superscript iii first strand synthesis supermix  (Thermo Fisher)


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    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
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    None
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    Kits and Assays
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
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    Structured Review

    Thermo Fisher superscript iii first strand synthesis supermix
    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 supermix/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superscript iii first strand synthesis supermix - by Bioz Stars, 2021-03
    99/100 stars

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    Related Articles

    Quantitative RT-PCR:

    Article Title: The MT1G Gene in LUHMES Neurons Is a Sensitive Biomarker of Neurotoxicity
    Article Snippet: .. qRT-PCR For gene expression assays, 3 × 105 cells were cultured per well in 12 well plates with 2 mL medium to enable RNA extraction and quantitative reverse transcriptase PCR (qRT-PCR). .. Cells were treated with each toxicant for 6 h in 2 mL of medium in 12-well microplates.

    Article Title: Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer’s disease
    Article Snippet: Small interfering RNA (siRNA) transfection studiesPrimary microglia were transfected with 40nM (final concentration) of either Msn siRNA (Santa Cruz Biotechnology, Cat. No. sc-35956) or nonspecific sham siRNA (Santa Cruz Biotechnology, Cat. No. sc-37007) using Lipofectamine™ RNAiMAX (Invitrogen, Cat. No. 13778100) and Opti-MEM (Invitrogen, Cat. No. 31985-062). .. After 48 hours, the efficiency of siRNA-mediated gene silencing was confirmed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) ( ). .. Quantitative reverse transcriptase PCR (qRT‐PCR)Total RNA from microglia was extracted using Trizol (Invitrogen, Cat. No. 15596026) and an RNeasy mini extraction kit (Qiagen, Cat. No. 74104) according to the manufacturer’s instructions.

    Article Title: Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility
    Article Snippet: The RNeasy mini kit (Qiagen, West Sussex, UK) was used according to the manufacturer’s instructions. .. RNA concentration was measured using an ND-100 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized using a Superscript III First-Strand Synthesis Supermix for quantitative reverse transcriptase PCR (qRT-PCR) kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. qRT-PCR was carried out using specific primers and fast SYBR Green and run on an ABI Prism 7900HT Sequence detection system. ..

    Article Title: Viral Determinants in H5N1 Influenza A Virus Enable Productive Infection of HeLa Cells
    Article Snippet: At 48 h posttransfection, either cells were infected, and productive virus growth was measured by plaque assay, or cell viability was determined using the CellTiter-Glo assay (Promega). .. RNA extraction and quantitative reverse transcription-PCR (qRT-PCR). .. At 48 h post-siRNA transfection, total RNA was extracted from HeLa cells using an RNeasy minikit (Qiagen).

    Article Title: Production and Functional Characterization of a Recombinant Predicted Pore-Forming Protein (TVSAPLIP12) of Trichomonas vaginalis in Nicotiana benthamiana Plants
    Article Snippet: Complementary DNA (cDNA) was obtained from 1 μg of total RNA using SuperScript IV Reverse Transcriptase and Random Hexamers (Invitrogen) as primers, following manufacturer's instructions. .. Transcription of selected TvSaplip genes 1–8, 11, 12 (GenBank accession no: TVAG_388060 , TVAG_209200 , TVAG_393030 , TVAG_473630 , TVAG_000220 , TVAG_453350 , TVAG_070250 , TVAG_213250 , TVAG_306610 , TVAG_183780 ) was evaluated by quantitative Reverse Transcription PCR (qRT-PCR). .. Specific TaqMan primer/probe sets have been designed by Beacon Designer Software (Premier Biosoft) for all TvSaplip genes except TvSaplip7 .

    Expressing:

    Article Title: The MT1G Gene in LUHMES Neurons Is a Sensitive Biomarker of Neurotoxicity
    Article Snippet: .. qRT-PCR For gene expression assays, 3 × 105 cells were cultured per well in 12 well plates with 2 mL medium to enable RNA extraction and quantitative reverse transcriptase PCR (qRT-PCR). .. Cells were treated with each toxicant for 6 h in 2 mL of medium in 12-well microplates.

    Cell Culture:

    Article Title: The MT1G Gene in LUHMES Neurons Is a Sensitive Biomarker of Neurotoxicity
    Article Snippet: .. qRT-PCR For gene expression assays, 3 × 105 cells were cultured per well in 12 well plates with 2 mL medium to enable RNA extraction and quantitative reverse transcriptase PCR (qRT-PCR). .. Cells were treated with each toxicant for 6 h in 2 mL of medium in 12-well microplates.

