one step rt pcr  (Thermo Fisher)


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    AgPath ID One Step RT PCR Reagents
    Description:
    AgPath ID One Step RT PCR Reagents are designed for sensitive robust amplification of RNA targets using a rapid single tube TaqMan real time reverse transcription PCR RT PCR strategy • Recommended for RNA pathogen amplification • Consistent amplification of RNA targets with high specificity and sensitivity • Optimized to work with your target specific primers and probes • Contains ROX for quantitative fluorescent signal normalization AgPath ID One Step RT PCR Reagents are configured for fast and simple reaction setup The reactions are assembled in a single tube minimizing sample handling errors and expediting set up time Once assembled results are available in approximately one hour The 25X RT PCR Enzyme Mix included in the kit contains highly efficient ArrayScript Reverse Transcriptase a mutant MMLV RT that produces high cDNA yields and AmpliTaq Gold polymerase the preferred hot start DNA polymerase for specific target amplification The 2X RT PCR Buffer has been optimized for efficient robust reverse transcription and PCR includes the passive reference dye ROX Dye for quantitative fluorescent signal normalization A Detection Enhancer is also provided as an optional reagent for amplification of templates with a high GC content or persistent secondary structure
    Catalog Number:
    4387391
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    Applications:
    Animal Health|Industrial & Applied Science|One-Step qRT-PCR|PCR & Real-Time PCR|Real Time PCR (qPCR)
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    Thermo Fisher one step rt pcr
    Adrenal gland phenotype in Trpm4 –/– mice. Increased rate of acetylcholine-induced catecholamine release from TRPM4-deficient chromaffin cells. ( A ) Representative examples of H E-stained adrenal gland sections from WT and Trpm4 –/– mice. Scale bars: 100 μm (upper panels) and 20 μm (lower panels). ( B ) Amplification of Trpm4 and Hprt transcripts by <t>RT-PCR</t> from <t>RNA</t> of cell clusters dissected from the adrenal medulla. neg Trpm4 and neg HPRT indicate amplifications without template cDNA. ( C ) Representative traces of carbon fiber amperometry and simultaneous measurements of intracellular calcium concentration ([Ca 2+ ] cyt ) in chromaffin cells from WT and Trpm4 –/– mice. ( D ) Averaged [Ca 2+ ] cyt before (baseline) and at indicated time points after application of acetylcholine (ACh; 10 μM) in chromaffin cells from WT ( n = 9) and Trpm4 –/– mice ( n = 14). ( E ) Number of amperometric events under basal conditions during 5 minutes before stimulation in WT ( n = 9) and Trpm4 –/– ( n = 14) chromaffin cells. ( F ) Box plot (left) and cumulative analysis (right) of amperometric events after stimulation with 10 μM acetylcholine in WT ( n = 9) and Trpm4 –/– ( n = 14) chromaffin cells. * P
    AgPath ID One Step RT PCR Reagents are designed for sensitive robust amplification of RNA targets using a rapid single tube TaqMan real time reverse transcription PCR RT PCR strategy • Recommended for RNA pathogen amplification • Consistent amplification of RNA targets with high specificity and sensitivity • Optimized to work with your target specific primers and probes • Contains ROX for quantitative fluorescent signal normalization AgPath ID One Step RT PCR Reagents are configured for fast and simple reaction setup The reactions are assembled in a single tube minimizing sample handling errors and expediting set up time Once assembled results are available in approximately one hour The 25X RT PCR Enzyme Mix included in the kit contains highly efficient ArrayScript Reverse Transcriptase a mutant MMLV RT that produces high cDNA yields and AmpliTaq Gold polymerase the preferred hot start DNA polymerase for specific target amplification The 2X RT PCR Buffer has been optimized for efficient robust reverse transcription and PCR includes the passive reference dye ROX Dye for quantitative fluorescent signal normalization A Detection Enhancer is also provided as an optional reagent for amplification of templates with a high GC content or persistent secondary structure
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    Images

    1) Product Images from "Increased catecholamine secretion contributes to hypertension in TRPM4-deficient mice"

    Article Title: Increased catecholamine secretion contributes to hypertension in TRPM4-deficient mice

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI41348

    Adrenal gland phenotype in Trpm4 –/– mice. Increased rate of acetylcholine-induced catecholamine release from TRPM4-deficient chromaffin cells. ( A ) Representative examples of H E-stained adrenal gland sections from WT and Trpm4 –/– mice. Scale bars: 100 μm (upper panels) and 20 μm (lower panels). ( B ) Amplification of Trpm4 and Hprt transcripts by RT-PCR from RNA of cell clusters dissected from the adrenal medulla. neg Trpm4 and neg HPRT indicate amplifications without template cDNA. ( C ) Representative traces of carbon fiber amperometry and simultaneous measurements of intracellular calcium concentration ([Ca 2+ ] cyt ) in chromaffin cells from WT and Trpm4 –/– mice. ( D ) Averaged [Ca 2+ ] cyt before (baseline) and at indicated time points after application of acetylcholine (ACh; 10 μM) in chromaffin cells from WT ( n = 9) and Trpm4 –/– mice ( n = 14). ( E ) Number of amperometric events under basal conditions during 5 minutes before stimulation in WT ( n = 9) and Trpm4 –/– ( n = 14) chromaffin cells. ( F ) Box plot (left) and cumulative analysis (right) of amperometric events after stimulation with 10 μM acetylcholine in WT ( n = 9) and Trpm4 –/– ( n = 14) chromaffin cells. * P
    Figure Legend Snippet: Adrenal gland phenotype in Trpm4 –/– mice. Increased rate of acetylcholine-induced catecholamine release from TRPM4-deficient chromaffin cells. ( A ) Representative examples of H E-stained adrenal gland sections from WT and Trpm4 –/– mice. Scale bars: 100 μm (upper panels) and 20 μm (lower panels). ( B ) Amplification of Trpm4 and Hprt transcripts by RT-PCR from RNA of cell clusters dissected from the adrenal medulla. neg Trpm4 and neg HPRT indicate amplifications without template cDNA. ( C ) Representative traces of carbon fiber amperometry and simultaneous measurements of intracellular calcium concentration ([Ca 2+ ] cyt ) in chromaffin cells from WT and Trpm4 –/– mice. ( D ) Averaged [Ca 2+ ] cyt before (baseline) and at indicated time points after application of acetylcholine (ACh; 10 μM) in chromaffin cells from WT ( n = 9) and Trpm4 –/– mice ( n = 14). ( E ) Number of amperometric events under basal conditions during 5 minutes before stimulation in WT ( n = 9) and Trpm4 –/– ( n = 14) chromaffin cells. ( F ) Box plot (left) and cumulative analysis (right) of amperometric events after stimulation with 10 μM acetylcholine in WT ( n = 9) and Trpm4 –/– ( n = 14) chromaffin cells. * P

    Techniques Used: Mouse Assay, Staining, Amplification, Reverse Transcription Polymerase Chain Reaction, Concentration Assay

    2) Product Images from "The Opuntia streptacantha OpsHSP18 Gene Confers Salt and Osmotic Stress Tolerance in Arabidopsis thaliana"

    Article Title: The Opuntia streptacantha OpsHSP18 Gene Confers Salt and Osmotic Stress Tolerance in Arabidopsis thaliana

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms130810154

    ( A ) Schematic diagram of the OpsHSP18 construction in pMDC32 binary vector. RB right border for T-DNA integration, 2 × 35S cauliflower mosaic virus (CaMV) promoter, attB1 and attB2 sites for recombination, OpsHSP18 (small heat shock protein 18) ORF from Opuntia streptacantha , NosT nopaline synthase (Nos) terminator region, HPTII hygromycin resistance gene, LB left border for T-DNA integration. ( B ) Detection of OpsHSP18 transcript in Arabidopsis thaliana 35S::OpsHSP18 -3, -6, and -7 over-expressing lines by semi-quantitative RT-PCR. Total RNA was isolated from 15 day-old A. thaliana seedlings. One microgram of total RNA was used for RT-PCR analysis. A. thaliana Actin8 gene was used as loading control.
    Figure Legend Snippet: ( A ) Schematic diagram of the OpsHSP18 construction in pMDC32 binary vector. RB right border for T-DNA integration, 2 × 35S cauliflower mosaic virus (CaMV) promoter, attB1 and attB2 sites for recombination, OpsHSP18 (small heat shock protein 18) ORF from Opuntia streptacantha , NosT nopaline synthase (Nos) terminator region, HPTII hygromycin resistance gene, LB left border for T-DNA integration. ( B ) Detection of OpsHSP18 transcript in Arabidopsis thaliana 35S::OpsHSP18 -3, -6, and -7 over-expressing lines by semi-quantitative RT-PCR. Total RNA was isolated from 15 day-old A. thaliana seedlings. One microgram of total RNA was used for RT-PCR analysis. A. thaliana Actin8 gene was used as loading control.

    Techniques Used: Plasmid Preparation, Expressing, Quantitative RT-PCR, Isolation, Reverse Transcription Polymerase Chain Reaction

    3) Product Images from "Tobacco Mosaic Virus Regulates the Expression of Its Own Resistance Gene N 1"

    Article Title: Tobacco Mosaic Virus Regulates the Expression of Its Own Resistance Gene N 1

    Journal: Plant Physiology

    doi: 10.1104/pp.104.044859

    Quantitative RT-PCR of N -mRNA from noninfected and PVY (72 h postinoculation)-infected leaves. Left, PCR with 18S-rRNA-specific primers. Right, PCR with N -specific primers. In each section, the top row represents samples from noninfected tobacco NN and the bottom row those from PVY-infected leaves. Cycle numbers are shown above each section.
    Figure Legend Snippet: Quantitative RT-PCR of N -mRNA from noninfected and PVY (72 h postinoculation)-infected leaves. Left, PCR with 18S-rRNA-specific primers. Right, PCR with N -specific primers. In each section, the top row represents samples from noninfected tobacco NN and the bottom row those from PVY-infected leaves. Cycle numbers are shown above each section.

    Techniques Used: Quantitative RT-PCR, Infection, Polymerase Chain Reaction

    4) Product Images from "Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates"

    Article Title: Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.42.10.4759-4764.2004

    Time course of 23S rRNA and groEL mRNA stabilities in ethanol-treated E. coli cells. Gel electrophoresis of RT-PCR products of the 23S rRNA (A) and the groEL mRNA (B) of E. coli ATCC 35218 was performed after LightCycler RT-PCRs as described in Materials and Methods. E. coli cells (10 9 cells per ml, lane 9) were inactivated by incubation with 67% ethanol for 7 min at room temperature for 5 (lane 4), 15 (lane 5), 45 (lane 6), and 70 min (lane 7). Lane 3 contains the untreated 1-to-100 dilution of E. coli , lane 8 contains the no-template control, and lane 1 contains the DNA molecular weight marker.
    Figure Legend Snippet: Time course of 23S rRNA and groEL mRNA stabilities in ethanol-treated E. coli cells. Gel electrophoresis of RT-PCR products of the 23S rRNA (A) and the groEL mRNA (B) of E. coli ATCC 35218 was performed after LightCycler RT-PCRs as described in Materials and Methods. E. coli cells (10 9 cells per ml, lane 9) were inactivated by incubation with 67% ethanol for 7 min at room temperature for 5 (lane 4), 15 (lane 5), 45 (lane 6), and 70 min (lane 7). Lane 3 contains the untreated 1-to-100 dilution of E. coli , lane 8 contains the no-template control, and lane 1 contains the DNA molecular weight marker.

