host rna  (Thermo Fisher)


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    Name:
    Ambion DNase I RNase free
    Description:
    Ambion DNase I RNase free E C 3 1 21 1 is a nonspecific endonuclease that degrades double and single stranded DNA and chromatin It functions by hydrolyzing phosphodiester linkages producing mono and oligonucleotides with a 5 phosphate and a 3 hydroxyl group RNase free DNase I is of the highest purity available and is recommended to degrade DNA in the presence of RNA when the absence of RNase is critical to maintain the integrity of the RNA For example DNase I is frequently used to remove template DNA following in vitro transcription and to remove contaminating DNA in total RNA preparations especially those from transfected cells that may contain plasmid DNA used for ribonuclease protection assays cDNA library contraction and RT PCR DNase I requires bivalent cations Mg2 and Ca2 at approximately 5 mM and 0 5 mM respectively for maximal activity and has a pH optimum of 7 8 RNase free DNase I outperforms the competitionA research report in BioTechniques Matthews et al 32 1412 1417 2002 compared RNase contamination in DNase I preparations from Sigma Roche Applied Science Qiagen and Ambion The results revealed that with the exception of Ambion s RNase free DNase I the integrity of cRNA from in vitro transcription reactions was compromised and was still contaminated with DNA Ambion s DNase was used for the remaining experiments requiring DNase digestion Ambion DNase I is tested for contaminating RNase and protease activity Functionality is determined by digestion of human genomic DNA followed by quantitative real time PCR to detect undigested DNA Unit definitionOne unit is the amount of enzyme required to completely degrade 1 µg DNA in 10 min at 37°C and is equivalent to 0 04 Kunitz units Accessory productsFor an alternative to bovine DNase I please consider Recombinant DNase I Cat No AM2235 For a more active salt tolerant DNase please see the TURBO DNase products Cat Nos AM2239 and AM2238
    Catalog Number:
    am2222
    Price:
    None
    Applications:
    General Real-Time PCR Reagents|PCR & Real-Time PCR|Real Time PCR (qPCR)|Reverse Transcription
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher host rna
    Evaluation of the coextraction method (Method 1) (A) Image of the electrophoresis gel for three independent <t>RNA</t> samples extracted from mice infected with M. ulcerans . L, RNA ladder. (B) Electropherogram: example of sample RNA derived from lane 1. (C) Table with RNA concentrations (ng/μl), indicator of purity (ratio A260/A280), integrity of RNA samples (RQI, RNA Quality Indicator), and total quantity (μg) of RNA extracted with Method 1. (D) Electropherogram: example of sample RNA before and after <t>MicrobEnrich</t> (ME) treatment. The 18S and 28S (mouse), and 16S and 23S (bacterial) rRNA bands are indicated in red and green, respectively.
    Ambion DNase I RNase free E C 3 1 21 1 is a nonspecific endonuclease that degrades double and single stranded DNA and chromatin It functions by hydrolyzing phosphodiester linkages producing mono and oligonucleotides with a 5 phosphate and a 3 hydroxyl group RNase free DNase I is of the highest purity available and is recommended to degrade DNA in the presence of RNA when the absence of RNase is critical to maintain the integrity of the RNA For example DNase I is frequently used to remove template DNA following in vitro transcription and to remove contaminating DNA in total RNA preparations especially those from transfected cells that may contain plasmid DNA used for ribonuclease protection assays cDNA library contraction and RT PCR DNase I requires bivalent cations Mg2 and Ca2 at approximately 5 mM and 0 5 mM respectively for maximal activity and has a pH optimum of 7 8 RNase free DNase I outperforms the competitionA research report in BioTechniques Matthews et al 32 1412 1417 2002 compared RNase contamination in DNase I preparations from Sigma Roche Applied Science Qiagen and Ambion The results revealed that with the exception of Ambion s RNase free DNase I the integrity of cRNA from in vitro transcription reactions was compromised and was still contaminated with DNA Ambion s DNase was used for the remaining experiments requiring DNase digestion Ambion DNase I is tested for contaminating RNase and protease activity Functionality is determined by digestion of human genomic DNA followed by quantitative real time PCR to detect undigested DNA Unit definitionOne unit is the amount of enzyme required to completely degrade 1 µg DNA in 10 min at 37°C and is equivalent to 0 04 Kunitz units Accessory productsFor an alternative to bovine DNase I please consider Recombinant DNase I Cat No AM2235 For a more active salt tolerant DNase please see the TURBO DNase products Cat Nos AM2239 and AM2238
    https://www.bioz.com/result/host rna/product/Thermo Fisher
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    host rna - by Bioz Stars, 2021-01
    99/100 stars

