalNT1 transcripts, do not express Gb4, and proliferate normally. (A) Detection of B3GalNT1 mRNA. Total mRNA was isolated from WT cells and from two single cell-derived RFP-expressing clones (KO1 and KO2) and used to detect B3GalNT1 transcripts by <t>RT-qPCR.</t> The amplicons were used in a nested <t>PCR</t> to ensure sufficient sensitivity. Dilutions (1% and 10%) of the WT amplicons were loaded as a reference. GAPDH mRNA was used as a loading control. (B) Detection of Gb4 by immunofluorescence. WT and KO cells were stained with anti-Gb4 antibody, fixed, and visualized by confocal microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (C) Phase-contrast images of WT and KO cells showing no morphological differences. (D) Cell proliferation of WT and KO cells. Cells were incubated at 37°C and counted at the indicated days.
    Luna Universal One Step Reverse Transcription Quantitative Pcr Rt Qpcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luna universal one step reverse transcription quantitative pcr rt qpcr kit/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
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    luna universal one step reverse transcription quantitative pcr rt qpcr kit - by Bioz Stars, 2021-09
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    Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p

    Journal: bioRxiv

    Article Title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome

    doi: 10.1101/2021.03.23.436611

    Figure Lengend Snippet: Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p

    Article Snippet: Yields of viral RNA were quantified by real-time qPCR by using SARS-CoV-2 specific primers targeting the E gene with the Luna®Universal One-Step RT-qPCR Kit (New England Biolabs) in a LightCycler 480 thermocycler (Roche) according to the manufacturer’s protocol.

    Techniques: Activity Assay, Infection, Quantitative RT-PCR

    Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.

    Journal: bioRxiv

    Article Title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome

    doi: 10.1101/2021.03.23.436611

    Figure Lengend Snippet: Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.

    Article Snippet: Yields of viral RNA were quantified by real-time qPCR by using SARS-CoV-2 specific primers targeting the E gene with the Luna®Universal One-Step RT-qPCR Kit (New England Biolabs) in a LightCycler 480 thermocycler (Roche) according to the manufacturer’s protocol.

    Techniques: Functional Assay, Transfection, Infection, Quantitative RT-PCR, Flow Cytometry

    Plasma lncRNA TUG1 was upregulated in osteoporosis patients than in healthy participants. RT-qPCR results showed that plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients than in healthy participants (* p

    Journal: Journal of Orthopaedic Surgery and Research

    Article Title: LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts

    doi: 10.1186/s13018-019-1430-4

    Figure Lengend Snippet: Plasma lncRNA TUG1 was upregulated in osteoporosis patients than in healthy participants. RT-qPCR results showed that plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients than in healthy participants (* p

    Article Snippet: To detect the expression of lncRNA TUG1 and PTEN mRNA, Luna® Universal One-Step RT-qPCR Kit (NEB) was used to prepare PCR reaction systems.

    Techniques: Quantitative RT-PCR

    Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) PCR analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) RT-qPCR analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p

    Journal: PLoS Genetics

    Article Title: A ubiquitin-like protein encoded by the “noncoding” RNA TINCR promotes keratinocyte proliferation and wound healing

    doi: 10.1371/journal.pgen.1009686

    Figure Lengend Snippet: Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) PCR analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) RT-qPCR analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p

    Article Snippet: The resulting cDNA was subjected to real-time PCR analysis with Luna Universal qPCR Master Mix (New England BioLabs) and specific primers in a StepOnePlus Real-Time PCR System (Applied Biosystems).

    Techniques: Mouse Assay, Mutagenesis, Homologous Recombination, CRISPR, Generated, Polymerase Chain Reaction, Electrophoresis, Quantitative RT-PCR

    B3GalNT1 KO UT7/Epo cells lack B3GalNT1 transcripts, do not express Gb4, and proliferate normally. (A) Detection of B3GalNT1 mRNA. Total mRNA was isolated from WT cells and from two single cell-derived RFP-expressing clones (KO1 and KO2) and used to detect B3GalNT1 transcripts by RT-qPCR. The amplicons were used in a nested PCR to ensure sufficient sensitivity. Dilutions (1% and 10%) of the WT amplicons were loaded as a reference. GAPDH mRNA was used as a loading control. (B) Detection of Gb4 by immunofluorescence. WT and KO cells were stained with anti-Gb4 antibody, fixed, and visualized by confocal microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (C) Phase-contrast images of WT and KO cells showing no morphological differences. (D) Cell proliferation of WT and KO cells. Cells were incubated at 37°C and counted at the indicated days.