    Techniques Used: Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Incubation, Molecular Weight, Marker

    5) Product Images from "Targeting of highly conserved Dengue virus sequences with anti-Dengue virus trans-splicing group I introns"

    Article Title: Targeting of highly conserved Dengue virus sequences with anti-Dengue virus trans-splicing group I introns

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-11-84

    αDENV-GrpI -FL constructs effectively target and suppress DENV-2 in mosquito cells . A) Confirmation of representative αDENV-GrpI trans-splicing activities. Ae. Albopictus C6/36 cells were transiently transfected with trans -splicing αDENV-GrpI bicistronic vector constructs. Resulting RNAs were analyzed by RT-PCR in the presence (+Rt) or absence (-Rt) of reverse transcriptase to insure observed amplified products were derived from RNA. A PCR amplification product derived from a constructed spliced sequence control (Methods) is provided as a size standard for each gel (DNA+Ctrl). Δ9 and Δ96 refer to Pabc5 deletion mutations located in the trans -splicing domain of the group I intron. These deletions are designed to knock out function providing an adequate negative control [ 52 ]. Arrows indicate the predicted size of the principle splice products resulting from intron activity. The identity of spliced product was confirmed by sequencing. B) C6/36 cells were transformed with trans -splicing αDENV-GrpI bicistronic vector constructs and maintained under 10 mg/ml hygromycin selection for more than 30 doublings. Transformed cells were washed twice in serum free media at 15 hours post plating, infected with DENV-2 NGC (MOI = 0.1). RNAs were analyzed by RT-PCR as described in A). Arrows indicate the predicted size of the principle splice products. C) Ae. albopictus C6/36 cells were transformed with group I intron vector constructs and maintained under hygromycin selection. Transformed cells were washed three times and challenged with DENV 2-NGC (MOI 0.01). Infections were allowed to proceed for 4 days, supernatants were collected, and viral titers were determined by TCID 50 -IFA as described in Methods.
    Figure Legend Snippet: αDENV-GrpI -FL constructs effectively target and suppress DENV-2 in mosquito cells . A) Confirmation of representative αDENV-GrpI trans-splicing activities. Ae. Albopictus C6/36 cells were transiently transfected with trans -splicing αDENV-GrpI bicistronic vector constructs. Resulting RNAs were analyzed by RT-PCR in the presence (+Rt) or absence (-Rt) of reverse transcriptase to insure observed amplified products were derived from RNA. A PCR amplification product derived from a constructed spliced sequence control (Methods) is provided as a size standard for each gel (DNA+Ctrl). Δ9 and Δ96 refer to Pabc5 deletion mutations located in the trans -splicing domain of the group I intron. These deletions are designed to knock out function providing an adequate negative control [ 52 ]. Arrows indicate the predicted size of the principle splice products resulting from intron activity. The identity of spliced product was confirmed by sequencing. B) C6/36 cells were transformed with trans -splicing αDENV-GrpI bicistronic vector constructs and maintained under 10 mg/ml hygromycin selection for more than 30 doublings. Transformed cells were washed twice in serum free media at 15 hours post plating, infected with DENV-2 NGC (MOI = 0.1). RNAs were analyzed by RT-PCR as described in A). Arrows indicate the predicted size of the principle splice products. C) Ae. albopictus C6/36 cells were transformed with group I intron vector constructs and maintained under hygromycin selection. Transformed cells were washed three times and challenged with DENV 2-NGC (MOI 0.01). Infections were allowed to proceed for 4 days, supernatants were collected, and viral titers were determined by TCID 50 -IFA as described in Methods.

    Techniques Used: Construct, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, Derivative Assay, Polymerase Chain Reaction, Sequencing, Knock-Out, Negative Control, Activity Assay, Transformation Assay, Selection, Infection, Immunofluorescence

    6) Product Images from "Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards"

    Article Title: Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.04034-14

    Viral shedding in feces from mallards was quantified by RRT-PCR of the IAV matrix gene. C T values of > 45 were considered to indicate a negative sample. Whiskers indicate standard errors of the means for two samples. The number of days after inoculation
    Figure Legend Snippet: Viral shedding in feces from mallards was quantified by RRT-PCR of the IAV matrix gene. C T values of > 45 were considered to indicate a negative sample. Whiskers indicate standard errors of the means for two samples. The number of days after inoculation

    Techniques Used: Quantitative RT-PCR

    7) Product Images from "Frequency of D222G and Q223R Hemagglutinin Mutants of Pandemic (H1N1) 2009 Influenza Virus in Japan between 2009 and 2010"

    Article Title: Frequency of D222G and Q223R Hemagglutinin Mutants of Pandemic (H1N1) 2009 Influenza Virus in Japan between 2009 and 2010

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030946

    Alignment of the amino acid sequences including D187E, D222G, and Q223R mutants within the receptor binding site of H1N1pdm hemagglutinin. E187, G222, and R223 variants obtained from three clinical specimens (#1, #2, and #3) from the first wave of the outbreak. Three clinical nasal swabs were each subjected to RNA extraction, RT-PCR, and TA cloning. More than one hundred clones per specimen were sequenced using conventional Sanger technology. Positions 187, 222, and 223 are shown in bold and are underlined.
    Figure Legend Snippet: Alignment of the amino acid sequences including D187E, D222G, and Q223R mutants within the receptor binding site of H1N1pdm hemagglutinin. E187, G222, and R223 variants obtained from three clinical specimens (#1, #2, and #3) from the first wave of the outbreak. Three clinical nasal swabs were each subjected to RNA extraction, RT-PCR, and TA cloning. More than one hundred clones per specimen were sequenced using conventional Sanger technology. Positions 187, 222, and 223 are shown in bold and are underlined.

    Techniques Used: Binding Assay, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, TA Cloning, Clone Assay

    8) Product Images from "A Relationship between Carotenoid Accumulation and the Distribution of Species of the Fungus Neurospora in Spain"

    Article Title: A Relationship between Carotenoid Accumulation and the Distribution of Species of the Fungus Neurospora in Spain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033658

    Activation by light of the albino genes in Neurospora species. Quantitative RT-PCR experiments were performed to measure the relative accumulation of al-1 or al-2 mRNA in mycelia of wild-type strains exposed to white light (2 W/m 2 blue light) or kept in the dark. The plots show the average and standard error of the mean of the relative mRNA accumulation in four independent experiments, each with three replicates. The results from each PCR for each gene were normalized to the corresponding PCR for tub-2 to correct for sampling errors and normalized to the result obtained with mycelia kept in the dark. The amount of carotenoids accumulated by each wild-type strain in cultures exposed to light is shown under each strain name. The initials describe each Neurospora species: Nc Neurospora crassa , Nt Neurospora tetrasperma , and Nd Neurospora discreta .
    Figure Legend Snippet: Activation by light of the albino genes in Neurospora species. Quantitative RT-PCR experiments were performed to measure the relative accumulation of al-1 or al-2 mRNA in mycelia of wild-type strains exposed to white light (2 W/m 2 blue light) or kept in the dark. The plots show the average and standard error of the mean of the relative mRNA accumulation in four independent experiments, each with three replicates. The results from each PCR for each gene were normalized to the corresponding PCR for tub-2 to correct for sampling errors and normalized to the result obtained with mycelia kept in the dark. The amount of carotenoids accumulated by each wild-type strain in cultures exposed to light is shown under each strain name. The initials describe each Neurospora species: Nc Neurospora crassa , Nt Neurospora tetrasperma , and Nd Neurospora discreta .

    Techniques Used: Activation Assay, Quantitative RT-PCR, Polymerase Chain Reaction, Sampling

    9) Product Images from "Proteomics reveals novel components of the Anopheles gambiae eggshell"

    Article Title: Proteomics reveals novel components of the Anopheles gambiae eggshell

    Journal: Journal of insect physiology

    doi: 10.1016/j.jinsphys.2010.04.013

    Accumulation of eggshell-related transcripts in Anopheles gambiae ovaries. Total RNA samples extracted from dissected ovaries or carcasses (body without ovaries) were utilized as templates in RT-PCR reactions. Amplified products were resolved by electrophoresis
    Figure Legend Snippet: Accumulation of eggshell-related transcripts in Anopheles gambiae ovaries. Total RNA samples extracted from dissected ovaries or carcasses (body without ovaries) were utilized as templates in RT-PCR reactions. Amplified products were resolved by electrophoresis

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Electrophoresis

    10) Product Images from "Evaluation of Efficacy, Biodistribution, and Inflammation for a Potent siRNA Nanoparticle: Effect of Dexamethasone Co-treatment"

    Article Title: Evaluation of Efficacy, Biodistribution, and Inflammation for a Potent siRNA Nanoparticle: Effect of Dexamethasone Co-treatment

    Journal: Molecular Therapy

    doi: 10.1038/mt.2009.208

    Administration of LNP201 in vivo results in target mRNA silencing. ( a ) Reduction of target mRNA in liver after a single intravenous dose of LNP201. Mice were treated with LNP201/Ssb siRNA at the indicated siRNA doses. At the indicated time points, the medial lobe of the liver was sampled and total RNA was isolated. quantitative PCR was performed to measure Ssb mRNA expression relative to a housekeeping gene, Ppib . Values from mice treated with vehicle only (PBS) were fixed to 100. Values are presented as the mean (five/group) ± SEM. ( b ) LNP potency increases after multiple weekly doses. Mice were treated with LNP201/Ssb siRNA once weekly for 4 weeks. At the indicated time points after the final of 4 doses, liver was collected and [mRNA] was measured as described above. ( c ), LNP201 loaded with a control siRNA sequence does not cause changes in the target mRNA. Mice were treated with either vehicle only (“PBS”) or LNP201/Control siRNA (“CTRL”) for either 48 hours or 1 week, and relative [Ssb mRNA] was measured as described above. ( d ), Activity of LNP201 in the spleen. Spleens were collected from mice (five/group) 48 hours after LNP201/Ssb siRNA or LNP201/CTRL siRNA dosed at 9 mg/kg, and mRNA was measured as described above. LNP, lipid nanoparticle; mRNA, messenger RNA; PBS, phosphate buffered saline; siRNA, small inhibitory RNA.
    Figure Legend Snippet: Administration of LNP201 in vivo results in target mRNA silencing. ( a ) Reduction of target mRNA in liver after a single intravenous dose of LNP201. Mice were treated with LNP201/Ssb siRNA at the indicated siRNA doses. At the indicated time points, the medial lobe of the liver was sampled and total RNA was isolated. quantitative PCR was performed to measure Ssb mRNA expression relative to a housekeeping gene, Ppib . Values from mice treated with vehicle only (PBS) were fixed to 100. Values are presented as the mean (five/group) ± SEM. ( b ) LNP potency increases after multiple weekly doses. Mice were treated with LNP201/Ssb siRNA once weekly for 4 weeks. At the indicated time points after the final of 4 doses, liver was collected and [mRNA] was measured as described above. ( c ), LNP201 loaded with a control siRNA sequence does not cause changes in the target mRNA. Mice were treated with either vehicle only (“PBS”) or LNP201/Control siRNA (“CTRL”) for either 48 hours or 1 week, and relative [Ssb mRNA] was measured as described above. ( d ), Activity of LNP201 in the spleen. Spleens were collected from mice (five/group) 48 hours after LNP201/Ssb siRNA or LNP201/CTRL siRNA dosed at 9 mg/kg, and mRNA was measured as described above. LNP, lipid nanoparticle; mRNA, messenger RNA; PBS, phosphate buffered saline; siRNA, small inhibitory RNA.