    Images

    1) Product Images from "An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans"

    Article Title: An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00512

    Evaluation of the coextraction method (Method 1) (A) Image of the electrophoresis gel for three independent RNA samples extracted from mice infected with M. ulcerans . L, RNA ladder. (B) Electropherogram: example of sample RNA derived from lane 1. (C) Table with RNA concentrations (ng/μl), indicator of purity (ratio A260/A280), integrity of RNA samples (RQI, RNA Quality Indicator), and total quantity (μg) of RNA extracted with Method 1. (D) Electropherogram: example of sample RNA before and after MicrobEnrich (ME) treatment. The 18S and 28S (mouse), and 16S and 23S (bacterial) rRNA bands are indicated in red and green, respectively.
    Figure Legend Snippet: Evaluation of the coextraction method (Method 1) (A) Image of the electrophoresis gel for three independent RNA samples extracted from mice infected with M. ulcerans . L, RNA ladder. (B) Electropherogram: example of sample RNA derived from lane 1. (C) Table with RNA concentrations (ng/μl), indicator of purity (ratio A260/A280), integrity of RNA samples (RQI, RNA Quality Indicator), and total quantity (μg) of RNA extracted with Method 1. (D) Electropherogram: example of sample RNA before and after MicrobEnrich (ME) treatment. The 18S and 28S (mouse), and 16S and 23S (bacterial) rRNA bands are indicated in red and green, respectively.

    Techniques Used: Electrophoresis, Mouse Assay, Infection, Derivative Assay

    Percentage of reads aligned with the M. ulcerans and mouse genomes. M1, Total RNA sample obtained with Method 1; M1_ME, Total RNA sample obtained with Method 1 after enrichment with MICROBEnrich (ME); M2, Bacterial RNA-enriched sample obtained by differential lysis (Method 2). M2-1, M2-2, M2-3 represent three replicates for Method 2.
    Figure Legend Snippet: Percentage of reads aligned with the M. ulcerans and mouse genomes. M1, Total RNA sample obtained with Method 1; M1_ME, Total RNA sample obtained with Method 1 after enrichment with MICROBEnrich (ME); M2, Bacterial RNA-enriched sample obtained by differential lysis (Method 2). M2-1, M2-2, M2-3 represent three replicates for Method 2.

    Techniques Used: Lysis

    2) Product Images from "Boceprevir, calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses in cell culture"

    Article Title: Boceprevir, calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses in cell culture

    Journal: bioRxiv

    doi: 10.1101/2020.10.30.362335

    Dose-dependent inhibitory effect of boceprevir, calpain inhibitors II and XII, and GC-376 on HCoV-NL63 viral RNA synthesis in RT-qPCR assay. Positive control remdesivir (A); GC-376 (B); Calpain inhibitor II (C); Calpain inhibitor XII (D); and Boceprevir (E). The left figures represent the normalized RNA levels of the average of three repeats from each concentration tested, and EC 50 curve fittings using log (concentration of inhibitors) vs normalized RNA levels with variable slopes in prism 8 were shown in the right figures. Data are mean ± standard deviation of three replicates.
    Figure Legend Snippet: Dose-dependent inhibitory effect of boceprevir, calpain inhibitors II and XII, and GC-376 on HCoV-NL63 viral RNA synthesis in RT-qPCR assay. Positive control remdesivir (A); GC-376 (B); Calpain inhibitor II (C); Calpain inhibitor XII (D); and Boceprevir (E). The left figures represent the normalized RNA levels of the average of three repeats from each concentration tested, and EC 50 curve fittings using log (concentration of inhibitors) vs normalized RNA levels with variable slopes in prism 8 were shown in the right figures. Data are mean ± standard deviation of three replicates.