    RNA Extraction:

    Article Title: The MT1G Gene in LUHMES Neurons Is a Sensitive Biomarker of Neurotoxicity
    Article Snippet: .. qRT-PCR For gene expression assays, 3 × 105 cells were cultured per well in 12 well plates with 2 mL medium to enable RNA extraction and quantitative reverse transcriptase PCR (qRT-PCR). .. Cells were treated with each toxicant for 6 h in 2 mL of medium in 12-well microplates.

    Article Title: Viral Determinants in H5N1 Influenza A Virus Enable Productive Infection of HeLa Cells
    Article Snippet: At 48 h posttransfection, either cells were infected, and productive virus growth was measured by plaque assay, or cell viability was determined using the CellTiter-Glo assay (Promega). .. RNA extraction and quantitative reverse transcription-PCR (qRT-PCR). .. At 48 h post-siRNA transfection, total RNA was extracted from HeLa cells using an RNeasy minikit (Qiagen).

    Polymerase Chain Reaction:

    Article Title: The MT1G Gene in LUHMES Neurons Is a Sensitive Biomarker of Neurotoxicity
    Article Snippet: .. qRT-PCR For gene expression assays, 3 × 105 cells were cultured per well in 12 well plates with 2 mL medium to enable RNA extraction and quantitative reverse transcriptase PCR (qRT-PCR). .. Cells were treated with each toxicant for 6 h in 2 mL of medium in 12-well microplates.

    Article Title: Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer’s disease
    Article Snippet: Small interfering RNA (siRNA) transfection studiesPrimary microglia were transfected with 40nM (final concentration) of either Msn siRNA (Santa Cruz Biotechnology, Cat. No. sc-35956) or nonspecific sham siRNA (Santa Cruz Biotechnology, Cat. No. sc-37007) using Lipofectamine™ RNAiMAX (Invitrogen, Cat. No. 13778100) and Opti-MEM (Invitrogen, Cat. No. 31985-062). .. After 48 hours, the efficiency of siRNA-mediated gene silencing was confirmed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) ( ). .. Quantitative reverse transcriptase PCR (qRT‐PCR)Total RNA from microglia was extracted using Trizol (Invitrogen, Cat. No. 15596026) and an RNeasy mini extraction kit (Qiagen, Cat. No. 74104) according to the manufacturer’s instructions.

    Article Title: Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility
    Article Snippet: The RNeasy mini kit (Qiagen, West Sussex, UK) was used according to the manufacturer’s instructions. .. RNA concentration was measured using an ND-100 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized using a Superscript III First-Strand Synthesis Supermix for quantitative reverse transcriptase PCR (qRT-PCR) kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. qRT-PCR was carried out using specific primers and fast SYBR Green and run on an ABI Prism 7900HT Sequence detection system. ..

    Article Title: Production and Functional Characterization of a Recombinant Predicted Pore-Forming Protein (TVSAPLIP12) of Trichomonas vaginalis in Nicotiana benthamiana Plants
    Article Snippet: Complementary DNA (cDNA) was obtained from 1 μg of total RNA using SuperScript IV Reverse Transcriptase and Random Hexamers (Invitrogen) as primers, following manufacturer's instructions. .. Transcription of selected TvSaplip genes 1–8, 11, 12 (GenBank accession no: TVAG_388060 , TVAG_209200 , TVAG_393030 , TVAG_473630 , TVAG_000220 , TVAG_453350 , TVAG_070250 , TVAG_213250 , TVAG_306610 , TVAG_183780 ) was evaluated by quantitative Reverse Transcription PCR (qRT-PCR). .. Specific TaqMan primer/probe sets have been designed by Beacon Designer Software (Premier Biosoft) for all TvSaplip genes except TvSaplip7 .

    Article Title: HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells
    Article Snippet: 8 h prior to RNA extraction and purification using the Invitrap spin universal RNA mini kit (Stratec biomedical, Germany), medium was changed for medium containing 1-25 μM flavopiridol (Sigma Aldrich) or 1-25 μM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB; Sigma Aldrich) as indicated. .. Quantitative reverse transcription PCR was performed as previously described ( ). .. Briefly, messenger RNA (mRNA) levels were quantified by TaqMan quantitative RT-PCR (qRT-PCR).

    Concentration Assay:

    Article Title: Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility
    Article Snippet: The RNeasy mini kit (Qiagen, West Sussex, UK) was used according to the manufacturer’s instructions. .. RNA concentration was measured using an ND-100 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized using a Superscript III First-Strand Synthesis Supermix for quantitative reverse transcriptase PCR (qRT-PCR) kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. qRT-PCR was carried out using specific primers and fast SYBR Green and run on an ABI Prism 7900HT Sequence detection system. ..