    Techniques Used: In Vivo, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Expressing, Sequencing, Activity Assay

    Lipid composition, siRNA design, and mouse biodistribution profile of LNP201. ( a ) Lipid components and ratio of the LNP201 assembly. ( b ) Sequences and chemical modification schemes for Ssb and control siRNAs. ( c ) Biodistribution of siRNA in mice. CD-1 mice (five/group) were dosed intravenously with LNP201/Ssb siRNA; tissues were collected 2 hours later and [Ssb siRNA] was measured by quantitative PCR as described in Materials and Methods. Values on the chart are reported as mean pg [siRNA] per mg of tissue ± SEM. ( d ) siRNA kinetics in mouse tissues after intravenous administration of LNP201. Liver and spleen tissue from mice (5/group) was collected at time points ranging from 2 hours to 1 week postdose; [Ssb siRNA] was measured as described. siRNA, small inhibitory RNA.
    Figure Legend Snippet: Lipid composition, siRNA design, and mouse biodistribution profile of LNP201. ( a ) Lipid components and ratio of the LNP201 assembly. ( b ) Sequences and chemical modification schemes for Ssb and control siRNAs. ( c ) Biodistribution of siRNA in mice. CD-1 mice (five/group) were dosed intravenously with LNP201/Ssb siRNA; tissues were collected 2 hours later and [Ssb siRNA] was measured by quantitative PCR as described in Materials and Methods. Values on the chart are reported as mean pg [siRNA] per mg of tissue ± SEM. ( d ) siRNA kinetics in mouse tissues after intravenous administration of LNP201. Liver and spleen tissue from mice (5/group) was collected at time points ranging from 2 hours to 1 week postdose; [Ssb siRNA] was measured as described. siRNA, small inhibitory RNA.

    Techniques Used: Modification, Mouse Assay, Real-time Polymerase Chain Reaction

    11) Product Images from "Roxithromycin Favorably Modifies the Initial Phase of Resistance against Infection with Macrolide-Resistant Streptococcus pneumoniae in a Murine Pneumonia Model ▿"

    Article Title: Roxithromycin Favorably Modifies the Initial Phase of Resistance against Infection with Macrolide-Resistant Streptococcus pneumoniae in a Murine Pneumonia Model ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01459-06

    Effect of RXM on the expression and activation of MMP-7 in the lung. (A) Expression of MMP-7 mRNA in the lungs after infection. cDNA was generated by one-step RT-PCR using total RNA and specific primers for mouse MMP-7 and GAPDH genes, and PCR products were evaluated by Southern hybridization using cDNA probes specific for MMP-7 and GAPDH genes. At each time point, RNA was extracted from three mice and individual RNA samples were assayed. The same experiments were performed twice. Representative results are shown. (B) Casein zymography using NOVEX 4 to 16% zymogram blue casein gels was performed with the extract (100 μg) of lungs from mice at 12 h postinfection; activated recombinant human MMP-7 (Mat; R D Systems) was included as a positive control. A precursor (28 kDa) and a mature form (19 kDa) were identified by Western blotting using anti-MMP-7 polyclonal antiserum. Extracts of lungs from three mice were prepared, and individual samples were assayed. Representative results are shown. (C) Casein zymography was performed at the indicated time points after infection. The zymograms were inverted and scanned, and caseinolytic activity was determined by scanning the lysis band in the area corresponding to a mature form (19 kDa) of MMP-7 by using a computerized densitometer. Data are representative of results from three separate experiments and are expressed as mean percentages of the activity in the lungs of infected control mice. The error bars represent SEM ( n , 5 to 6 at each time point). *, P of
    Figure Legend Snippet: Effect of RXM on the expression and activation of MMP-7 in the lung. (A) Expression of MMP-7 mRNA in the lungs after infection. cDNA was generated by one-step RT-PCR using total RNA and specific primers for mouse MMP-7 and GAPDH genes, and PCR products were evaluated by Southern hybridization using cDNA probes specific for MMP-7 and GAPDH genes. At each time point, RNA was extracted from three mice and individual RNA samples were assayed. The same experiments were performed twice. Representative results are shown. (B) Casein zymography using NOVEX 4 to 16% zymogram blue casein gels was performed with the extract (100 μg) of lungs from mice at 12 h postinfection; activated recombinant human MMP-7 (Mat; R D Systems) was included as a positive control. A precursor (28 kDa) and a mature form (19 kDa) were identified by Western blotting using anti-MMP-7 polyclonal antiserum. Extracts of lungs from three mice were prepared, and individual samples were assayed. Representative results are shown. (C) Casein zymography was performed at the indicated time points after infection. The zymograms were inverted and scanned, and caseinolytic activity was determined by scanning the lysis band in the area corresponding to a mature form (19 kDa) of MMP-7 by using a computerized densitometer. Data are representative of results from three separate experiments and are expressed as mean percentages of the activity in the lungs of infected control mice. The error bars represent SEM ( n , 5 to 6 at each time point). *, P of

    Techniques Used: Expressing, Activation Assay, Infection, Generated, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Hybridization, Mouse Assay, Zymography, Recombinant, Positive Control, Western Blot, Activity Assay, Lysis

    12) Product Images from "Inhibition of the AIF/CypA complex protects against intrinsic death pathways induced by oxidative stress"

    Article Title: Inhibition of the AIF/CypA complex protects against intrinsic death pathways induced by oxidative stress

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.518

    Deletion of CypA prevents the apoptotic process. ( a ) HT-22 cells were transfected with scrambled siRNA (siCtrl) or with siRNA against mouse CypA (siCypA). Total cell lysates from siRNA-treated cells were prepared and the mRNA levels and expression levels of CypA were assessed by RT-PCR (upper panel) and immunoblotting (lower panel), respectively. Expression levels of CypA were significantly reduced in cells transfected with the specific siRNA against the protein. ( b ) Immunofluorescent labeling of AIF (red) and nuclei (DAPI, blue) in HT-22 cells transfected with siCtrl or siCypA, followed by exposure to 2 mM glutamate for 12 h (Glut) as indicated. CypA-downregulated HT-22 cells showed very weak nuclear AIF staining in injured cells. ( c ) Cell viability assessed by the MTT assay in wild-type and CypA knockdown HT-22 cells treated or untreated (control) with 2 mM glutamate for 12 h. Data show that wild-type cells (treated with vehicle) or with the siCtrl underwent apoptosis on glutamate insult, whereas those expressing reduced levels of CypA were more resistant to the glutamate challenge. MTT results are presented as percentage of control, considered to be 100%, and represent mean±S.D. of three independent experiments. ( d ) Cell mortality in CypA-downregulated HT-22 cells on glutamate treatment (2 mM at 0 h, n =8) was also assessed over a time period of about 15 h by impedance measurement. Data show again that cells underwent apoptosis by glutamate only in the presence of CypA. Cell death in wild-type and CypA-blunted cells was also analyzed by Annexin-V-FITC/PI double staining ( e ), showing that apoptosis was abrogated when CypA expression was silenced by specific siRNA. Data are reported as mean±S.D. ** P
    Figure Legend Snippet: Deletion of CypA prevents the apoptotic process. ( a ) HT-22 cells were transfected with scrambled siRNA (siCtrl) or with siRNA against mouse CypA (siCypA). Total cell lysates from siRNA-treated cells were prepared and the mRNA levels and expression levels of CypA were assessed by RT-PCR (upper panel) and immunoblotting (lower panel), respectively. Expression levels of CypA were significantly reduced in cells transfected with the specific siRNA against the protein. ( b ) Immunofluorescent labeling of AIF (red) and nuclei (DAPI, blue) in HT-22 cells transfected with siCtrl or siCypA, followed by exposure to 2 mM glutamate for 12 h (Glut) as indicated. CypA-downregulated HT-22 cells showed very weak nuclear AIF staining in injured cells. ( c ) Cell viability assessed by the MTT assay in wild-type and CypA knockdown HT-22 cells treated or untreated (control) with 2 mM glutamate for 12 h. Data show that wild-type cells (treated with vehicle) or with the siCtrl underwent apoptosis on glutamate insult, whereas those expressing reduced levels of CypA were more resistant to the glutamate challenge. MTT results are presented as percentage of control, considered to be 100%, and represent mean±S.D. of three independent experiments. ( d ) Cell mortality in CypA-downregulated HT-22 cells on glutamate treatment (2 mM at 0 h, n =8) was also assessed over a time period of about 15 h by impedance measurement. Data show again that cells underwent apoptosis by glutamate only in the presence of CypA. Cell death in wild-type and CypA-blunted cells was also analyzed by Annexin-V-FITC/PI double staining ( e ), showing that apoptosis was abrogated when CypA expression was silenced by specific siRNA. Data are reported as mean±S.D. ** P

    Techniques Used: Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Labeling, Staining, MTT Assay, Double Staining

    13) Product Images from "Transcriptional Suppression of miR-181c by Hepatitis C Virus Enhances Homeobox A1 Expression"

    Article Title: Transcriptional Suppression of miR-181c by Hepatitis C Virus Enhances Homeobox A1 Expression

    Journal: Journal of Virology

    doi: 10.1128/JVI.00787-14

    HCV infection suppresses miR-181c expression. (A) Primary human hepatocytes were mock treated or HCV infected for 96 h. Total RNA from mock- or virus-infected cells was extracted, and the expression level of miR-181c was measured by qRT-PCR and normalized
    Figure Legend Snippet: HCV infection suppresses miR-181c expression. (A) Primary human hepatocytes were mock treated or HCV infected for 96 h. Total RNA from mock- or virus-infected cells was extracted, and the expression level of miR-181c was measured by qRT-PCR and normalized

    Techniques Used: Infection, Expressing, Quantitative RT-PCR

    14) Product Images from "Genes affected by mouse mammary tumor virus (MMTV) proviral insertions in mouse mammary tumors are deregulated or mutated in primary human mammary tumors"

    Article Title: Genes affected by mouse mammary tumor virus (MMTV) proviral insertions in mouse mammary tumors are deregulated or mutated in primary human mammary tumors

    Journal: Oncotarget

    doi:

    Agarose Gel Analysis of Intragenic RT PCR Products RT-PCR verification of MMTV CIS intragenic expression of MMTV LTR U5-host chimeric mRNA from MMTV induced mammary tumors. Agarose gel (2%) electrophoresis of the PCR product using nested MMTV LTR U5 and host exon primers ( Supplementary Table 2 ). The size of the fragments, in base pairs (bp) is indicated on the Y-axis and is 350 bp for Usp31 , 500 bp for Nckap5 , 100 bp for Cadm2 and 600 bp for Notch4 . The name of the gene and a H 2 O control is indicated.
    Figure Legend Snippet: Agarose Gel Analysis of Intragenic RT PCR Products RT-PCR verification of MMTV CIS intragenic expression of MMTV LTR U5-host chimeric mRNA from MMTV induced mammary tumors. Agarose gel (2%) electrophoresis of the PCR product using nested MMTV LTR U5 and host exon primers ( Supplementary Table 2 ). The size of the fragments, in base pairs (bp) is indicated on the Y-axis and is 350 bp for Usp31 , 500 bp for Nckap5 , 100 bp for Cadm2 and 600 bp for Notch4 . The name of the gene and a H 2 O control is indicated.