    Techniques Used: Quantitative RT-PCR, Positive Control, Concentration Assay, Standard Deviation

    3) Product Images from "Boceprevir, calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses in cell culture"

    Article Title: Boceprevir, calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses in cell culture

    Journal: bioRxiv

    doi: 10.1101/2020.10.30.362335

    Dose-dependent inhibitory effect of boceprevir, calpain inhibitors II and XII, and GC-376 on HCoV-NL63 viral RNA synthesis in RT-qPCR assay. Positive control remdesivir (A); GC-376 (B); Calpain inhibitor II (C); Calpain inhibitor XII (D); and Boceprevir (E). The left figures represent the normalized RNA levels of the average of three repeats from each concentration tested, and EC 50 curve fittings using log (concentration of inhibitors) vs normalized RNA levels with variable slopes in prism 8 were shown in the right figures. Data are mean ± standard deviation of three replicates.
    Figure Legend Snippet: Dose-dependent inhibitory effect of boceprevir, calpain inhibitors II and XII, and GC-376 on HCoV-NL63 viral RNA synthesis in RT-qPCR assay. Positive control remdesivir (A); GC-376 (B); Calpain inhibitor II (C); Calpain inhibitor XII (D); and Boceprevir (E). The left figures represent the normalized RNA levels of the average of three repeats from each concentration tested, and EC 50 curve fittings using log (concentration of inhibitors) vs normalized RNA levels with variable slopes in prism 8 were shown in the right figures. Data are mean ± standard deviation of three replicates.

    Techniques Used: Quantitative RT-PCR, Positive Control, Concentration Assay, Standard Deviation

    4) Product Images from "An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans"

    Article Title: An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00512

    Evaluation of the coextraction method (Method 1) (A) Image of the electrophoresis gel for three independent RNA samples extracted from mice infected with M. ulcerans . L, RNA ladder. (B) Electropherogram: example of sample RNA derived from lane 1. (C) Table with RNA concentrations (ng/μl), indicator of purity (ratio A260/A280), integrity of RNA samples (RQI, RNA Quality Indicator), and total quantity (μg) of RNA extracted with Method 1. (D) Electropherogram: example of sample RNA before and after MicrobEnrich (ME) treatment. The 18S and 28S (mouse), and 16S and 23S (bacterial) rRNA bands are indicated in red and green, respectively.
    Figure Legend Snippet: Evaluation of the coextraction method (Method 1) (A) Image of the electrophoresis gel for three independent RNA samples extracted from mice infected with M. ulcerans . L, RNA ladder. (B) Electropherogram: example of sample RNA derived from lane 1. (C) Table with RNA concentrations (ng/μl), indicator of purity (ratio A260/A280), integrity of RNA samples (RQI, RNA Quality Indicator), and total quantity (μg) of RNA extracted with Method 1. (D) Electropherogram: example of sample RNA before and after MicrobEnrich (ME) treatment. The 18S and 28S (mouse), and 16S and 23S (bacterial) rRNA bands are indicated in red and green, respectively.

    Techniques Used: Electrophoresis, Mouse Assay, Infection, Derivative Assay

    Percentage of reads aligned with the M. ulcerans and mouse genomes. M1, Total RNA sample obtained with Method 1; M1_ME, Total RNA sample obtained with Method 1 after enrichment with MICROBEnrich (ME); M2, Bacterial RNA-enriched sample obtained by differential lysis (Method 2). M2-1, M2-2, M2-3 represent three replicates for Method 2.
    Figure Legend Snippet: Percentage of reads aligned with the M. ulcerans and mouse genomes. M1, Total RNA sample obtained with Method 1; M1_ME, Total RNA sample obtained with Method 1 after enrichment with MICROBEnrich (ME); M2, Bacterial RNA-enriched sample obtained by differential lysis (Method 2). M2-1, M2-2, M2-3 represent three replicates for Method 2.

