superscript iii first strand synthesis system for rt pcr  (Thermo Fisher)


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    Name:
    SuperScript III First Strand Synthesis SuperMix for qRT PCR
    Description:
    SuperScript III First Strand Synthesis SuperMix for qRT PCR provides the high temperature capability of SuperScript III Reverse Transcriptase in an optimized SuperMix format for the synthesis of first strand cDNA for use in real time quantitative RT PCR qRT PCR The simple time saving reaction set up uses just two tubes a 2X Reaction Mix and an Enzyme Mix Enzyme mixSuperScript III Reverse Transcriptase included in the RT Enzyme Mix is a version of M MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability The enzyme can be used to synthesize cDNA at a temperature range of 42 60°C providing increased specificity higher yields of cDNA and more full length product than other reverse transcriptases Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA it can be used to synthesize cDNA from total RNA RNaseOUT Recombinant Ribonuclease Inhibitor also included in the enzyme mix is an RNase inhibitor protein that safeguards against the degradation of target RNA due to ribonuclease contamination of the RNA preparation Reaction mixThe 2X RT Reaction Mix includes oligo dT 20 random hexamers MgCl2 and dNTPs in a buffer formulation that has been optimized for qRT PCR E coli RNase H is provided as a separate tube in the kit to remove the RNA template from the cDNA RNA hybrid molecule after first strand synthesis This has been shown to increase sensitivity in qRT PCR Using SuperScript III First Strand Synthesis SuperMixThis SuperMix formulation can be used to quantify fewer than 10 copies of a target gene in qRT PCR with a broad dynamic range that supports accurate quantification of high copy mRNA from up to 1 µg of total RNA Reagents are provided for 50 or 250 RT reactions of 20 µL each
    Catalog Number:
    11752050
    Price:
    None
    Category:
    Kits and Assays
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
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    Structured Review

    Thermo Fisher superscript iii first strand synthesis system for rt pcr
    HIV-Tat gene expression in the kidney of newborn wild type (WT) and HIV-Tg 26 mice infected with adenoviral vectors carrying Lac-Z or HIV-Tat coding sequences. A. The protein sequence of the HIV-Tat gene derived from a child with HIVAN is aligned with HIV-Tat derived from the lymphotropic HIV-IIIB virus or the monocyte-tropic HIV-1 virus ADA, using the Clustal Omega multiple sequence alignment program ( http://www.ebi.ac.uk/Tools/msa/clustalo/ ). The basic domain and RGD motifs are indicated in brackets. The Tat sequencing procedure was repeated <t>three</t> times to rule out the possibility of a sequencing error. B. Newborn HIV-Tg 26 mice were infected with recombinant adenoviral (rAd) vectors carrying either Lac-Z ( rAd- LacZ) or HIV-Tat (rAd-Tat) vectors. Seven days later, all mice were sacrificed, and the kidneys were harvested and processed for the <t>RT-PCR</t> studies using specific Tat primers as described in detail in the methods section. The upper panel shows representative RT-PCR results corresponding to HIV-Tat mRNA expression in the kidney of young wild type (WT) HIV-Tg 26 mice infected either with rAd- LacZ or rAd-Tat vectors. HIV-Tg 26 Tg mice infected with rAd- LacZ vectors showed Tat mRNA levels transcribed from the HIV-1 proviral DNA construct d1443 used to make the HIV-Tg 26 mice. The lower panel shows representative Western blots Tat results in kidney and liver sections derived from wild type mice infected with rAd Lac-Z or rAd-Tat vectors.
    SuperScript III First Strand Synthesis SuperMix for qRT PCR provides the high temperature capability of SuperScript III Reverse Transcriptase in an optimized SuperMix format for the synthesis of first strand cDNA for use in real time quantitative RT PCR qRT PCR The simple time saving reaction set up uses just two tubes a 2X Reaction Mix and an Enzyme Mix Enzyme mixSuperScript III Reverse Transcriptase included in the RT Enzyme Mix is a version of M MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability The enzyme can be used to synthesize cDNA at a temperature range of 42 60°C providing increased specificity higher yields of cDNA and more full length product than other reverse transcriptases Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA it can be used to synthesize cDNA from total RNA RNaseOUT Recombinant Ribonuclease Inhibitor also included in the enzyme mix is an RNase inhibitor protein that safeguards against the degradation of target RNA due to ribonuclease contamination of the RNA preparation Reaction mixThe 2X RT Reaction Mix includes oligo dT 20 random hexamers MgCl2 and dNTPs in a buffer formulation that has been optimized for qRT PCR E coli RNase H is provided as a separate tube in the kit to remove the RNA template from the cDNA RNA hybrid molecule after first strand synthesis This has been shown to increase sensitivity in qRT PCR Using SuperScript III First Strand Synthesis SuperMixThis SuperMix formulation can be used to quantify fewer than 10 copies of a target gene in qRT PCR with a broad dynamic range that supports accurate quantification of high copy mRNA from up to 1 µg of total RNA Reagents are provided for 50 or 250 RT reactions of 20 µL each
    https://www.bioz.com/result/superscript iii first strand synthesis system for rt pcr/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superscript iii first strand synthesis system for rt pcr - by Bioz Stars, 2021-03
    99/100 stars