    Techniques Used: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Expressing, Electrophoresis, Polymerase Chain Reaction

    15) Product Images from "Preliminary evaluation on the efficiency of the kit Platelia Dengue NS1 Ag-ELISA to detect dengue virus in dried Aedes aegypti: a potential tool to improve dengue surveillance"

    Article Title: Preliminary evaluation on the efficiency of the kit Platelia Dengue NS1 Ag-ELISA to detect dengue virus in dried Aedes aegypti: a potential tool to improve dengue surveillance

    Journal: Parasites & Vectors

    doi: 10.1186/1756-3305-7-155

    Comparison between the median optical density revealed by the Platelia Dengue NS1 Ag-ELISA (bars) and the median number of RNA copies determined by qRT-PCR (line) of Aedes aegypti mosquitoes killed naturally and analyzed for dengue virus detection between 0–72 h after death.
    Figure Legend Snippet: Comparison between the median optical density revealed by the Platelia Dengue NS1 Ag-ELISA (bars) and the median number of RNA copies determined by qRT-PCR (line) of Aedes aegypti mosquitoes killed naturally and analyzed for dengue virus detection between 0–72 h after death.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    16) Product Images from "Temporal expression and tissue distribution of interleukin-1? in two strains of guinea pigs with varying propensity for spontaneous knee osteoarthritis"

    Article Title: Temporal expression and tissue distribution of interleukin-1? in two strains of guinea pigs with varying propensity for spontaneous knee osteoarthritis

    Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

    doi: 10.1016/j.joca.2011.01.004

    Median (range provided) immunohistochemistry (IHC) indices (A) and corresponding photomicrographs (measure bar = 40μm) demonstrating significant differences (B) for IL-1β expression in weight-bearing knee cartilage of Hartley and Strain 13 guinea pigs. RT-PCR results (C) performed on cartilage samples on days 60, 120, and 180 days of age confirmed the reported IHC IL-1β protein expression. Within strain IHC indices for Hartley guinea pigs were not statistically different at the time points investigated. Significant decreases in index values were found within Strain 13 animals over time at 120 and 180 days of age (p=0.01†). Significant differences in positive cells and/or matrix staining were noted between the two strains at 60, 120, 180 days of age, as indicated (NS=not significant, p > 0.05).
    Figure Legend Snippet: Median (range provided) immunohistochemistry (IHC) indices (A) and corresponding photomicrographs (measure bar = 40μm) demonstrating significant differences (B) for IL-1β expression in weight-bearing knee cartilage of Hartley and Strain 13 guinea pigs. RT-PCR results (C) performed on cartilage samples on days 60, 120, and 180 days of age confirmed the reported IHC IL-1β protein expression. Within strain IHC indices for Hartley guinea pigs were not statistically different at the time points investigated. Significant decreases in index values were found within Strain 13 animals over time at 120 and 180 days of age (p=0.01†). Significant differences in positive cells and/or matrix staining were noted between the two strains at 60, 120, 180 days of age, as indicated (NS=not significant, p > 0.05).

    Techniques Used: Immunohistochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining

    17) Product Images from "One-Step Reverse Transcription-Polymerase Chain Reaction for Ebola and Marburg Viruses"

    Article Title: One-Step Reverse Transcription-Polymerase Chain Reaction for Ebola and Marburg Viruses

    Journal: Osong Public Health and Research Perspectives

    doi: 10.1016/j.phrp.2016.04.004

    Sensitivity of conventional RT-PCR, amplified with specific primers for filoviruses. Determination of the detection limit of filoviral RNAs using RT-PCR and 10-fold serial dilutions of RNA transcripts. Input RNA dilutions are indicated above the lanes. BEBOV = Bundibugyo EBOV; EBOV = Ebola virus; Lane M = 1-kb DNA ladder; Lane NC = negative control; MARV = Marburg virus; MMARV = MARV-Musoke; REBOV = Reston EBOV; RMARV = Ravn virus; RT-PCR = reverse transcription-polymerase chain reaction; SEBOV = Sudan EBOV; TEBOV = Taï Forest EBOV; ZEBOV = Zaire EBOV.
    Figure Legend Snippet: Sensitivity of conventional RT-PCR, amplified with specific primers for filoviruses. Determination of the detection limit of filoviral RNAs using RT-PCR and 10-fold serial dilutions of RNA transcripts. Input RNA dilutions are indicated above the lanes. BEBOV = Bundibugyo EBOV; EBOV = Ebola virus; Lane M = 1-kb DNA ladder; Lane NC = negative control; MARV = Marburg virus; MMARV = MARV-Musoke; REBOV = Reston EBOV; RMARV = Ravn virus; RT-PCR = reverse transcription-polymerase chain reaction; SEBOV = Sudan EBOV; TEBOV = Taï Forest EBOV; ZEBOV = Zaire EBOV.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control

    18) Product Images from "Complete study demonstrating the absence of rhabdovirus in a distinct Sf9 cell line"

    Article Title: Complete study demonstrating the absence of rhabdovirus in a distinct Sf9 cell line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175633

    (A). RT-PCR of total RNA from Sf9 cells. Primers were designed to amplify 14 target sequences that cover 99.1% of the reported Sf-rhabdovirus RNA in an overlapping manner as indicated by the gene schematic. Sizes of 13 target sequences ranged from ~1.0 to 1.3 kb (lanes 1–13) and one target sequence was ~0.5 kb (lane 14). The sizes of amplicons matched the expected sizes of target sequences except for one amplicon (*, lane 7), which was amplified by primers targeting bp 6965–7134. (B). Sequencing of the amplicon revealed a deletion of 320 bp at position 6371–6690 in Sf-rhabdovirus RNA. The 320 bp deletion contains the 3’ region of ORF-X and a part of the intergenic region between ORF-X and ORF-L that includes rhabdovirus conserved transcription motifs for the X and L genes.
    Figure Legend Snippet: (A). RT-PCR of total RNA from Sf9 cells. Primers were designed to amplify 14 target sequences that cover 99.1% of the reported Sf-rhabdovirus RNA in an overlapping manner as indicated by the gene schematic. Sizes of 13 target sequences ranged from ~1.0 to 1.3 kb (lanes 1–13) and one target sequence was ~0.5 kb (lane 14). The sizes of amplicons matched the expected sizes of target sequences except for one amplicon (*, lane 7), which was amplified by primers targeting bp 6965–7134. (B). Sequencing of the amplicon revealed a deletion of 320 bp at position 6371–6690 in Sf-rhabdovirus RNA. The 320 bp deletion contains the 3’ region of ORF-X and a part of the intergenic region between ORF-X and ORF-L that includes rhabdovirus conserved transcription motifs for the X and L genes.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification

    19) Product Images from "The CFTR frameshift mutation 3905insT and its effect at transcript and protein level"

    Article Title: The CFTR frameshift mutation 3905insT and its effect at transcript and protein level

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2009.140

    RT–PCR results obtained for the amplification of a region encompassing exons 19–24 of the CFTR mRNA derived from EBV-transformed lymphocytes of F508del/3905insT patients ( a ), nasal epithelial cells of F508del/3905insT patients ( b ), and
    Figure Legend Snippet: RT–PCR results obtained for the amplification of a region encompassing exons 19–24 of the CFTR mRNA derived from EBV-transformed lymphocytes of F508del/3905insT patients ( a ), nasal epithelial cells of F508del/3905insT patients ( b ), and

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Derivative Assay, Transformation Assay

    20) Product Images from "Replication competence of virions induced from CD4+ lymphocytes latently infected with HIV"

    Article Title: Replication competence of virions induced from CD4+ lymphocytes latently infected with HIV

    Journal: Retrovirology

    doi: 10.1186/s12977-019-0466-1

    Parallel QVOA results in the presence or absence of raltegravir with CD4 lymphocytes from subject 219. Six replicates were performed for each serial threefold dilution of CD4 cells in the QVOA assay with ( a , left row) or without ( a right row) raltegravir in the culture medium throughout the assay shown on a log 10 scale. Culture supernatants were assayed for HIV RNA by real time (RT)-PCR. The asterisks on the lines of the replicates without raltegravir on day 14 indicate values for each well without raltegravir that exceeded the mean plus 5 standard deviations of the 6 replicates performed with raltegravir. b Displays the same data for day 14 to indicate visually that virions are induced in the presence of raltegravir (upper figure) and that amplification occurs in almost as many wells (lower figure)
    Figure Legend Snippet: Parallel QVOA results in the presence or absence of raltegravir with CD4 lymphocytes from subject 219. Six replicates were performed for each serial threefold dilution of CD4 cells in the QVOA assay with ( a , left row) or without ( a right row) raltegravir in the culture medium throughout the assay shown on a log 10 scale. Culture supernatants were assayed for HIV RNA by real time (RT)-PCR. The asterisks on the lines of the replicates without raltegravir on day 14 indicate values for each well without raltegravir that exceeded the mean plus 5 standard deviations of the 6 replicates performed with raltegravir. b Displays the same data for day 14 to indicate visually that virions are induced in the presence of raltegravir (upper figure) and that amplification occurs in almost as many wells (lower figure)

    Techniques Used: Quantitative RT-PCR, Amplification

    21) Product Images from "Expression and Function of Serotonin 2A and 2B Receptors in the Mammalian Respiratory Network"

    Article Title: Expression and Function of Serotonin 2A and 2B Receptors in the Mammalian Respiratory Network