    Techniques Used: Lysis

    Related Articles

    Agarose Gel Electrophoresis:

    Article Title: Selected imprinting of INS in the marsupial
    Article Snippet: .. Total RNA was DNase treated (DNA-free™; Ambion) to remove contaminating DNA, run on a 1% agarose gel to assess the quality, quantified with a nano-spectrometer (NanoDrop ND-1000 Spectrophotometer; NanoDrop Technologies Inc., Wilmington, DE, USA) and cDNA was synthesised using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carisbad, CA, USA). .. Typically, 2 μg or a maximum of 8 μl total RNA was used in each cDNA synthesis reaction, with 1 μl Oligo (dT)12-18 (50 μM). cDNA integrity was immediately assessed with GAPDH RT-PCR.

    Isolation:

    Article Title: Effect of adenovirus infection on transgene expression under the adenoviral MLP/TPL and the CMVie promoter/enhancer in CHO cells
    Article Snippet: .. 2.6 DNase treatment of RNA The digestion of residual DNA in the isolated RNA samples was performed using TURBO DNase (Ambion). .. Each 50 µL of sample was digested in a 100 µL reaction mixture containing the provided buffer and four units of TURBO DNase, with incubation at 37 °C for 30 min.

    Generated:

    Article Title: KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome
    Article Snippet: .. Eluted RNA was treated with DNase using Turbo DNA-Free (Ambion). cDNA was generated by reverse-transcriptase PCR using iScript (BioRad). cDNA was then used for qPCR using Taqman probes specific for PAN and cyclophilin (ABi). .. The enrichment of PAN RNA was calculated by comparing PAN RNA eluted from the beads to PAN RNA depleted from the hybridization supernatant relative to LacZ control oligonucleotides.

    Spectrophotometry:

    Article Title: Selected imprinting of INS in the marsupial
    Article Snippet: .. Total RNA was DNase treated (DNA-free™; Ambion) to remove contaminating DNA, run on a 1% agarose gel to assess the quality, quantified with a nano-spectrometer (NanoDrop ND-1000 Spectrophotometer; NanoDrop Technologies Inc., Wilmington, DE, USA) and cDNA was synthesised using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carisbad, CA, USA). .. Typically, 2 μg or a maximum of 8 μl total RNA was used in each cDNA synthesis reaction, with 1 μl Oligo (dT)12-18 (50 μM). cDNA integrity was immediately assessed with GAPDH RT-PCR.

    Purification:

    Article Title: Identification of Differentially Expressed Genes in the Pheromone Glands of Mated and Virgin Bombyx mori by Digital Gene Expression Profiling
    Article Snippet: .. After removing the template DNA and single-stranded RNA by DNase and RNase treatments respectively, dsRNA was purified using MEGAclearTM columns (Ambion) and then eluted in diethyl pyrocarbonate-treated nuclease-free water. .. The dsRNA concentrations were measured using a biophotometer (Eppendorf).

    Real-time Polymerase Chain Reaction:

    Article Title: KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome
    Article Snippet: .. Eluted RNA was treated with DNase using Turbo DNA-Free (Ambion). cDNA was generated by reverse-transcriptase PCR using iScript (BioRad). cDNA was then used for qPCR using Taqman probes specific for PAN and cyclophilin (ABi). .. The enrichment of PAN RNA was calculated by comparing PAN RNA eluted from the beads to PAN RNA depleted from the hybridization supernatant relative to LacZ control oligonucleotides.