    Spectrophotometry:

    Article Title: Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility
    Article Snippet: The RNeasy mini kit (Qiagen, West Sussex, UK) was used according to the manufacturer’s instructions. .. RNA concentration was measured using an ND-100 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized using a Superscript III First-Strand Synthesis Supermix for quantitative reverse transcriptase PCR (qRT-PCR) kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. qRT-PCR was carried out using specific primers and fast SYBR Green and run on an ABI Prism 7900HT Sequence detection system. ..

    Synthesized:

    Article Title: Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility
    Article Snippet: The RNeasy mini kit (Qiagen, West Sussex, UK) was used according to the manufacturer’s instructions. .. RNA concentration was measured using an ND-100 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized using a Superscript III First-Strand Synthesis Supermix for quantitative reverse transcriptase PCR (qRT-PCR) kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. qRT-PCR was carried out using specific primers and fast SYBR Green and run on an ABI Prism 7900HT Sequence detection system. ..

    SYBR Green Assay:

    Article Title: Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility
    Article Snippet: The RNeasy mini kit (Qiagen, West Sussex, UK) was used according to the manufacturer’s instructions. .. RNA concentration was measured using an ND-100 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized using a Superscript III First-Strand Synthesis Supermix for quantitative reverse transcriptase PCR (qRT-PCR) kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. qRT-PCR was carried out using specific primers and fast SYBR Green and run on an ABI Prism 7900HT Sequence detection system. ..

    Sequencing:

    Article Title: Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility
    Article Snippet: The RNeasy mini kit (Qiagen, West Sussex, UK) was used according to the manufacturer’s instructions. .. RNA concentration was measured using an ND-100 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized using a Superscript III First-Strand Synthesis Supermix for quantitative reverse transcriptase PCR (qRT-PCR) kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. qRT-PCR was carried out using specific primers and fast SYBR Green and run on an ABI Prism 7900HT Sequence detection system. ..

    Isolation:

    Article Title: Rsp promotes the transcription of virulence factors in an agr-independent manner in Staphylococcus aureus
    Article Snippet: The combinant plasmids were first electroporated into S. aureus strain RN4220 for modification and subsequently transformed into the WT and agr mutant strains to derive the overexpressed strains. .. 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. .. S. aureus cells were collected by centrifugation and processed with 1 ml of RNAiso plus (TaKaRa) in combination with 0.1-mm-diameter silica-zirconia beads in a FastPrep-24 automated system (MP Biomedicals), and the residual DNA was removed with RNase-free DNase I (TransGen, 3 U/μl).

    Article Title: Latrunculin A-Induced Perturbation of the Actin Cytoskeleton Mediates Pap1p-Dependent Induction of the Caf5p Efflux Pump in Schizosaccharomyces pombe
    Article Snippet: S. pombe cells expressing Pap1-GFP fusions were observed live (GFP filter set) using a Leica DMI6000B inverted microscope equipped with a 100 × Plan Apochromat 1.4 NA oil objective and a Photometrics QuantEM:512SC EMCCD camera driven by Metamorph software. .. qRT-PCR Total RNA was isolated using TRIzol Reagent (Life Technologies) according to the supplier’s protocol. cDNA synthesis was performed with 1 µg of total RNA using the SuperScript III First Strand Synthesis Supermix for qRT-PCR kit (Invitrogen), according to the supplier’s protocol. .. Real-time PCR was performed using a Bio-Rad CFX Connect Real-Time PCR Detection System in conjunction with the Maxima SYBR Green qPCR Master Mix kit (Thermo Scientific).