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021395

    Quantification of expression levels and co-expression of 5-HT 2A and 5-HT 2B Rs within the pontine respiratory network. ( A ) ( a ) shows a schematic representation of the dissected pre-BötC and its landmarks: pre-Bötzinger complex (pre-BötC), nucleus of the solitary tract (Sol), nucleus ambiguus (NA), hypoglossal nucleus (XII), principal nucleus of the inferior olive (IO Pr ). ( b ) shows the specific mRNA of both receptors detected in the pre-BötC. ( B ) Double labeling of 5-HT 2A and 5-HT 2B Rs. 5-HT 2A (Cy5, red) and 5-HT 2B Rs (Cy2, green) are strongly co-expressed in pre-BötC-neurons. Immunohistochemical analysis does not reveal the ratio of co-expressed proteins. Therefore, we performed quantitative real-time RT-PCR on four selected nuclei of the respiratory network ( C ). The bar diagram represents results of quantitative real-time RT-PCR analysis of 5-HT 2 R genes ( Htr2a , Htr2b ) of spinal cord (Spc), inferior olive (IO), pre-Bötzinger complex (pre-BötC), and parabrachial complex (PB). At the RNA level 5-HT 2A R is significantly stronger expressed compared to Htr2b in all regions analyzed. Asterisks indicate significance (*** = p
    Figure Legend Snippet: Quantification of expression levels and co-expression of 5-HT 2A and 5-HT 2B Rs within the pontine respiratory network. ( A ) ( a ) shows a schematic representation of the dissected pre-BötC and its landmarks: pre-Bötzinger complex (pre-BötC), nucleus of the solitary tract (Sol), nucleus ambiguus (NA), hypoglossal nucleus (XII), principal nucleus of the inferior olive (IO Pr ). ( b ) shows the specific mRNA of both receptors detected in the pre-BötC. ( B ) Double labeling of 5-HT 2A and 5-HT 2B Rs. 5-HT 2A (Cy5, red) and 5-HT 2B Rs (Cy2, green) are strongly co-expressed in pre-BötC-neurons. Immunohistochemical analysis does not reveal the ratio of co-expressed proteins. Therefore, we performed quantitative real-time RT-PCR on four selected nuclei of the respiratory network ( C ). The bar diagram represents results of quantitative real-time RT-PCR analysis of 5-HT 2 R genes ( Htr2a , Htr2b ) of spinal cord (Spc), inferior olive (IO), pre-Bötzinger complex (pre-BötC), and parabrachial complex (PB). At the RNA level 5-HT 2A R is significantly stronger expressed compared to Htr2b in all regions analyzed. Asterisks indicate significance (*** = p

    Techniques Used: Expressing, Labeling, Immunohistochemistry, Quantitative RT-PCR

    22) Product Images from "Ectopic expression of Triticum aestivum SERK genes (TaSERKs) control plant growth and development in Arabidopsis"

    Article Title: Ectopic expression of Triticum aestivum SERK genes (TaSERKs) control plant growth and development in Arabidopsis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10038-1

    Expression profiles of TaSERK1 , 2 , 3 , 4 and 5 by qRT-PCR. cDNAs normalized to housekeeping gene, ACTIN , in different tissues. The error bars represent mean ± SD of two biological replicates, each analysed with three technical replicates. Asterisks above error bars represent the significance levels (Students t -test; *p value ≤ 0.05).
    Figure Legend Snippet: Expression profiles of TaSERK1 , 2 , 3 , 4 and 5 by qRT-PCR. cDNAs normalized to housekeeping gene, ACTIN , in different tissues. The error bars represent mean ± SD of two biological replicates, each analysed with three technical replicates. Asterisks above error bars represent the significance levels (Students t -test; *p value ≤ 0.05).

    Techniques Used: Expressing, Quantitative RT-PCR

    23) Product Images from "Analysis of Multiply Spliced Transcripts in Lymphoid Tissue Reservoirs of Rhesus Macaques Infected with RT-SHIV during HAART"

    Article Title: Analysis of Multiply Spliced Transcripts in Lymphoid Tissue Reservoirs of Rhesus Macaques Infected with RT-SHIV during HAART

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087914

    Validation of TaqMan PCR for MS mRNA, multiplex preamplification PCR, and distribution of GAPDH RNA in macaque tissues. A) Vectors containing a portion of the MS transcript with or without the CAG nucleotides at the coding exon-1 and coding exon-2 splice junction were used as standards for real-time TaqMan PCR. The plasmids were quantified by A260 spectrophotometric readings and serially diluted for quantification by TaqMan PCR. Data shown are the average threshold cycle (C t ) +/− standard deviation of triplicate experiments, with some standard deviations too small to appear. The lines indicate regression analysis used to obtain line equations for calculating the amplification efficiencies and for the quantification of MS mRNA in samples. B) cDNA was generated from total RNA from RT-SHIV-infected CEMx174 cells isolated 48 hours post-infection using random hexamers primers. Multiplex preamplification PCR was performed for viral MS mRNA and gag RNA in addition to the cellular housekeeping gene GAPDH RNA. Individual reactions were removed from the cycler every five cycles, followed by quantification of the three transcripts by TaqMan PCR to determine whether the preamplification step was linear. Data shown are the average +/− standard deviation of triplicate experiments, with some standard deviations too small to appear. The lines indicate regression analysis. C) Total RNA was isolated from tissues and quantified by two-step TaqMan RT-PCR. Total cDNA was preamplified for 25 cycles in multiplex PCR before TaqMan PCR quantification. Cellular GAPDH RNA was measured in these six tissues from all nine macaques in order to determine whether it could serve as a standard. The solid symbols indicate samples from animals that received only HAART while the open symbols indicate samples from animals that received HAART plus induction therapy. The horizontal lines represent the averages with 95% confidence intervals.
    Figure Legend Snippet: Validation of TaqMan PCR for MS mRNA, multiplex preamplification PCR, and distribution of GAPDH RNA in macaque tissues. A) Vectors containing a portion of the MS transcript with or without the CAG nucleotides at the coding exon-1 and coding exon-2 splice junction were used as standards for real-time TaqMan PCR. The plasmids were quantified by A260 spectrophotometric readings and serially diluted for quantification by TaqMan PCR. Data shown are the average threshold cycle (C t ) +/− standard deviation of triplicate experiments, with some standard deviations too small to appear. The lines indicate regression analysis used to obtain line equations for calculating the amplification efficiencies and for the quantification of MS mRNA in samples. B) cDNA was generated from total RNA from RT-SHIV-infected CEMx174 cells isolated 48 hours post-infection using random hexamers primers. Multiplex preamplification PCR was performed for viral MS mRNA and gag RNA in addition to the cellular housekeeping gene GAPDH RNA. Individual reactions were removed from the cycler every five cycles, followed by quantification of the three transcripts by TaqMan PCR to determine whether the preamplification step was linear. Data shown are the average +/− standard deviation of triplicate experiments, with some standard deviations too small to appear. The lines indicate regression analysis. C) Total RNA was isolated from tissues and quantified by two-step TaqMan RT-PCR. Total cDNA was preamplified for 25 cycles in multiplex PCR before TaqMan PCR quantification. Cellular GAPDH RNA was measured in these six tissues from all nine macaques in order to determine whether it could serve as a standard. The solid symbols indicate samples from animals that received only HAART while the open symbols indicate samples from animals that received HAART plus induction therapy. The horizontal lines represent the averages with 95% confidence intervals.

    Techniques Used: Polymerase Chain Reaction, Mass Spectrometry, Multiplex Assay, Standard Deviation, Amplification, Generated, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction

    24) Product Images from "Influenza A(H7N9) Virus Acquires Resistance-Related Neuraminidase I222T Substitution When Infected Mallards Are Exposed to Low Levels of Oseltamivir in Water"

    Article Title: Influenza A(H7N9) Virus Acquires Resistance-Related Neuraminidase I222T Substitution When Infected Mallards Are Exposed to Low Levels of Oseltamivir in Water

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00886-15

    Experimental mallard model with IAV shedding and NA 222 amino acid residues. The values 2.5 ± 0.28, 7.2 ± 0.79, and 24 ±1.4 μg/liter are the means ± standard deviations of 11, 9, and 7 water samples, respectively. The dashed horizontal lines indicate changes in OC concentrations. G1 represents generation one consisting of two mallards, G2 represents the second pair of mallards introduced in the experimental room, etc. Closed rectangles represent the presence of mallards in the experimental room and dotted lines indicate days. Note that birds were introduced into and removed from the experiment room in the daytime such that each bird was part of the experiment during 5 ′ 24 h, with a 24-h gap between every second generation. Shading indicates detection of IAV by RRT-PCR of the matrix gene from daily fecal samples ( C T values of ≥45 were determined to be negative). I, isoleucine at NA residue 222; T*, threonine in shared proportions with isoleucine at NA residue 222; T, threonine at NA residue 222, as determined by Sanger sequencing of fecal samples; W, water from the 170-liter pool in the experimental room. Closed rectangles represent change of water and shading indicates detection of IAV by RRT-PCR of the matrix gene.
    Figure Legend Snippet: Experimental mallard model with IAV shedding and NA 222 amino acid residues. The values 2.5 ± 0.28, 7.2 ± 0.79, and 24 ±1.4 μg/liter are the means ± standard deviations of 11, 9, and 7 water samples, respectively. The dashed horizontal lines indicate changes in OC concentrations. G1 represents generation one consisting of two mallards, G2 represents the second pair of mallards introduced in the experimental room, etc. Closed rectangles represent the presence of mallards in the experimental room and dotted lines indicate days. Note that birds were introduced into and removed from the experiment room in the daytime such that each bird was part of the experiment during 5 ′ 24 h, with a 24-h gap between every second generation. Shading indicates detection of IAV by RRT-PCR of the matrix gene from daily fecal samples ( C T values of ≥45 were determined to be negative). I, isoleucine at NA residue 222; T*, threonine in shared proportions with isoleucine at NA residue 222; T, threonine at NA residue 222, as determined by Sanger sequencing of fecal samples; W, water from the 170-liter pool in the experimental room. Closed rectangles represent change of water and shading indicates detection of IAV by RRT-PCR of the matrix gene.

    Techniques Used: Quantitative RT-PCR, Sequencing

    Shedding of A(H7N9) virus determined by RRT-PCR of the matrix gene from daily fecal samples. The x axis displays the number of days that each pair of birds had been present in the experimental room at the time of sampling. The y axis displays cycle threshold ( C T ) values of the RRT-PCR as a semiquantitative measure of excreted IAV. C T values of ≥45 were determined to be negative. G1 represents mallard generation one of the experiment, consisting of two mallards, G2 represents the second pair of mallards introduced in the experimental room, etc. Values in the graph indicate mean C T values from two samples, error bars indicate standard errors of the mean (SEM). For clarity, if one of the two samples of a generation from 1 day was IAV negative ( C T of ≥45), only the positive sample was included in the graph, i.e., G1, day 1; G2, day 5; G3, day 2; G4, day 3; G5, day 1 (see also Fig. 1 ).
    Figure Legend Snippet: Shedding of A(H7N9) virus determined by RRT-PCR of the matrix gene from daily fecal samples. The x axis displays the number of days that each pair of birds had been present in the experimental room at the time of sampling. The y axis displays cycle threshold ( C T ) values of the RRT-PCR as a semiquantitative measure of excreted IAV. C T values of ≥45 were determined to be negative. G1 represents mallard generation one of the experiment, consisting of two mallards, G2 represents the second pair of mallards introduced in the experimental room, etc. Values in the graph indicate mean C T values from two samples, error bars indicate standard errors of the mean (SEM). For clarity, if one of the two samples of a generation from 1 day was IAV negative ( C T of ≥45), only the positive sample was included in the graph, i.e., G1, day 1; G2, day 5; G3, day 2; G4, day 3; G5, day 1 (see also Fig. 1 ).