    Article Title: Optimization of Feline Immunodeficiency Virus Vectors for RNA Interference
    Article Snippet: .. To perform QPCR, 1 μg total RNA was DNase treated (DNA-free, Ambion) for 1 h at 37°C. .. Following DNase inactivation, 500 ng was used to synthesize cDNA, per the manufacturer's protocols but with a 1-h extension time, using Taqman reverse transcription reagents (Applied Biosystems).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Selected imprinting of INS in the marsupial
    Article Snippet: .. Total RNA was DNase treated (DNA-free™; Ambion) to remove contaminating DNA, run on a 1% agarose gel to assess the quality, quantified with a nano-spectrometer (NanoDrop ND-1000 Spectrophotometer; NanoDrop Technologies Inc., Wilmington, DE, USA) and cDNA was synthesised using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carisbad, CA, USA). .. Typically, 2 μg or a maximum of 8 μl total RNA was used in each cDNA synthesis reaction, with 1 μl Oligo (dT)12-18 (50 μM). cDNA integrity was immediately assessed with GAPDH RT-PCR.

    Article Title: Physiological stressors and invasive plant infections alter the small RNA transcriptome of the rice blast fungus, Magnaporthe oryzae
    Article Snippet: .. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Genomic DNA was removed from total RNA by TURBO® DNase treatment (Ambion, New York, USA). .. Reverse transcription reactions were performed with 500 ng of RNA in 20 μl reaction using High Capacity RNA-to-cDNA Kit (Applied Biosystems, California, USA) by following the manufacturer’s instructions.

    Incubation:

    Article Title: The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function—in vivo and in vitro evaluation
    Article Snippet: .. To remove contaminating DNA and DNase from RNA preparations, the RNA samples were incubated with reagents from the TURBO DNA-free™ Kit (Ambion, Austin, TX). .. The yield and quality of the RNA were assessed using a NanoDrop ND2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

    Quantitative RT-PCR:

    Article Title: Physiological stressors and invasive plant infections alter the small RNA transcriptome of the rice blast fungus, Magnaporthe oryzae
    Article Snippet: .. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Genomic DNA was removed from total RNA by TURBO® DNase treatment (Ambion, New York, USA). .. Reverse transcription reactions were performed with 500 ng of RNA in 20 μl reaction using High Capacity RNA-to-cDNA Kit (Applied Biosystems, California, USA) by following the manufacturer’s instructions.

    Polymerase Chain Reaction:

    Article Title: KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome
    Article Snippet: .. Eluted RNA was treated with DNase using Turbo DNA-Free (Ambion). cDNA was generated by reverse-transcriptase PCR using iScript (BioRad). cDNA was then used for qPCR using Taqman probes specific for PAN and cyclophilin (ABi). .. The enrichment of PAN RNA was calculated by comparing PAN RNA eluted from the beads to PAN RNA depleted from the hybridization supernatant relative to LacZ control oligonucleotides.

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  • 90
    Thermo Fisher host rna levels
    Genes identified are required for virus entry by regulating the expression of receptor ACE2. A. The effect on virion binding and internalization in gene-edited cells. A549-ACE2 cells were incubated with SARS-CoV-2 Sfull infectious virus on ice for binding or then switched to 37°C for internalization. Viral <t>RNA</t> was extracted for <t>RT-qPCR</t> analysis. Data shown are an average of two independent experiments performed in duplicate and are normalized to the controls of individual experiments. B-C. Surface expression of receptor ACE2 was decreased in gene-edited cells as measured by flow cytometry using S1-Fc recombinant protein or anti-ACE2 antibody. D-E. Surface and total expression of receptor ACE2 were decreased in gene-edited cells. The plasma membrane proteins were biotin-labeled and immunoprecipitated by streptavidin beads for Western blotting. One representative blot was shown (D) and Data are pooled from four independent experiments, quantified, and normalized to the controls of individual experiments (E). One-way ANOVA with Dunnett’s test (A, B, C, E); *P
    Host Rna Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/host rna levels/product/Thermo Fisher
    Average 90 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    host rna levels - by Bioz Stars, 2021-01
    90/100 stars
      Buy from Supplier