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    Thermo Fisher superscript iii first strand synthesis supermix for qrt pcr
    Transcriptional responses of <t>three</t> biomarker genes to 15 toxicants in six cell lines. The six cell lines indicated at left were treated with vehicle or one of 15 toxicants. Transcriptional responses by DDIT3 , MT1G , PDK4 , and GAPDH were quantified by <t>qRT-PCR</t> at 6 h, and cell viability was assessed at 24 h as intracellular [ATP]. Below the four genes for each cell line is a row of cells that indicate cell viability. White indicates no detectable loss of viability whereas blue color saturation indicates the % of cell death 24 h post-treatment. Toxicant treatments were HCP, Hexachlorophene 10 μM; CAP, captan 10 μM; 6HD, 6-hydroxydopamine 3 μM; MAP, 4-(methylamino)phenol hemisulfate salt 5 μM; CR2, sodium dichromate 5 μM; CLM, chlorambucil 10 μM; MPP, 1-methyl-4-phenylpyridinium 10 μM; MNC, manganese chloride 10 μM; PQ, paraquat 10 μM; STA, staurosporine 2 μM; ROT, rotenone 3 μM; MS5, MS275 10 μM; TER, terfenadine 10 μM; MHG, methylmercury chloride 2 μM; ZIR, ziram, 6 μM
    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
    superscript iii first strand synthesis supermix for qrt pcr - by Bioz Stars, 2021-03
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    Transcriptional responses of three biomarker genes to 15 toxicants in six cell lines. The six cell lines indicated at left were treated with vehicle or one of 15 toxicants. Transcriptional responses by DDIT3 , MT1G , PDK4 , and GAPDH were quantified by qRT-PCR at 6 h, and cell viability was assessed at 24 h as intracellular [ATP]. Below the four genes for each cell line is a row of cells that indicate cell viability. White indicates no detectable loss of viability whereas blue color saturation indicates the % of cell death 24 h post-treatment. Toxicant treatments were HCP, Hexachlorophene 10 μM; CAP, captan 10 μM; 6HD, 6-hydroxydopamine 3 μM; MAP, 4-(methylamino)phenol hemisulfate salt 5 μM; CR2, sodium dichromate 5 μM; CLM, chlorambucil 10 μM; MPP, 1-methyl-4-phenylpyridinium 10 μM; MNC, manganese chloride 10 μM; PQ, paraquat 10 μM; STA, staurosporine 2 μM; ROT, rotenone 3 μM; MS5, MS275 10 μM; TER, terfenadine 10 μM; MHG, methylmercury chloride 2 μM; ZIR, ziram, 6 μM

    Journal: Neurotoxicity research

    Article Title: The MT1G Gene in LUHMES Neurons Is a Sensitive Biomarker of Neurotoxicity

    doi: 10.1007/s12640-020-00272-3

    Figure Lengend Snippet: Transcriptional responses of three biomarker genes to 15 toxicants in six cell lines. The six cell lines indicated at left were treated with vehicle or one of 15 toxicants. Transcriptional responses by DDIT3 , MT1G , PDK4 , and GAPDH were quantified by qRT-PCR at 6 h, and cell viability was assessed at 24 h as intracellular [ATP]. Below the four genes for each cell line is a row of cells that indicate cell viability. White indicates no detectable loss of viability whereas blue color saturation indicates the % of cell death 24 h post-treatment. Toxicant treatments were HCP, Hexachlorophene 10 μM; CAP, captan 10 μM; 6HD, 6-hydroxydopamine 3 μM; MAP, 4-(methylamino)phenol hemisulfate salt 5 μM; CR2, sodium dichromate 5 μM; CLM, chlorambucil 10 μM; MPP, 1-methyl-4-phenylpyridinium 10 μM; MNC, manganese chloride 10 μM; PQ, paraquat 10 μM; STA, staurosporine 2 μM; ROT, rotenone 3 μM; MS5, MS275 10 μM; TER, terfenadine 10 μM; MHG, methylmercury chloride 2 μM; ZIR, ziram, 6 μM

    Article Snippet: qRT-PCR For gene expression assays, 3 × 105 cells were cultured per well in 12 well plates with 2 mL medium to enable RNA extraction and quantitative reverse transcriptase PCR (qRT-PCR).

    Techniques: Biomarker Assay, Quantitative RT-PCR

    eGFP.OR3 punctae in infected cell nuclei are detected independent of HIV-1 transcription. ( a ) eGFP.OR3 and eBFP2.LMNB1 expressing TZM-bl cells were infected with 30 μU RT/cell VSV-G pseudotyped HIV ANCH for 47 h, after which 5 μM of the pTEF-b transcription initiation inhibitor Flavopiridol was added for another 8 h. Number of nuclear IN.SNAP and eGFP.OR3 punctae were quantified. One of three independent experiments is shown with n > 25 cells per sample, error bars represent 95 % CI. ( b,c ) qRT-PCR of viral RNA products specific for gag (u1a) ( b ) and the transcription of the first nucleosome nuc1a ( c ) quantified at 55 h p.i‥ eBFP2.LMNB1 and eGFP.OR3 expressing TZM-bl cells were infected with 5 μU RT/cell VSV-G pseudotyped HIV ANCH. Flavopiridol and the transcription elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) were added 8 h prior RNA extraction. The experiment was performed in biological triplicates and error bars represent SD.