    Images

    1) Product Images from "An HIV-Tat inducible mouse model system of childhood HIV-associated nephropathy"

    Article Title: An HIV-Tat inducible mouse model system of childhood HIV-associated nephropathy

    Journal: bioRxiv

    doi: 10.1101/2020.05.06.081851

    HIV-Tat gene expression in the kidney of newborn wild type (WT) and HIV-Tg 26 mice infected with adenoviral vectors carrying Lac-Z or HIV-Tat coding sequences. A. The protein sequence of the HIV-Tat gene derived from a child with HIVAN is aligned with HIV-Tat derived from the lymphotropic HIV-IIIB virus or the monocyte-tropic HIV-1 virus ADA, using the Clustal Omega multiple sequence alignment program ( http://www.ebi.ac.uk/Tools/msa/clustalo/ ). The basic domain and RGD motifs are indicated in brackets. The Tat sequencing procedure was repeated three times to rule out the possibility of a sequencing error. B. Newborn HIV-Tg 26 mice were infected with recombinant adenoviral (rAd) vectors carrying either Lac-Z ( rAd- LacZ) or HIV-Tat (rAd-Tat) vectors. Seven days later, all mice were sacrificed, and the kidneys were harvested and processed for the RT-PCR studies using specific Tat primers as described in detail in the methods section. The upper panel shows representative RT-PCR results corresponding to HIV-Tat mRNA expression in the kidney of young wild type (WT) HIV-Tg 26 mice infected either with rAd- LacZ or rAd-Tat vectors. HIV-Tg 26 Tg mice infected with rAd- LacZ vectors showed Tat mRNA levels transcribed from the HIV-1 proviral DNA construct d1443 used to make the HIV-Tg 26 mice. The lower panel shows representative Western blots Tat results in kidney and liver sections derived from wild type mice infected with rAd Lac-Z or rAd-Tat vectors.
    Figure Legend Snippet: HIV-Tat gene expression in the kidney of newborn wild type (WT) and HIV-Tg 26 mice infected with adenoviral vectors carrying Lac-Z or HIV-Tat coding sequences. A. The protein sequence of the HIV-Tat gene derived from a child with HIVAN is aligned with HIV-Tat derived from the lymphotropic HIV-IIIB virus or the monocyte-tropic HIV-1 virus ADA, using the Clustal Omega multiple sequence alignment program ( http://www.ebi.ac.uk/Tools/msa/clustalo/ ). The basic domain and RGD motifs are indicated in brackets. The Tat sequencing procedure was repeated three times to rule out the possibility of a sequencing error. B. Newborn HIV-Tg 26 mice were infected with recombinant adenoviral (rAd) vectors carrying either Lac-Z ( rAd- LacZ) or HIV-Tat (rAd-Tat) vectors. Seven days later, all mice were sacrificed, and the kidneys were harvested and processed for the RT-PCR studies using specific Tat primers as described in detail in the methods section. The upper panel shows representative RT-PCR results corresponding to HIV-Tat mRNA expression in the kidney of young wild type (WT) HIV-Tg 26 mice infected either with rAd- LacZ or rAd-Tat vectors. HIV-Tg 26 Tg mice infected with rAd- LacZ vectors showed Tat mRNA levels transcribed from the HIV-1 proviral DNA construct d1443 used to make the HIV-Tg 26 mice. The lower panel shows representative Western blots Tat results in kidney and liver sections derived from wild type mice infected with rAd Lac-Z or rAd-Tat vectors.