    91
    Thermo Fisher host gapdh rna levels
    CP11 inhibits release of p6 HEV from Huh7 cells. (A) Viability measurement of p6 HEV-expressing Huh7 cells, treated with 10 μM CP11, as indicated. Values are means ± SEM of data for triplicate samples. (B) QRT-PCR measurement of the intracellular level of p6 HEV sense-strand <t>RNA</t> in the samples shown in panel A. p6 HEV sense RNA values were normalized to that of <t>GAPDH</t> and are represented as means ± SEM of data from triplicate samples. (C) QRT-PCR estimation of p6 HEV genome copy numbers in the culture medium of Huh7 cells shown in panel A. Values are means ± SEM of data for triplicate samples. (D) ELISA of ORF2 VLPs using anti-ORF2 antibody. Values are represented as means ± SEM of the corrected absorbances ( A 450–650 [absorbance at 450 nm minus the absorbance at 650 nm]) for triplicate samples. (E) ELISA to quantitate ORF2 levels in the culture medium of Huh7 cells expressing p6 HEV and treated with 10 μM CP11, as indicated. Values are means ± SEM of the corrected absorbances ( A 450–650 ) for triplicate samples. (F) Measurement of secreted Gaussia luciferase in the culture medium of Huh7 cells expressing p6 HEV-Luc and treated with 10 μM CP11, as indicated. Luc values were normalized to cell viability values and are represented as means ± SEM. (G) IC 50 estimation for CP11 by QRT-PCR-mediated quantitation of p6 HEV sense RNA levels in the culture medium of Huh7 cells expressing p6 HEV and treated for 24 h with the indicated quantities of CP11 (final concentration). Values are means ± SEM of data from triplicate samples. (H) QRT-PCR measurement of p6 HEV sense-strand RNA in S10-3 cells infected with the PEG-precipitated virus obtained from the culture medium of p6 HEV-electroporated cells treated with CP11 or mock treated. Note that equal amounts of virus (10 6 genome copies) from mock- and CP11-treated culture media were used for infection. As a positive control, 10 7 genome copies of the virus were used for infection. p6 HEV sense RNA values were normalized to that of GAPDH and are represented as means ± SEM of data from triplicate samples. (I) QRT-PCR estimation of the p6 HEV genome copy numbers in the culture medium of S10-3 cells shown in panel H. Media from all three time points were pooled, PEG precipitated, and processed for RNA isolation. Values are means ± SEM. Bars in the graph are the same as for panel H.
    Host Gapdh Rna Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/host gapdh rna levels/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    host gapdh rna levels - by Bioz Stars, 2021-01
    91/100 stars
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    Genes identified are required for virus entry by regulating the expression of receptor ACE2. A. The effect on virion binding and internalization in gene-edited cells. A549-ACE2 cells were incubated with SARS-CoV-2 Sfull infectious virus on ice for binding or then switched to 37°C for internalization. Viral RNA was extracted for RT-qPCR analysis. Data shown are an average of two independent experiments performed in duplicate and are normalized to the controls of individual experiments. B-C. Surface expression of receptor ACE2 was decreased in gene-edited cells as measured by flow cytometry using S1-Fc recombinant protein or anti-ACE2 antibody. D-E. Surface and total expression of receptor ACE2 were decreased in gene-edited cells. The plasma membrane proteins were biotin-labeled and immunoprecipitated by streptavidin beads for Western blotting. One representative blot was shown (D) and Data are pooled from four independent experiments, quantified, and normalized to the controls of individual experiments (E). One-way ANOVA with Dunnett’s test (A, B, C, E); *P

    Journal: bioRxiv

    Article Title: The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

    doi: 10.1101/2020.08.25.266775

    Figure Lengend Snippet: Genes identified are required for virus entry by regulating the expression of receptor ACE2. A. The effect on virion binding and internalization in gene-edited cells. A549-ACE2 cells were incubated with SARS-CoV-2 Sfull infectious virus on ice for binding or then switched to 37°C for internalization. Viral RNA was extracted for RT-qPCR analysis. Data shown are an average of two independent experiments performed in duplicate and are normalized to the controls of individual experiments. B-C. Surface expression of receptor ACE2 was decreased in gene-edited cells as measured by flow cytometry using S1-Fc recombinant protein or anti-ACE2 antibody. D-E. Surface and total expression of receptor ACE2 were decreased in gene-edited cells. The plasma membrane proteins were biotin-labeled and immunoprecipitated by streptavidin beads for Western blotting. One representative blot was shown (D) and Data are pooled from four independent experiments, quantified, and normalized to the controls of individual experiments (E). One-way ANOVA with Dunnett’s test (A, B, C, E); *P