    Journal: bioRxiv

    Article Title: HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells

    doi: 10.1101/2020.11.13.380030

    Figure Lengend Snippet: eGFP.OR3 punctae in infected cell nuclei are detected independent of HIV-1 transcription. ( a ) eGFP.OR3 and eBFP2.LMNB1 expressing TZM-bl cells were infected with 30 μU RT/cell VSV-G pseudotyped HIV ANCH for 47 h, after which 5 μM of the pTEF-b transcription initiation inhibitor Flavopiridol was added for another 8 h. Number of nuclear IN.SNAP and eGFP.OR3 punctae were quantified. One of three independent experiments is shown with n > 25 cells per sample, error bars represent 95 % CI. ( b,c ) qRT-PCR of viral RNA products specific for gag (u1a) ( b ) and the transcription of the first nucleosome nuc1a ( c ) quantified at 55 h p.i‥ eBFP2.LMNB1 and eGFP.OR3 expressing TZM-bl cells were infected with 5 μU RT/cell VSV-G pseudotyped HIV ANCH. Flavopiridol and the transcription elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) were added 8 h prior RNA extraction. The experiment was performed in biological triplicates and error bars represent SD.

    Article Snippet: Quantitative reverse transcription PCR was performed as previously described ( ).

    Techniques: Infection, Expressing, Quantitative RT-PCR, RNA Extraction

    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

    Microbiota independent inflammatory and stress response phenotypes of EPI-/- skin. ( a ) qPCR of 16s RNA gene in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- ear skin samples. ( b ) Number of CD4 + CD3 + cells per mm 2 dermis. ( c ) Number of γδTCR + cells per mm epidermis. ( d ) Number of Vγ3 + cells per mm epidermis. ( e ) qRT-PCR of Rae-1 in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- epidermis. ( f ) Quantification of MPO in WT, EPI-/-, and flora-deficient skin lysates. Data are means ± SEM from at least four ( a ) or three ( b–f ) mice per group. * P

    Journal: The Journal of Investigative Dermatology

    Article Title: Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility

    doi: 10.1038/JID.2015.383

    Figure Lengend Snippet: Microbiota independent inflammatory and stress response phenotypes of EPI-/- skin. ( a ) qPCR of 16s RNA gene in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- ear skin samples. ( b ) Number of CD4 + CD3 + cells per mm 2 dermis. ( c ) Number of γδTCR + cells per mm epidermis. ( d ) Number of Vγ3 + cells per mm epidermis. ( e ) qRT-PCR of Rae-1 in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- epidermis. ( f ) Quantification of MPO in WT, EPI-/-, and flora-deficient skin lysates. Data are means ± SEM from at least four ( a ) or three ( b–f ) mice per group. * P

    Article Snippet: RNA concentration was measured using an ND-100 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized using a Superscript III First-Strand Synthesis Supermix for quantitative reverse transcriptase PCR (qRT-PCR) kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. qRT-PCR was carried out using specific primers and fast SYBR Green and run on an ABI Prism 7900HT Sequence detection system.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mouse Assay

    AMP expression and protease activity in EPI-/- epidermis after microbiota modulation. ( a–k ) qRT-PCR of Camp ( a ) , Defb1 ( b ), Defb2 ( c ), Defb3 ( d ), Defb6 ( e ), S100A9 ( f ), Pla2g2a ( g ), Reg3b ( h ), Reg3g ( i ), and Serpina1b ( j ) in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- epidermis. Data are means ± SEM from at least four mice per group. * P

    Journal: The Journal of Investigative Dermatology

    Article Title: Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility

    doi: 10.1038/JID.2015.383

    Figure Lengend Snippet: AMP expression and protease activity in EPI-/- epidermis after microbiota modulation. ( a–k ) qRT-PCR of Camp ( a ) , Defb1 ( b ), Defb2 ( c ), Defb3 ( d ), Defb6 ( e ), S100A9 ( f ), Pla2g2a ( g ), Reg3b ( h ), Reg3g ( i ), and Serpina1b ( j ) in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- epidermis. Data are means ± SEM from at least four mice per group. * P

    Article Snippet: RNA concentration was measured using an ND-100 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized using a Superscript III First-Strand Synthesis Supermix for quantitative reverse transcriptase PCR (qRT-PCR) kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. qRT-PCR was carried out using specific primers and fast SYBR Green and run on an ABI Prism 7900HT Sequence detection system.

    Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Mouse Assay