    Techniques Used: Expressing, Mouse Assay, Infection, Sequencing, Derivative Assay, Recombinant, Reverse Transcription Polymerase Chain Reaction, Construct, Western Blot

    2) Product Images from "Epigenetic reprogramming towards mesenchymal-epithelial transition in ovarian cancer-associated mesenchymal stem cells drives metastasis"

    Article Title: Epigenetic reprogramming towards mesenchymal-epithelial transition in ovarian cancer-associated mesenchymal stem cells drives metastasis

    Journal: bioRxiv

    doi: 10.1101/2020.02.25.964197

    Verification of differential DNA methylation loci demonstrating methylation differences persist with MSC differentiation and are acquired during CA-MSC reprogramming. A. DNA methylation heatmap showing six different categories of CpG probes hyper- or hypo-methylated in different combinations of CA-MSC, FTE and MSC. Probe categories and the number of probes included are labelled on the left side of the heatmap. A blue-to-red gradient indicates a beta value of 0-1 (DNA methylation level of 0% to 100%) in all heatmaps. Corresponding cell types or cancer types are labelled on top of each heatmap panel. B. Top DMRs within five different genes chosen for quantitative methylation specific PCR (qMSP) validation. Mean beta values for CA-MSCs and MSCs are plotted on the y-axis against probe locations as plotted on the x-axis. Within-group variability is indicated by standard error bars for each group at each probe locus. C. qMSP validation of differential methylation between groups of CA-MSCs and normal MSCs shown in B, using independent CA-MSC and MSC samples. D. Both CA-MSCs and MSCs that undergo lineage differentiation (adipose and fibroblast differentiation) do not demonstrate significant DNA methylation changes at the selected loci as shown in C. Mean SEM of 3 independent samples are represented. E. Cancer stimulation of normal MSCs in vitro induces a partial CA-MSC methylation pattern. MSCs that underwent partial conversion to a CA-MSC phenotype via hypoxic co-culture with ovarian cancer cells acquire CA-MSC-related methylation changes in three ( SASH1 , ELN , MARVELD2 ) out of the five loci tested. Mean SEM of 3 independent samples are represented.
    Figure Legend Snippet: Verification of differential DNA methylation loci demonstrating methylation differences persist with MSC differentiation and are acquired during CA-MSC reprogramming. A. DNA methylation heatmap showing six different categories of CpG probes hyper- or hypo-methylated in different combinations of CA-MSC, FTE and MSC. Probe categories and the number of probes included are labelled on the left side of the heatmap. A blue-to-red gradient indicates a beta value of 0-1 (DNA methylation level of 0% to 100%) in all heatmaps. Corresponding cell types or cancer types are labelled on top of each heatmap panel. B. Top DMRs within five different genes chosen for quantitative methylation specific PCR (qMSP) validation. Mean beta values for CA-MSCs and MSCs are plotted on the y-axis against probe locations as plotted on the x-axis. Within-group variability is indicated by standard error bars for each group at each probe locus. C. qMSP validation of differential methylation between groups of CA-MSCs and normal MSCs shown in B, using independent CA-MSC and MSC samples. D. Both CA-MSCs and MSCs that undergo lineage differentiation (adipose and fibroblast differentiation) do not demonstrate significant DNA methylation changes at the selected loci as shown in C. Mean SEM of 3 independent samples are represented. E. Cancer stimulation of normal MSCs in vitro induces a partial CA-MSC methylation pattern. MSCs that underwent partial conversion to a CA-MSC phenotype via hypoxic co-culture with ovarian cancer cells acquire CA-MSC-related methylation changes in three ( SASH1 , ELN , MARVELD2 ) out of the five loci tested. Mean SEM of 3 independent samples are represented.

    Techniques Used: DNA Methylation Assay, Methylation, Polymerase Chain Reaction, In Vitro, Co-Culture Assay

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    Quantitative RT-PCR:

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    Expressing:

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    Cell Culture:

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    RNA Extraction:

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    Polymerase Chain Reaction:

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    Concentration Assay:

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    Spectrophotometry:

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    Synthesized:

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    SYBR Green Assay:

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    Sequencing:

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    Isolation:

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    Thermo Fisher superscript iii first strand synthesis supermix for qrt pcr
    HeLa cells show reduced nuclear import, replication, and translation, as well as deficient budding of H1N1 IAV. (A and B) HeLa cells were synchronously infected with either A/WSN/33 or H5N1-HaLo at an MOI of 10, treated with CHX (100 mg/ml), and then incubated for 3 h at 37°C to allow entry (A) or for 4.5 h at 37°C to allow nuclear import (B). Cells were fixed and stained for IAV NP, and immunofluorescence images were acquired using the IC200 imaging system and were analyzed for cytoplasmic (A) or nuclear (B) NP staining. At least <t>three</t> wells per condition were used, and four fields per well were analyzed for quantification. Circles and squares indicate individual wells assayed, and crossbars indicate mean ± standard deviation from two independent experiments. Rel., relative; n.s., not significant. (C) An IAV infection-driven minigenome luciferase assay was used to measure polymerase activity in order to monitor infection with either H5N1-HaLo (left) or A/WSN/33 (right) at the indicated time points postinfection in HeLa (solid red lines) or 293T (solid black lines) cells. The firefly luciferase signal was normalized to the expression of Renilla luciferase, used as a transfection control. Data are mean ± standard error of the mean from three independent biological experiments. (D and E) Representative Western blots of protein lysates from HeLa cells (D) or A549 cells (E) infected with A/WSN/33 or H5N1-HaLo at an MOI of 3, collected at 3, 6, 9, and 12 hpi and probed for expression of IAV PA, NP, and M2 proteins and the loading control β-actin. Three independent biological experiments were performed. The number sign (#) indicates a nonspecific band seen under all conditions. (F) HA cell surface staining in nonpermeabilized HeLa (red bars) or A549 (black bars) cells following infection with A/WSN/33 at an MOI of 0.5. The percentage of HA-positive (HA+) cells, determined by gating of live, single cells and analysis via flow cytometry, is shown. Data are mean ± standard deviation for three independent experiments. (G) 293T or HeLa cells were transfected with plasmids expressing either A/WSN/33 (H1N1) HA or A/Vietnam/1203/04 (H5N1) HA. Cell surface staining of HA was assessed at 48 h posttransfection by flow cytometry. The percentages of HA+ cells are graphed, and histograms of HA+ populations following the gating of live, single cells are shown. A plasmid expressing eGFP was transfected alongside the virus-expressing plasmids as a positive control, and mock-transfected cells served as negative controls. Data are mean ± standard deviation from three independent experiments. (H) HeLa cells were either mock infected or infected at an MOI of 1 with A/WSN/33 or H5N1-HaLo. At 10 hpi, cells were fixed, negatively stained with uranyl acetate, and visualized by transmission electron microscopy. Arrows indicate newly released influenza virions. (I) A549 or HeLa cells were infected with A/WSN/33 at an MOI of 0.01, and supernatant samples were collected at 72 hpi for extraction of total viral RNA. Genomic copies of NP were quantified by <t>qRT-PCR</t> in three independent biological experiments. Asterisks indicate significant differences by multiple Student t tests ( * , P ≤ 0.05; * * , P ≤ 0.001; ** * , P ≤ 0.0001).
    Superscript Iii First Strand Synthesis Supermix For Qrt Pcr, supplied by Thermo Fisher, 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/superscript iii first strand synthesis supermix for qrt pcr/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Thermo Fisher superscript iii first strand synthesis kit
    Effect of perilipin 2 siRNA on lipolysis product-induced perilipin 2 expression. THP-1 monocytes were pre-incubated with perilipin 2 siRNA or scramble siRNA prior to incubation with media control or lipolysis products for 3 hours. A. Perilipin 2 siRNA reduced perilipin 2 gene expression in THP-1 cells treated with media and lipolysis products. Gene expression was measured by <t>qRT-PCR,</t> and normalized to GAPDH expression. Fold change was determined relative to the scramble siRNA media control group, n=9 separate samples per treatment group obtained from <t>three</t> experimental runs with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. B. Perilipin 2 siRNA reduced lipolysis product-induced perilipin 2 protein expression. i. Representative image of western blot. ii. Densitometry analysis of western blot, protein expression determined relative to β-actin, n=3 separate samples per treatment group obtained from one experimental run with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. All data shown is mean +/− standard deviation. Statistical differences are indicated by **(p