    Article Snippet: Viral or host RNA levels were determined using the TaqPath™ 1-Step RT-qPCR Master Mix (ThermoFisher # A15299) on CFX Connect Real-Time System (Bio-Rad) instrument.

    Techniques: Expressing, Binding Assay, Incubation, Quantitative RT-PCR, Flow Cytometry, Recombinant, Labeling, Immunoprecipitation, Western Blot

    Dose-dependent inhibitory effect of boceprevir, calpain inhibitors II and XII, and GC-376 on HCoV-NL63 viral RNA synthesis in RT-qPCR assay. Positive control remdesivir (A); GC-376 (B); Calpain inhibitor II (C); Calpain inhibitor XII (D); and Boceprevir (E). The left figures represent the normalized RNA levels of the average of three repeats from each concentration tested, and EC 50 curve fittings using log (concentration of inhibitors) vs normalized RNA levels with variable slopes in prism 8 were shown in the right figures. Data are mean ± standard deviation of three replicates.

    Journal: bioRxiv

    Article Title: Boceprevir, calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses in cell culture

    doi: 10.1101/2020.10.30.362335

    Figure Lengend Snippet: Dose-dependent inhibitory effect of boceprevir, calpain inhibitors II and XII, and GC-376 on HCoV-NL63 viral RNA synthesis in RT-qPCR assay. Positive control remdesivir (A); GC-376 (B); Calpain inhibitor II (C); Calpain inhibitor XII (D); and Boceprevir (E). The left figures represent the normalized RNA levels of the average of three repeats from each concentration tested, and EC 50 curve fittings using log (concentration of inhibitors) vs normalized RNA levels with variable slopes in prism 8 were shown in the right figures. Data are mean ± standard deviation of three replicates.

    Article Snippet: 2.0□μg of total RNA was used to synthesize the first strand cDNA of viral RNA and host RNA using SuperScript III reverse transcriptase (Thermo Fisher Scientific) and Random Hexamer primer.

    Techniques: Quantitative RT-PCR, Positive Control, Concentration Assay, Standard Deviation

    CP11 inhibits release of p6 HEV from Huh7 cells. (A) Viability measurement of p6 HEV-expressing Huh7 cells, treated with 10 μM CP11, as indicated. Values are means ± SEM of data for triplicate samples. (B) QRT-PCR measurement of the intracellular level of p6 HEV sense-strand RNA in the samples shown in panel A. p6 HEV sense RNA values were normalized to that of GAPDH and are represented as means ± SEM of data from triplicate samples. (C) QRT-PCR estimation of p6 HEV genome copy numbers in the culture medium of Huh7 cells shown in panel A. Values are means ± SEM of data for triplicate samples. (D) ELISA of ORF2 VLPs using anti-ORF2 antibody. Values are represented as means ± SEM of the corrected absorbances ( A 450–650 [absorbance at 450 nm minus the absorbance at 650 nm]) for triplicate samples. (E) ELISA to quantitate ORF2 levels in the culture medium of Huh7 cells expressing p6 HEV and treated with 10 μM CP11, as indicated. Values are means ± SEM of the corrected absorbances ( A 450–650 ) for triplicate samples. (F) Measurement of secreted Gaussia luciferase in the culture medium of Huh7 cells expressing p6 HEV-Luc and treated with 10 μM CP11, as indicated. Luc values were normalized to cell viability values and are represented as means ± SEM. (G) IC 50 estimation for CP11 by QRT-PCR-mediated quantitation of p6 HEV sense RNA levels in the culture medium of Huh7 cells expressing p6 HEV and treated for 24 h with the indicated quantities of CP11 (final concentration). Values are means ± SEM of data from triplicate samples. (H) QRT-PCR measurement of p6 HEV sense-strand RNA in S10-3 cells infected with the PEG-precipitated virus obtained from the culture medium of p6 HEV-electroporated cells treated with CP11 or mock treated. Note that equal amounts of virus (10 6 genome copies) from mock- and CP11-treated culture media were used for infection. As a positive control, 10 7 genome copies of the virus were used for infection. p6 HEV sense RNA values were normalized to that of GAPDH and are represented as means ± SEM of data from triplicate samples. (I) QRT-PCR estimation of the p6 HEV genome copy numbers in the culture medium of S10-3 cells shown in panel H. Media from all three time points were pooled, PEG precipitated, and processed for RNA isolation. Values are means ± SEM. Bars in the graph are the same as for panel H.