    Techniques Used: Quantitative RT-PCR, Sampling

    25) Product Images from "Wnt/Wingless signaling through ?-catenin requires the function of both LRP/Arrow and frizzled classes of receptors"

    Article Title: Wnt/Wingless signaling through ?-catenin requires the function of both LRP/Arrow and frizzled classes of receptors

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-4-4

    The expression of Wg dual receptors and their activities in S2 cells. One-step RT-PCR was performed to study the expression of Arr and DFz2 in mbn-2 cells and S2 cells (A). 0.1 μg RNA from each cell line served as the template, and primers for Arr and DFz1, 2, 3 and 4 (see Methods) were used for the RT-PCR reactions. (B) S2 cells were transfected with the indicated plasmids and the plasmids as described in Methods. Fold-activation values were measured relative to the levels of luciferase activity in cells transfected with empty vectors and normalized by Renilla luciferase activities. These values are plotted as a log function. Averages of the fold-activation are indicated above each column. All experiments were done in triplicate; error bars represent standard deviations.
    Figure Legend Snippet: The expression of Wg dual receptors and their activities in S2 cells. One-step RT-PCR was performed to study the expression of Arr and DFz2 in mbn-2 cells and S2 cells (A). 0.1 μg RNA from each cell line served as the template, and primers for Arr and DFz1, 2, 3 and 4 (see Methods) were used for the RT-PCR reactions. (B) S2 cells were transfected with the indicated plasmids and the plasmids as described in Methods. Fold-activation values were measured relative to the levels of luciferase activity in cells transfected with empty vectors and normalized by Renilla luciferase activities. These values are plotted as a log function. Averages of the fold-activation are indicated above each column. All experiments were done in triplicate; error bars represent standard deviations.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Activation Assay, Luciferase, Activity Assay

    26) Product Images from "Ero1L, a thiol oxidase, is required for Notch signaling through cysteine bridge formation of the Lin12-Notch repeats in Drosophila melanogaster"

    Article Title: Ero1L, a thiol oxidase, is required for Notch signaling through cysteine bridge formation of the Lin12-Notch repeats in Drosophila melanogaster

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200805001

    Ero1L is involved in secretion and disulfide bond formation of the LNR domain of Notch. (A) Secretion of the LNR domain into the medium is affected when Ero1L is reduced (lane 4, bottom, Western blot). S2 cells treated with either dsRNA against EGFP (lanes 1 and 3) or Ero1L (lanes 2 and 4) were followed by transfection with EGF-V5 (lanes 1 and 2) and LNR-V5 (lanes 3 and 4). Western blots of the cell lysate (top) and the medium from these samples are shown (bottom). (B) Secretion of the EGF domain into the medium is affected when QSOX1 (CG4670) is knocked down. S2 cells treated with various dsRNA are indicated in the table above the Western blotting. The bottom panel shows the relative abundance of mRNA of Arp66B (control), Ero1L, and QSOX1, which was measured by RT-PCR. (C) Disulfide bonds in the LNR domain of Notch fail to form properly in Ero1L knockdown S2 cells. An AMS thiol-modifying analysis was used to reveal disulfide bond formation under various conditions. As a positive control, lysate was treated with DTT followed by AMS incubation. The expected molecular weight shift of the LNR domain is observed in lane 6 (*). A similar molecular weight shift is observed in Ero1L knockdown lysate without prior DTT treatment (lane 4), indicating a loss of disulfide bond formation in a portion of the LNR domain. Note that the LNR domain can form homodimers without AMS treatment (**) that can be removed in a high concentration of urea (not depicted).
    Figure Legend Snippet: Ero1L is involved in secretion and disulfide bond formation of the LNR domain of Notch. (A) Secretion of the LNR domain into the medium is affected when Ero1L is reduced (lane 4, bottom, Western blot). S2 cells treated with either dsRNA against EGFP (lanes 1 and 3) or Ero1L (lanes 2 and 4) were followed by transfection with EGF-V5 (lanes 1 and 2) and LNR-V5 (lanes 3 and 4). Western blots of the cell lysate (top) and the medium from these samples are shown (bottom). (B) Secretion of the EGF domain into the medium is affected when QSOX1 (CG4670) is knocked down. S2 cells treated with various dsRNA are indicated in the table above the Western blotting. The bottom panel shows the relative abundance of mRNA of Arp66B (control), Ero1L, and QSOX1, which was measured by RT-PCR. (C) Disulfide bonds in the LNR domain of Notch fail to form properly in Ero1L knockdown S2 cells. An AMS thiol-modifying analysis was used to reveal disulfide bond formation under various conditions. As a positive control, lysate was treated with DTT followed by AMS incubation. The expected molecular weight shift of the LNR domain is observed in lane 6 (*). A similar molecular weight shift is observed in Ero1L knockdown lysate without prior DTT treatment (lane 4), indicating a loss of disulfide bond formation in a portion of the LNR domain. Note that the LNR domain can form homodimers without AMS treatment (**) that can be removed in a high concentration of urea (not depicted).

    Techniques Used: Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, Affinity Magnetic Separation, Positive Control, Incubation, Molecular Weight, Concentration Assay

    Notch is a major target in Ero1L mutant cells. (A–D') Confocal sections through wing imaginal discs harboring Ubx-FLP –induced mutant clones (lack of GFP expression). (A–A'”) Cells without Notch and Ero1L have lower levels of Hsc3 expression compared with cells that lack only Ero1L . A z section through a wing imaginal disc harboring Ero1L and/or Notch mutant clones (marked by dashed lines) is shown. It was stained for Notch (green) and Hsc3 (red). In A”, wild-type cells expressing GFP (blue) show low levels of expression for both Hsc3 and Notch. In A', high levels of Hsc3 and Notch are observed in cells that are only mutant for Ero1L . In A”', a group of Notch and Ero1L double mutant cells, indicated by the loss of GFP (blue) and Notch (green) expression, show lower levels of Hsc3 when compared with Ero1L mutant cells in the left panel (compare A'” with A'). (B) Quantification of Hsc3 expression in the cells of different genotypes is shown in the bar graph (a.u., arbitrary unit). Error bars indicate SEM. (C–D') Normal expression levels and localization of various membrane proteins are observed in Ero1L mutant clones. Dl (C–C', red) and Drosophila EGF receptor (D and D', red) are localized normally and are not up-regulated in Ero1L mutant cells. (E) Knockdown of CG4670 revealed by semiquantitative RT-PCR. In larva with ubiquitous expression of CG4670 RNAi (left), the mRNA of CG4670 is significantly lower than control larva (right). (F–H) Genetic interaction between QSOX protein and Ero1L. Wings from adult female flies incubated at 25°C. The genotype of each fly is indicated near the figure, and the arrows indicate the wing vein-thickening phenotypes. Bars, 10 μm.
    Figure Legend Snippet: Notch is a major target in Ero1L mutant cells. (A–D') Confocal sections through wing imaginal discs harboring Ubx-FLP –induced mutant clones (lack of GFP expression). (A–A'”) Cells without Notch and Ero1L have lower levels of Hsc3 expression compared with cells that lack only Ero1L . A z section through a wing imaginal disc harboring Ero1L and/or Notch mutant clones (marked by dashed lines) is shown. It was stained for Notch (green) and Hsc3 (red). In A”, wild-type cells expressing GFP (blue) show low levels of expression for both Hsc3 and Notch. In A', high levels of Hsc3 and Notch are observed in cells that are only mutant for Ero1L . In A”', a group of Notch and Ero1L double mutant cells, indicated by the loss of GFP (blue) and Notch (green) expression, show lower levels of Hsc3 when compared with Ero1L mutant cells in the left panel (compare A'” with A'). (B) Quantification of Hsc3 expression in the cells of different genotypes is shown in the bar graph (a.u., arbitrary unit). Error bars indicate SEM. (C–D') Normal expression levels and localization of various membrane proteins are observed in Ero1L mutant clones. Dl (C–C', red) and Drosophila EGF receptor (D and D', red) are localized normally and are not up-regulated in Ero1L mutant cells. (E) Knockdown of CG4670 revealed by semiquantitative RT-PCR. In larva with ubiquitous expression of CG4670 RNAi (left), the mRNA of CG4670 is significantly lower than control larva (right). (F–H) Genetic interaction between QSOX protein and Ero1L. Wings from adult female flies incubated at 25°C. The genotype of each fly is indicated near the figure, and the arrows indicate the wing vein-thickening phenotypes. Bars, 10 μm.

    Techniques Used: Mutagenesis, Clone Assay, Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Incubation

    27) Product Images from "DNA methylation directs microRNA biogenesis in mammalian cells"

    Article Title: DNA methylation directs microRNA biogenesis in mammalian cells

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13527-1

    Drosha genomic occupancy is sensitive to DNA methylation. a Plot of expression of transcripts encoding miRNA biogenesis proteins ( n = 40) and other RNA-binding proteins ( n = 166) in WT vs. TKO cells. R - and p- values of Pearson’s product-moment correlation are given. b Drosha mRNA levels in WT and TKO mouse ESCs quantified by qRT-PCR. Bars represent means of three independent experiments. Values were normalized to tubulin . c Relative Drosha occupancy over miRNA genomic regions in WT and TKO mouse ESCs determined by ChIP with an antibody against Drosha. Immunoprecipitated DNA was quantified by qRT-PCR with primers spanning the indicated pre-miRNA sequences. Data were normalized to input DNA. d Efficiency of miRNA biogenesis measured as the ratio of mature miRNA to pri-miRNA expression levels for the indicated miRNAs in WT mouse ESCs upon treatment with 2.5 µM 5-Aza relative to vehicle control. Data were normalized to RPLP0 for pri-miRNAs and RNU6 for mature miRNAs. e Drosha protein levels in untreated control WT mouse ESCs and cells treated with 2.5 µM 5-Aza. GAPDH was used as the loading control. f Relative Drosha occupancy over miRNA genomic regions in untreated WT mouse ESCs and cells treated with 2.5 µM 5-Aza determined by ChIP with an antibody against Drosha. Immunoprecipitated DNA was quantified by qRT-PCR with primers spanning the indicated pre-miRNA sequences of the methylated and unmethylated groups. Data were normalized to input DNA. All Error bars represent ± SEM ( n = 3). * represents p
    Figure Legend Snippet: Drosha genomic occupancy is sensitive to DNA methylation. a Plot of expression of transcripts encoding miRNA biogenesis proteins ( n = 40) and other RNA-binding proteins ( n = 166) in WT vs. TKO cells. R - and p- values of Pearson’s product-moment correlation are given. b Drosha mRNA levels in WT and TKO mouse ESCs quantified by qRT-PCR. Bars represent means of three independent experiments. Values were normalized to tubulin . c Relative Drosha occupancy over miRNA genomic regions in WT and TKO mouse ESCs determined by ChIP with an antibody against Drosha. Immunoprecipitated DNA was quantified by qRT-PCR with primers spanning the indicated pre-miRNA sequences. Data were normalized to input DNA. d Efficiency of miRNA biogenesis measured as the ratio of mature miRNA to pri-miRNA expression levels for the indicated miRNAs in WT mouse ESCs upon treatment with 2.5 µM 5-Aza relative to vehicle control. Data were normalized to RPLP0 for pri-miRNAs and RNU6 for mature miRNAs. e Drosha protein levels in untreated control WT mouse ESCs and cells treated with 2.5 µM 5-Aza. GAPDH was used as the loading control. f Relative Drosha occupancy over miRNA genomic regions in untreated WT mouse ESCs and cells treated with 2.5 µM 5-Aza determined by ChIP with an antibody against Drosha. Immunoprecipitated DNA was quantified by qRT-PCR with primers spanning the indicated pre-miRNA sequences of the methylated and unmethylated groups. Data were normalized to input DNA. All Error bars represent ± SEM ( n = 3). * represents p