    Superscript Iii First Strand Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Thermo Fisher superscript iii first strand synthesis system
    Transcript levels of AGO and other small RNA biogenesis genes. <t>Three</t> different life stages (myc, mycelium; zoo, zoospores; gc, germinated cysts) and an infection time series (3, 6, 12, 24, and 48 h post infection) were assayed in replicate by <t>qRT-PCR</t> to determine transcript levels of (A) DCL 1, DCL 2, RDR , and (B,C) the AGO homologs in P. sojae as compared to housekeeping genes b-tubulin and WS41 . Each life stage or time series point is normalized to the housekeeping gene(s). Standard error bars are included, but in most cases the standard error bars are blocked by the symbol due to low value.
    Superscript Iii First Strand Synthesis System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HeLa cells show reduced nuclear import, replication, and translation, as well as deficient budding of H1N1 IAV. (A and B) HeLa cells were synchronously infected with either A/WSN/33 or H5N1-HaLo at an MOI of 10, treated with CHX (100 mg/ml), and then incubated for 3 h at 37°C to allow entry (A) or for 4.5 h at 37°C to allow nuclear import (B). Cells were fixed and stained for IAV NP, and immunofluorescence images were acquired using the IC200 imaging system and were analyzed for cytoplasmic (A) or nuclear (B) NP staining. At least three wells per condition were used, and four fields per well were analyzed for quantification. Circles and squares indicate individual wells assayed, and crossbars indicate mean ± standard deviation from two independent experiments. Rel., relative; n.s., not significant. (C) An IAV infection-driven minigenome luciferase assay was used to measure polymerase activity in order to monitor infection with either H5N1-HaLo (left) or A/WSN/33 (right) at the indicated time points postinfection in HeLa (solid red lines) or 293T (solid black lines) cells. The firefly luciferase signal was normalized to the expression of Renilla luciferase, used as a transfection control. Data are mean ± standard error of the mean from three independent biological experiments. (D and E) Representative Western blots of protein lysates from HeLa cells (D) or A549 cells (E) infected with A/WSN/33 or H5N1-HaLo at an MOI of 3, collected at 3, 6, 9, and 12 hpi and probed for expression of IAV PA, NP, and M2 proteins and the loading control β-actin. Three independent biological experiments were performed. The number sign (#) indicates a nonspecific band seen under all conditions. (F) HA cell surface staining in nonpermeabilized HeLa (red bars) or A549 (black bars) cells following infection with A/WSN/33 at an MOI of 0.5. The percentage of HA-positive (HA+) cells, determined by gating of live, single cells and analysis via flow cytometry, is shown. Data are mean ± standard deviation for three independent experiments. (G) 293T or HeLa cells were transfected with plasmids expressing either A/WSN/33 (H1N1) HA or A/Vietnam/1203/04 (H5N1) HA. Cell surface staining of HA was assessed at 48 h posttransfection by flow cytometry. The percentages of HA+ cells are graphed, and histograms of HA+ populations following the gating of live, single cells are shown. A plasmid expressing eGFP was transfected alongside the virus-expressing plasmids as a positive control, and mock-transfected cells served as negative controls. Data are mean ± standard deviation from three independent experiments. (H) HeLa cells were either mock infected or infected at an MOI of 1 with A/WSN/33 or H5N1-HaLo. At 10 hpi, cells were fixed, negatively stained with uranyl acetate, and visualized by transmission electron microscopy. Arrows indicate newly released influenza virions. (I) A549 or HeLa cells were infected with A/WSN/33 at an MOI of 0.01, and supernatant samples were collected at 72 hpi for extraction of total viral RNA. Genomic copies of NP were quantified by qRT-PCR in three independent biological experiments. Asterisks indicate significant differences by multiple Student t tests ( * , P ≤ 0.05; * * , P ≤ 0.001; ** * , P ≤ 0.0001).