    Journal: Journal of Virology

    Article Title: Potent Inhibition of Hepatitis E Virus Release by a Cyclic Peptide Inhibitor of the Interaction between Viral Open Reading Frame 3 Protein and Host Tumor Susceptibility Gene 101

    doi: 10.1128/JVI.00684-18

    Figure Lengend Snippet: CP11 inhibits release of p6 HEV from Huh7 cells. (A) Viability measurement of p6 HEV-expressing Huh7 cells, treated with 10 μM CP11, as indicated. Values are means ± SEM of data for triplicate samples. (B) QRT-PCR measurement of the intracellular level of p6 HEV sense-strand RNA in the samples shown in panel A. p6 HEV sense RNA values were normalized to that of GAPDH and are represented as means ± SEM of data from triplicate samples. (C) QRT-PCR estimation of p6 HEV genome copy numbers in the culture medium of Huh7 cells shown in panel A. Values are means ± SEM of data for triplicate samples. (D) ELISA of ORF2 VLPs using anti-ORF2 antibody. Values are represented as means ± SEM of the corrected absorbances ( A 450–650 [absorbance at 450 nm minus the absorbance at 650 nm]) for triplicate samples. (E) ELISA to quantitate ORF2 levels in the culture medium of Huh7 cells expressing p6 HEV and treated with 10 μM CP11, as indicated. Values are means ± SEM of the corrected absorbances ( A 450–650 ) for triplicate samples. (F) Measurement of secreted Gaussia luciferase in the culture medium of Huh7 cells expressing p6 HEV-Luc and treated with 10 μM CP11, as indicated. Luc values were normalized to cell viability values and are represented as means ± SEM. (G) IC 50 estimation for CP11 by QRT-PCR-mediated quantitation of p6 HEV sense RNA levels in the culture medium of Huh7 cells expressing p6 HEV and treated for 24 h with the indicated quantities of CP11 (final concentration). Values are means ± SEM of data from triplicate samples. (H) QRT-PCR measurement of p6 HEV sense-strand RNA in S10-3 cells infected with the PEG-precipitated virus obtained from the culture medium of p6 HEV-electroporated cells treated with CP11 or mock treated. Note that equal amounts of virus (10 6 genome copies) from mock- and CP11-treated culture media were used for infection. As a positive control, 10 7 genome copies of the virus were used for infection. p6 HEV sense RNA values were normalized to that of GAPDH and are represented as means ± SEM of data from triplicate samples. (I) QRT-PCR estimation of the p6 HEV genome copy numbers in the culture medium of S10-3 cells shown in panel H. Media from all three time points were pooled, PEG precipitated, and processed for RNA isolation. Values are means ± SEM. Bars in the graph are the same as for panel H.

    Article Snippet: For measuring p6 HEV, g1-HEV sense, and host GAPDH RNA levels, cDNA was synthesized using random hexamers and Superscript III reverse transcriptase (Thermo Fisher Scientific, MA, USA).

    Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Luciferase, Quantitation Assay, Concentration Assay, Infection, Positive Control, Isolation