    Techniques Used: DNA Methylation Assay, Expressing, RNA Binding Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunoprecipitation, Methylation

    28) Product Images from "Increased Early RNA Replication by Chimeric West Nile Virus W956IC Leads to IPS-1-Mediated Activation of NF-?B and Insufficient Virus-Mediated Counteraction of the Resulting Canonical Type I Interferon Signaling"

    Article Title: Increased Early RNA Replication by Chimeric West Nile Virus W956IC Leads to IPS-1-Mediated Activation of NF-?B and Insufficient Virus-Mediated Counteraction of the Resulting Canonical Type I Interferon Signaling

    Journal: Journal of Virology

    doi: 10.1128/JVI.02842-12

    Induction and activation of PKR in MEFs after infection with WNV Eg101 or W956IC. (A) C3H/He MEFs were infected with WNV Eg101 or W956IC at an MOI of 1. Changes in Eif2ak2 (PKR) mRNA levels were assessed by real-time qRT-PCR at the indicated times after
    Figure Legend Snippet: Induction and activation of PKR in MEFs after infection with WNV Eg101 or W956IC. (A) C3H/He MEFs were infected with WNV Eg101 or W956IC at an MOI of 1. Changes in Eif2ak2 (PKR) mRNA levels were assessed by real-time qRT-PCR at the indicated times after

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

    29) Product Images from "Reduced Susceptibility of Haemophilus influenzae to the Peptide Deformylase Inhibitor LBM415 Can Result from Target Protein Overexpression Due to Amplified Chromosomal def Gene Copy Number ▿"

    Article Title: Reduced Susceptibility of Haemophilus influenzae to the Peptide Deformylase Inhibitor LBM415 Can Result from Target Protein Overexpression Due to Amplified Chromosomal def Gene Copy Number ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01103-06

    RNA isolation, microarray analysis, and real-time RT-PCR.
    Figure Legend Snippet: RNA isolation, microarray analysis, and real-time RT-PCR.

    Techniques Used: Isolation, Microarray, Quantitative RT-PCR

    30) Product Images from "Full genome characterization of Laem Singh virus (LSNV) in shrimp Penaeus monodon"

    Article Title: Full genome characterization of Laem Singh virus (LSNV) in shrimp Penaeus monodon

    Journal: bioRxiv

    doi: 10.1101/2020.07.25.221432

    cDNA sequences of LSNV RT-PCR amplicons and predicted open reading frames. ( A ) Sequence of LSNV genome segment 1 with two predicted-ORF of 453 and 382 deduced amino acids related to a protease-like protein and an RdRP respectively, based on BlastP analysis. The shaded box indicates a conserved region corresponding to the reverse transcriptase-RNA-dependent RNA polymerase-like superfamily. ( B ) DNA sequence of LSNV genome segment 2 with a single predicted-ORF of 585 deduced amino acids and showing sequence similarity to capsid proteins. The shaded box indicates a conserved region corresponding to viral capsid protein family. ( C ) Schematic diagram showing the structure of the LSNV genome segments and predicted proteins compositions (figure not to scale).
    Figure Legend Snippet: cDNA sequences of LSNV RT-PCR amplicons and predicted open reading frames. ( A ) Sequence of LSNV genome segment 1 with two predicted-ORF of 453 and 382 deduced amino acids related to a protease-like protein and an RdRP respectively, based on BlastP analysis. The shaded box indicates a conserved region corresponding to the reverse transcriptase-RNA-dependent RNA polymerase-like superfamily. ( B ) DNA sequence of LSNV genome segment 2 with a single predicted-ORF of 585 deduced amino acids and showing sequence similarity to capsid proteins. The shaded box indicates a conserved region corresponding to viral capsid protein family. ( C ) Schematic diagram showing the structure of the LSNV genome segments and predicted proteins compositions (figure not to scale).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing

    Purification of LSNV from infected P. monodon gill tissue. ( A ) Sucrose density gradient centrifugation tube showing 2 layers between 54 and 58%. ( B ) TEM showing icosahedral viral-like particles with diameters ranging from 23-33 nm. ( C ) Agarose gel showing RT-PCR amplicons using templates with and without RNase treatment for both
    Figure Legend Snippet: Purification of LSNV from infected P. monodon gill tissue. ( A ) Sucrose density gradient centrifugation tube showing 2 layers between 54 and 58%. ( B ) TEM showing icosahedral viral-like particles with diameters ranging from 23-33 nm. ( C ) Agarose gel showing RT-PCR amplicons using templates with and without RNase treatment for both

    Techniques Used: Purification, Infection, Gradient Centrifugation, Transmission Electron Microscopy, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    RT-PCR detection of LSNV genome segments. (A) Agarose gels showing full-length amplification of LSNV genome segments of 2,684 bp for segment 1 and 1,846 bp using specific primers using template RNA from LSNV-infected shrimp. (B) RT-PCR testing by using overlapping primers between two LSNV genome segments. Absence of amplicons was observed when using different pairs of primer flanking between genome segment 1 and segment 2 (primer set 1 (P1); LSNVS1_Luteo-F + LSNVS2-1810R, and primer set 2 (P2); LSNVS1_941F + LSNVS2-471R).
    Figure Legend Snippet: RT-PCR detection of LSNV genome segments. (A) Agarose gels showing full-length amplification of LSNV genome segments of 2,684 bp for segment 1 and 1,846 bp using specific primers using template RNA from LSNV-infected shrimp. (B) RT-PCR testing by using overlapping primers between two LSNV genome segments. Absence of amplicons was observed when using different pairs of primer flanking between genome segment 1 and segment 2 (primer set 1 (P1); LSNVS1_Luteo-F + LSNVS2-1810R, and primer set 2 (P2); LSNVS1_941F + LSNVS2-471R).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Infection

    31) Product Images from "Sudden unexpected death related to enterovirus myocarditis: histopathology, immunohistochemistry and molecular pathology diagnosis at post-mortem"

    Article Title: Sudden unexpected death related to enterovirus myocarditis: histopathology, immunohistochemistry and molecular pathology diagnosis at post-mortem

    Journal: BMC Infectious Diseases

    doi: 10.1186/1471-2334-12-212

    Detection of Coxsackie B enterovirus RNA by RT-PCR in post-mortem myocardial samples from Sudden Unexpected Death victims (1 and 10: molecular size marker 100-bp DNA ladder; 2: Negative control RNA extraction; 3: Negative control RT-PCR mixture; 4 to 6 and 8: Samples from coxsackie B enterovirus-positive cases; 7: Samples from a coxsackie B enterovirus-negative case; 9: A positive control; coxsackievirus B3: 155 bp).
    Figure Legend Snippet: Detection of Coxsackie B enterovirus RNA by RT-PCR in post-mortem myocardial samples from Sudden Unexpected Death victims (1 and 10: molecular size marker 100-bp DNA ladder; 2: Negative control RNA extraction; 3: Negative control RT-PCR mixture; 4 to 6 and 8: Samples from coxsackie B enterovirus-positive cases; 7: Samples from a coxsackie B enterovirus-negative case; 9: A positive control; coxsackievirus B3: 155 bp).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Marker, Negative Control, RNA Extraction, Positive Control

    Related Articles

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Optimization of Multiple Pathogen Detection Using the TaqMan Array Card: Application for a Population-Based Study of Neonatal Infection
    Article Snippet: .. Each reaction consisted of 1× AgPath-ID One-step RT-PCR buffer and 1× AgPath-ID One-step RT-PCR enzyme mix (Applied Biosystems, Foster City, CA, USA) or 1× qScript XLT One-step RT-qPCR ToughMix, low ROX (Quanta Biosciences, Gaithersburg, MD, USA), forward and reverse primers and FAM-labeled hydrolysis probe at the concentrations listed in Table S1 in , and nuclease-free water to final volume of 20 µL. ..

    Article Title: Dietary zinc modulates gene expression in murine thymus: Results from a comprehensive differential display screening
    Article Snippet: .. The 18S rRNA assay, used for total RNA normalization, and all one-step RT-PCR reagents were purchased from Applied Biosystems, and all assays were performed on a GeneAmp 5700 Sequence Detection System (Applied Biosystems). .. Relative quantitation was determined from 4 log10 -range standard curves with pooled samples run in triplicate.

    Article Title: Acute hepatotoxicity of 2′ fluoro-modified 5–10–5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins
    Article Snippet: .. TaqMan One-step qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents (Thermo Fisher Scientific). .. Expression levels of target RNA were normalized to total RNA quantified using Quant-iT RiboGreen RNA Reagent (Thermo Fisher Scientific).

    Article Title: Equine Mx1 Restricts Influenza A Virus Replication by Targeting at Distinct Site of its Nucleoprotein
    Article Snippet: .. Measurement of Gene Expression Using RT-qPCR Total RNA from the collected supernatants was extracted using the RNeasy plus Minikit (Qiagen, Venlo, Netherland) and were subjected to a one-step real-time quantitative PCR (RT-qPCR) analysis using the AgPath-ID™ One-Step RT-PCR reagents (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s protocol. .. FAM-labeled probes (EIV-Tq-P) 5′-TCAGGCCCCCTCAAAGCCGA-TAMRA and specific primers targeting the M-gene of IAV, 5′-AGATGAGYCTTCTAACCGAGGTCG-3′ (EIV-Tq-forward) and 5′-TGCAAANACATCYTCAAGTCTCTG-3′ (EIV-Tq-reverse) were used for the amplification of RNA, and relative mRNA expression levels were determined using double-standard curve methods.