    Journal: Journal of Virology

    Article Title: Viral Determinants in H5N1 Influenza A Virus Enable Productive Infection of HeLa Cells

    doi: 10.1128/JVI.01410-19

    Figure Lengend Snippet: HeLa cells show reduced nuclear import, replication, and translation, as well as deficient budding of H1N1 IAV. (A and B) HeLa cells were synchronously infected with either A/WSN/33 or H5N1-HaLo at an MOI of 10, treated with CHX (100 mg/ml), and then incubated for 3 h at 37°C to allow entry (A) or for 4.5 h at 37°C to allow nuclear import (B). Cells were fixed and stained for IAV NP, and immunofluorescence images were acquired using the IC200 imaging system and were analyzed for cytoplasmic (A) or nuclear (B) NP staining. At least three wells per condition were used, and four fields per well were analyzed for quantification. Circles and squares indicate individual wells assayed, and crossbars indicate mean ± standard deviation from two independent experiments. Rel., relative; n.s., not significant. (C) An IAV infection-driven minigenome luciferase assay was used to measure polymerase activity in order to monitor infection with either H5N1-HaLo (left) or A/WSN/33 (right) at the indicated time points postinfection in HeLa (solid red lines) or 293T (solid black lines) cells. The firefly luciferase signal was normalized to the expression of Renilla luciferase, used as a transfection control. Data are mean ± standard error of the mean from three independent biological experiments. (D and E) Representative Western blots of protein lysates from HeLa cells (D) or A549 cells (E) infected with A/WSN/33 or H5N1-HaLo at an MOI of 3, collected at 3, 6, 9, and 12 hpi and probed for expression of IAV PA, NP, and M2 proteins and the loading control β-actin. Three independent biological experiments were performed. The number sign (#) indicates a nonspecific band seen under all conditions. (F) HA cell surface staining in nonpermeabilized HeLa (red bars) or A549 (black bars) cells following infection with A/WSN/33 at an MOI of 0.5. The percentage of HA-positive (HA+) cells, determined by gating of live, single cells and analysis via flow cytometry, is shown. Data are mean ± standard deviation for three independent experiments. (G) 293T or HeLa cells were transfected with plasmids expressing either A/WSN/33 (H1N1) HA or A/Vietnam/1203/04 (H5N1) HA. Cell surface staining of HA was assessed at 48 h posttransfection by flow cytometry. The percentages of HA+ cells are graphed, and histograms of HA+ populations following the gating of live, single cells are shown. A plasmid expressing eGFP was transfected alongside the virus-expressing plasmids as a positive control, and mock-transfected cells served as negative controls. Data are mean ± standard deviation from three independent experiments. (H) HeLa cells were either mock infected or infected at an MOI of 1 with A/WSN/33 or H5N1-HaLo. At 10 hpi, cells were fixed, negatively stained with uranyl acetate, and visualized by transmission electron microscopy. Arrows indicate newly released influenza virions. (I) A549 or HeLa cells were infected with A/WSN/33 at an MOI of 0.01, and supernatant samples were collected at 72 hpi for extraction of total viral RNA. Genomic copies of NP were quantified by qRT-PCR in three independent biological experiments. Asterisks indicate significant differences by multiple Student t tests ( * , P ≤ 0.05; * * , P ≤ 0.001; ** * , P ≤ 0.0001).

    Article Snippet: RNA extraction and quantitative reverse transcription-PCR (qRT-PCR).

    Techniques: Infection, Incubation, Staining, Immunofluorescence, Imaging, Standard Deviation, Luciferase, Activity Assay, Expressing, Transfection, Western Blot, Flow Cytometry, Plasmid Preparation, Positive Control, Transmission Assay, Electron Microscopy, Quantitative RT-PCR

    Rsp positively regulates its own expression. (A) The transcript levels of rsp at different growth phases were measured by qRT-PCR. (B) The β-galactosidase activities of the WT and rsp mutant strains with a plasmid pOS rsp were detected in the indicated time points. (C) Analysis of mRNA half-lives of p rsp - lacZ and rsp . Cells were collected after rifampin (200 μg/ml) treatment for RNA isolation, and then the mRNA half-lives were measured by qRT-PCR. (D) SDS-PAGE analysis of Rsp purified from the pRSF-Duet expression vector. The gel was stained with Coomassie blue. (E) EMSA of purified Rsp with the biotin-labelled DNA fragment containing the putative promoter region of rsp . Increasing concentrations of purified Rsp and 4 fmol of the biotin-labelled probe were used in the reactions. The unlabelled probe was added as a specific competitor, and the unlabelled fragment of the hu ORF region was added as a nonspecific competitor. (F) Promoter sequence of the rsp gene. The start points of the truncated probes are marked by arrows. (G) EMSA assay of Rsp with rsp truncated probes. The nonspecific competitor was added to the reactions. The error bars indicate the standard errors of the means of three biological replicates. ** P

    Journal: Emerging Microbes & Infections

    Article Title: Rsp promotes the transcription of virulence factors in an agr-independent manner in Staphylococcus aureus

    doi: 10.1080/22221751.2020.1752116

    Figure Lengend Snippet: Rsp positively regulates its own expression. (A) The transcript levels of rsp at different growth phases were measured by qRT-PCR. (B) The β-galactosidase activities of the WT and rsp mutant strains with a plasmid pOS rsp were detected in the indicated time points. (C) Analysis of mRNA half-lives of p rsp - lacZ and rsp . Cells were collected after rifampin (200 μg/ml) treatment for RNA isolation, and then the mRNA half-lives were measured by qRT-PCR. (D) SDS-PAGE analysis of Rsp purified from the pRSF-Duet expression vector. The gel was stained with Coomassie blue. (E) EMSA of purified Rsp with the biotin-labelled DNA fragment containing the putative promoter region of rsp . Increasing concentrations of purified Rsp and 4 fmol of the biotin-labelled probe were used in the reactions. The unlabelled probe was added as a specific competitor, and the unlabelled fragment of the hu ORF region was added as a nonspecific competitor. (F) Promoter sequence of the rsp gene. The start points of the truncated probes are marked by arrows. (G) EMSA assay of Rsp with rsp truncated probes. The nonspecific competitor was added to the reactions. The error bars indicate the standard errors of the means of three biological replicates. ** P

    Article Snippet: Total RNA isolation, cDNA generation, and real-time quantitative reverse transcription-PCR Overnight cultures of S. aureus were diluted 1:100 in TSB and then grown to the indicated cell density until being collected.

    Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, Plasmid Preparation, Isolation, SDS Page, Purification, Staining, Sequencing

    Rsp positively regulates the transcription of virulence genes. (A) The transcript levels of 7 virulence-related genes were detected by qRT-PCR in the WT and rs p mutant strains. The transcript levels of hla (B), psmα (C), and psmβ (D) in the WT, rs p mutant, and rs p chromosomal-complemented strains at different growth phases. The β-galactosidase activities of hla (E), psmα (F), and psmβ (G) promoter in the WT and rs p mutant strains. Cells were collected at early stationary phase, and the β-galactosidase activity was detected with ONPG. (H) Hla protein levels of the WT, rs p mutant, and rs p chromosomal-complemented strains were measured by Western blot. (I) The transcript levels of hla , psmα , and psmβ in the WT, rs p mutant, and rs p chromosomal-complemented strains in the process of haemolysis. Cells were collected after incubated with 3% sheep red blood cells for 30 min, and the mRNA levels were analysed by qRT-PCR. The error bars indicate the standard errors of the means of three biological replicates. * P

    Journal: Emerging Microbes & Infections

    Article Title: Rsp promotes the transcription of virulence factors in an agr-independent manner in Staphylococcus aureus

    doi: 10.1080/22221751.2020.1752116

    Figure Lengend Snippet: Rsp positively regulates the transcription of virulence genes. (A) The transcript levels of 7 virulence-related genes were detected by qRT-PCR in the WT and rs p mutant strains. The transcript levels of hla (B), psmα (C), and psmβ (D) in the WT, rs p mutant, and rs p chromosomal-complemented strains at different growth phases. The β-galactosidase activities of hla (E), psmα (F), and psmβ (G) promoter in the WT and rs p mutant strains. Cells were collected at early stationary phase, and the β-galactosidase activity was detected with ONPG. (H) Hla protein levels of the WT, rs p mutant, and rs p chromosomal-complemented strains were measured by Western blot. (I) The transcript levels of hla , psmα , and psmβ in the WT, rs p mutant, and rs p chromosomal-complemented strains in the process of haemolysis. Cells were collected after incubated with 3% sheep red blood cells for 30 min, and the mRNA levels were analysed by qRT-PCR. The error bars indicate the standard errors of the means of three biological replicates. * P

    Article Snippet: Total RNA isolation, cDNA generation, and real-time quantitative reverse transcription-PCR Overnight cultures of S. aureus were diluted 1:100 in TSB and then grown to the indicated cell density until being collected.

    Techniques: Quantitative RT-PCR, Mutagenesis, Activity Assay, Western Blot, Incubation

    Effect of perilipin 2 siRNA on lipolysis product-induced perilipin 2 expression. THP-1 monocytes were pre-incubated with perilipin 2 siRNA or scramble siRNA prior to incubation with media control or lipolysis products for 3 hours. A. Perilipin 2 siRNA reduced perilipin 2 gene expression in THP-1 cells treated with media and lipolysis products. Gene expression was measured by qRT-PCR, and normalized to GAPDH expression. Fold change was determined relative to the scramble siRNA media control group, n=9 separate samples per treatment group obtained from three experimental runs with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. B. Perilipin 2 siRNA reduced lipolysis product-induced perilipin 2 protein expression. i. Representative image of western blot. ii. Densitometry analysis of western blot, protein expression determined relative to β-actin, n=3 separate samples per treatment group obtained from one experimental run with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. All data shown is mean +/− standard deviation. Statistical differences are indicated by **(p

    Journal: Food & function

    Article Title: Inhibition of perilipin 2 expression reduces pro-inflammatory gene expression and increases lipid droplet size

    doi: 10.1039/c8fo01420e

    Figure Lengend Snippet: Effect of perilipin 2 siRNA on lipolysis product-induced perilipin 2 expression. THP-1 monocytes were pre-incubated with perilipin 2 siRNA or scramble siRNA prior to incubation with media control or lipolysis products for 3 hours. A. Perilipin 2 siRNA reduced perilipin 2 gene expression in THP-1 cells treated with media and lipolysis products. Gene expression was measured by qRT-PCR, and normalized to GAPDH expression. Fold change was determined relative to the scramble siRNA media control group, n=9 separate samples per treatment group obtained from three experimental runs with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. B. Perilipin 2 siRNA reduced lipolysis product-induced perilipin 2 protein expression. i. Representative image of western blot. ii. Densitometry analysis of western blot, protein expression determined relative to β-actin, n=3 separate samples per treatment group obtained from one experimental run with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. All data shown is mean +/− standard deviation. Statistical differences are indicated by **(p

    Article Snippet: RNA from each sample was reverse transcribed using Superscript III First Strand Synthesis Kit (Life Technologies) according to manufacturer’s instructions. qRT-PCR was performed with FastStart Universal SYBR Green Master (Roche) to quantify the gene expression.

    Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Standard Deviation

    Effects of perilipin 2 siRNA on TGRL lipolysis product-induced gene expression. THP-1 monocytes were pre-incubated with perilipin 2 siRNA or scramble siRNA prior to incubation with media or TGRL lipolysis products for 3 hours. Gene expression for A. SGK1, B. MAFF, C. IL-8, and D. CCL3. Gene expression was analyzed by qRT-PCR, normalized to GAPDH. Fold change is relative to scramble siRNA media control group. All data shown is mean +/− standard deviation, n=9 separate samples per treatment group obtained from three experimental runs with triplicate cultures. For each gene, analysis by two-way ANOVA indicated an interaction of the two factors. Tukey’s post hoc test was used to determine differences between groups. Statistical differences are indicated by **(p

    Journal: Food & function

    Article Title: Inhibition of perilipin 2 expression reduces pro-inflammatory gene expression and increases lipid droplet size

    doi: 10.1039/c8fo01420e

    Figure Lengend Snippet: Effects of perilipin 2 siRNA on TGRL lipolysis product-induced gene expression. THP-1 monocytes were pre-incubated with perilipin 2 siRNA or scramble siRNA prior to incubation with media or TGRL lipolysis products for 3 hours. Gene expression for A. SGK1, B. MAFF, C. IL-8, and D. CCL3. Gene expression was analyzed by qRT-PCR, normalized to GAPDH. Fold change is relative to scramble siRNA media control group. All data shown is mean +/− standard deviation, n=9 separate samples per treatment group obtained from three experimental runs with triplicate cultures. For each gene, analysis by two-way ANOVA indicated an interaction of the two factors. Tukey’s post hoc test was used to determine differences between groups. Statistical differences are indicated by **(p

    Article Snippet: RNA from each sample was reverse transcribed using Superscript III First Strand Synthesis Kit (Life Technologies) according to manufacturer’s instructions. qRT-PCR was performed with FastStart Universal SYBR Green Master (Roche) to quantify the gene expression.

    Techniques: Expressing, Incubation, Quantitative RT-PCR, Standard Deviation

    Transcript levels of AGO and other small RNA biogenesis genes. Three different life stages (myc, mycelium; zoo, zoospores; gc, germinated cysts) and an infection time series (3, 6, 12, 24, and 48 h post infection) were assayed in replicate by qRT-PCR to determine transcript levels of (A) DCL 1, DCL 2, RDR , and (B,C) the AGO homologs in P. sojae as compared to housekeeping genes b-tubulin and WS41 . Each life stage or time series point is normalized to the housekeeping gene(s). Standard error bars are included, but in most cases the standard error bars are blocked by the symbol due to low value.

    Journal: Frontiers in Microbiology

    Article Title: Expansion and Divergence of Argonaute Genes in the Oomycete Genus Phytophthora

    doi: 10.3389/fmicb.2018.02841

    Figure Lengend Snippet: Transcript levels of AGO and other small RNA biogenesis genes. Three different life stages (myc, mycelium; zoo, zoospores; gc, germinated cysts) and an infection time series (3, 6, 12, 24, and 48 h post infection) were assayed in replicate by qRT-PCR to determine transcript levels of (A) DCL 1, DCL 2, RDR , and (B,C) the AGO homologs in P. sojae as compared to housekeeping genes b-tubulin and WS41 . Each life stage or time series point is normalized to the housekeeping gene(s). Standard error bars are included, but in most cases the standard error bars are blocked by the symbol due to low value.

    Article Snippet: The Superscript III First-Strand Synthesis System for RT-PCR kit (Invitrogen, Carlsbad, CA, United States) was used to produce cDNA, followed with purification by phenol:chloroform extraction. cDNA was quantified with a Nanodrop ND-1000 (Thermo Scientific, Waltham, MA, United States) to allow for equal quantities of cDNA template in subsequent reactions.

    Techniques: Infection, Quantitative RT-PCR