    Journal: Journal of Virology

    Article Title: Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection

    doi: 10.1128/JVI.00972-19

    Figure Lengend Snippet: B3GalNT1 KO UT7/Epo cells lack B3GalNT1 transcripts, do not express Gb4, and proliferate normally. (A) Detection of B3GalNT1 mRNA. Total mRNA was isolated from WT cells and from two single cell-derived RFP-expressing clones (KO1 and KO2) and used to detect B3GalNT1 transcripts by RT-qPCR. The amplicons were used in a nested PCR to ensure sufficient sensitivity. Dilutions (1% and 10%) of the WT amplicons were loaded as a reference. GAPDH mRNA was used as a loading control. (B) Detection of Gb4 by immunofluorescence. WT and KO cells were stained with anti-Gb4 antibody, fixed, and visualized by confocal microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (C) Phase-contrast images of WT and KO cells showing no morphological differences. (D) Cell proliferation of WT and KO cells. Cells were incubated at 37°C and counted at the indicated days.

    Article Snippet: For detection of the B3GalNT1 mRNA, the Luna Universal one-step reverse transcription-quantitative PCR (RT-qPCR) kit (New England BioLabs, Ipswich, MA) was used.

    Techniques: Isolation, Derivative Assay, Expressing, Clone Assay, Quantitative RT-PCR, Nested PCR, Immunofluorescence, Staining, Confocal Microscopy, Incubation

    Gb4 is dispensable for B19V cell attachment, internalization, and VP1u exposure. (A) Detection of B19V attachment by immunofluorescence. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. Cells were fixed, stained with antibody 860-55D against capsids, and visualized by confocal microscopy. (B) Detection of B19V internalization by immunofluorescence. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, and trypsinized to remove noninternalized viruses. Cells were fixed, stained with antibody 860-55D, and visualized by confocal microscopy. (C) Quantification of B19V attachment. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. The number of virions bound to the cells was quantified by PCR. (D) Quantification of B19V internalization. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, trypsinized to remove noninternalized viruses, and quantified by PCR. WT cells incubated at 4°C serve as negative controls (no internalization). (E) Quantification of VP1u exposure from free virus or bound to cells. Virions were immunoprecipitated with antibody 860-55D against capsids (total capsids) and a rabbit antibody against the PLA 2 region (α-VP1u), followed by qPCR. Normal rabbit IgG was used as a negative control. P values were calculated according to Student’s t test. *, P

    Journal: Journal of Virology

    Article Title: Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection

    doi: 10.1128/JVI.00972-19

    Figure Lengend Snippet: Gb4 is dispensable for B19V cell attachment, internalization, and VP1u exposure. (A) Detection of B19V attachment by immunofluorescence. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. Cells were fixed, stained with antibody 860-55D against capsids, and visualized by confocal microscopy. (B) Detection of B19V internalization by immunofluorescence. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, and trypsinized to remove noninternalized viruses. Cells were fixed, stained with antibody 860-55D, and visualized by confocal microscopy. (C) Quantification of B19V attachment. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. The number of virions bound to the cells was quantified by PCR. (D) Quantification of B19V internalization. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, trypsinized to remove noninternalized viruses, and quantified by PCR. WT cells incubated at 4°C serve as negative controls (no internalization). (E) Quantification of VP1u exposure from free virus or bound to cells. Virions were immunoprecipitated with antibody 860-55D against capsids (total capsids) and a rabbit antibody against the PLA 2 region (α-VP1u), followed by qPCR. Normal rabbit IgG was used as a negative control. P values were calculated according to Student’s t test. *, P

    Article Snippet: For detection of the B3GalNT1 mRNA, the Luna Universal one-step reverse transcription-quantitative PCR (RT-qPCR) kit (New England BioLabs, Ipswich, MA) was used.

    Techniques: Cell Attachment Assay, Immunofluorescence, Incubation, Staining, Confocal Microscopy, Polymerase Chain Reaction, Immunoprecipitation, Proximity Ligation Assay, Real-time Polymerase Chain Reaction, Negative Control