    Article Title: Induction of the Cellular MicroRNA, Hs_154, by West Nile Virus Contributes to Virus-Mediated Apoptosis through Repression of Antiapoptotic Factors
    Article Snippet: .. Portions (25 ng) of total RNA or RISC-associated RNA were subjected to quantitative reverse transcription-PCR (qRT-PCR) using CTCF (Hs00902008_m1)-, ECOP (Hs00697470_m1)-, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Hs99999905_m1)-specific TaqMan gene expression assays and One-Step RT-PCR reagents on a StepOne Plus real-time PCR machine (Applied Biosystems) according to the manufacturer's instructions. .. The relative expression in samples was calculated by the ΔΔ CT method using GAPDH as the endogenous control.

    Sequencing:

    Article Title: Dietary zinc modulates gene expression in murine thymus: Results from a comprehensive differential display screening
    Article Snippet: .. The 18S rRNA assay, used for total RNA normalization, and all one-step RT-PCR reagents were purchased from Applied Biosystems, and all assays were performed on a GeneAmp 5700 Sequence Detection System (Applied Biosystems). .. Relative quantitation was determined from 4 log10 -range standard curves with pooled samples run in triplicate.

    Real-time Polymerase Chain Reaction:

    Article Title: Equine Mx1 Restricts Influenza A Virus Replication by Targeting at Distinct Site of its Nucleoprotein
    Article Snippet: .. Measurement of Gene Expression Using RT-qPCR Total RNA from the collected supernatants was extracted using the RNeasy plus Minikit (Qiagen, Venlo, Netherland) and were subjected to a one-step real-time quantitative PCR (RT-qPCR) analysis using the AgPath-ID™ One-Step RT-PCR reagents (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s protocol. .. FAM-labeled probes (EIV-Tq-P) 5′-TCAGGCCCCCTCAAAGCCGA-TAMRA and specific primers targeting the M-gene of IAV, 5′-AGATGAGYCTTCTAACCGAGGTCG-3′ (EIV-Tq-forward) and 5′-TGCAAANACATCYTCAAGTCTCTG-3′ (EIV-Tq-reverse) were used for the amplification of RNA, and relative mRNA expression levels were determined using double-standard curve methods.

    Article Title: Persistence of the protective immunity and kinetics of the isotype specific antibody response against the viral nucleocapsid protein after experimental Schmallenberg virus infection of sheep
    Article Snippet: .. The presence of SBV S-segment (forward primer: 5′-tcagattgtcatgccccttgc-3′; reverse primer: 5′-ttcggccccaggtgcaaatc-3′; probe: FAM-5′-ttaagggatgcacctgggccgatg GT-3′-BHQ1 [ ]) and the presence of B-actin as internal control of extraction (forward primer: 5′-cagcacaatgaagatcaagatcatc-3′; reverse primer: 5′-cggactcatcgtactcctgctt-3′; probe: HEX-5′-tcgctgtccaccttccagcagcagatgt-3′-BHQ1 [ ]) were detected in a duplex qRT-PCR carried out with the AgPath-ID one-step qRT-PCR kit (Life Technologies, California, USA) and measured with the LightCycler 480 Real-Time PCR system (Roche Applied Science, Indianapolis, USA). .. One positive control and one negative control of PCR amplification were added to each run of qRT-PCR.

    Article Title: Induction of the Cellular MicroRNA, Hs_154, by West Nile Virus Contributes to Virus-Mediated Apoptosis through Repression of Antiapoptotic Factors
    Article Snippet: .. Portions (25 ng) of total RNA or RISC-associated RNA were subjected to quantitative reverse transcription-PCR (qRT-PCR) using CTCF (Hs00902008_m1)-, ECOP (Hs00697470_m1)-, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Hs99999905_m1)-specific TaqMan gene expression assays and One-Step RT-PCR reagents on a StepOne Plus real-time PCR machine (Applied Biosystems) according to the manufacturer's instructions. .. The relative expression in samples was calculated by the ΔΔ CT method using GAPDH as the endogenous control.

    Quantitative RT-PCR:

    Article Title: Optimization of Multiple Pathogen Detection Using the TaqMan Array Card: Application for a Population-Based Study of Neonatal Infection
    Article Snippet: .. Each reaction consisted of 1× AgPath-ID One-step RT-PCR buffer and 1× AgPath-ID One-step RT-PCR enzyme mix (Applied Biosystems, Foster City, CA, USA) or 1× qScript XLT One-step RT-qPCR ToughMix, low ROX (Quanta Biosciences, Gaithersburg, MD, USA), forward and reverse primers and FAM-labeled hydrolysis probe at the concentrations listed in Table S1 in , and nuclease-free water to final volume of 20 µL. ..

    Article Title: CONTAIN: An open-source shipping container laboratory optimised for automated COVID-19 diagnostics
    Article Snippet: .. The samples were run through the CONTAIN workflow, this time using the AgPath-ID™ One-Step RT-qPCR mix from ThermoFisher. .. The RT-qPCR results showed that for the N1 assay, 8 reactions returned positive, however, one of these had not been detected as positive in the initial clinical testing of the samples, additionally, one sample identified as positive in the initial clinical test but did not amplify in the CONTAIN test ( ).

    Article Title: Acute hepatotoxicity of 2′ fluoro-modified 5–10–5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins
    Article Snippet: .. TaqMan One-step qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents (Thermo Fisher Scientific). .. Expression levels of target RNA were normalized to total RNA quantified using Quant-iT RiboGreen RNA Reagent (Thermo Fisher Scientific).

    Article Title: Equine Mx1 Restricts Influenza A Virus Replication by Targeting at Distinct Site of its Nucleoprotein
    Article Snippet: .. Measurement of Gene Expression Using RT-qPCR Total RNA from the collected supernatants was extracted using the RNeasy plus Minikit (Qiagen, Venlo, Netherland) and were subjected to a one-step real-time quantitative PCR (RT-qPCR) analysis using the AgPath-ID™ One-Step RT-PCR reagents (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s protocol. .. FAM-labeled probes (EIV-Tq-P) 5′-TCAGGCCCCCTCAAAGCCGA-TAMRA and specific primers targeting the M-gene of IAV, 5′-AGATGAGYCTTCTAACCGAGGTCG-3′ (EIV-Tq-forward) and 5′-TGCAAANACATCYTCAAGTCTCTG-3′ (EIV-Tq-reverse) were used for the amplification of RNA, and relative mRNA expression levels were determined using double-standard curve methods.

    Article Title: Persistence of the protective immunity and kinetics of the isotype specific antibody response against the viral nucleocapsid protein after experimental Schmallenberg virus infection of sheep
    Article Snippet: .. The presence of SBV S-segment (forward primer: 5′-tcagattgtcatgccccttgc-3′; reverse primer: 5′-ttcggccccaggtgcaaatc-3′; probe: FAM-5′-ttaagggatgcacctgggccgatg GT-3′-BHQ1 [ ]) and the presence of B-actin as internal control of extraction (forward primer: 5′-cagcacaatgaagatcaagatcatc-3′; reverse primer: 5′-cggactcatcgtactcctgctt-3′; probe: HEX-5′-tcgctgtccaccttccagcagcagatgt-3′-BHQ1 [ ]) were detected in a duplex qRT-PCR carried out with the AgPath-ID one-step qRT-PCR kit (Life Technologies, California, USA) and measured with the LightCycler 480 Real-Time PCR system (Roche Applied Science, Indianapolis, USA). .. One positive control and one negative control of PCR amplification were added to each run of qRT-PCR.

    Article Title: Induction of the Cellular MicroRNA, Hs_154, by West Nile Virus Contributes to Virus-Mediated Apoptosis through Repression of Antiapoptotic Factors
    Article Snippet: .. Portions (25 ng) of total RNA or RISC-associated RNA were subjected to quantitative reverse transcription-PCR (qRT-PCR) using CTCF (Hs00902008_m1)-, ECOP (Hs00697470_m1)-, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Hs99999905_m1)-specific TaqMan gene expression assays and One-Step RT-PCR reagents on a StepOne Plus real-time PCR machine (Applied Biosystems) according to the manufacturer's instructions. .. The relative expression in samples was calculated by the ΔΔ CT method using GAPDH as the endogenous control.

    Expressing:

    Article Title: Equine Mx1 Restricts Influenza A Virus Replication by Targeting at Distinct Site of its Nucleoprotein
    Article Snippet: .. Measurement of Gene Expression Using RT-qPCR Total RNA from the collected supernatants was extracted using the RNeasy plus Minikit (Qiagen, Venlo, Netherland) and were subjected to a one-step real-time quantitative PCR (RT-qPCR) analysis using the AgPath-ID™ One-Step RT-PCR reagents (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s protocol. .. FAM-labeled probes (EIV-Tq-P) 5′-TCAGGCCCCCTCAAAGCCGA-TAMRA and specific primers targeting the M-gene of IAV, 5′-AGATGAGYCTTCTAACCGAGGTCG-3′ (EIV-Tq-forward) and 5′-TGCAAANACATCYTCAAGTCTCTG-3′ (EIV-Tq-reverse) were used for the amplification of RNA, and relative mRNA expression levels were determined using double-standard curve methods.

    Article Title: Induction of the Cellular MicroRNA, Hs_154, by West Nile Virus Contributes to Virus-Mediated Apoptosis through Repression of Antiapoptotic Factors
    Article Snippet: .. Portions (25 ng) of total RNA or RISC-associated RNA were subjected to quantitative reverse transcription-PCR (qRT-PCR) using CTCF (Hs00902008_m1)-, ECOP (Hs00697470_m1)-, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Hs99999905_m1)-specific TaqMan gene expression assays and One-Step RT-PCR reagents on a StepOne Plus real-time PCR machine (Applied Biosystems) according to the manufacturer's instructions. .. The relative expression in samples was calculated by the ΔΔ CT method using GAPDH as the endogenous control.

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    Transfusion of pregnant A129 mice with early-stage (high dose) ZIKV + plasma resulted in significant viremia and broad tissue tropism. Pregnant (E10–12) A129 mice were transfused with ZIKV + plasma from early, middle, and late stages, respectively, and plasma and tissues were collected 6 days post-transfusion for below tests. Plasma ZIKV RNA (A) and ZIKV titers (B) were detected by <t>qRT-PCR</t> and plaque assay, respectively. Tissue ZIKV RNA (C) and viral titers (D) were detected by qRT-PCR and plaque assay, respectively. ∗ , ∗∗ , and ∗∗∗ indicate significant differences between early stage (high dose) and early stage (low dose) post-transfusion with ZIKV + plasma. The data are presented as means ± s.e.m ( n = 6 mice/group). In (A) and (C) , the limit of detection, shown as the horizontal lines, was about 2.5 × 10 3 copies/ml (for A ) or 2.5 × 10 3 copies/g (for C ) of ZIKV RNA. This was determined based on a detection limit of 10 2 copies in a linear standard curve (at serial dilutions of 10 2 to 10 10 copies) of ZIKV RNA, as described in Materials and Methods. In (B) and (D) , the detection limit was about 1 PFU/ml (for B ) or 1 PFU/g (for D ). The experiments were repeated twice, and similar results were obtained. Normal plasma: control plasma from A129 mice transfused with normal plasma without ZIKV.
    One Step Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ct one step rt qpcr kit
    <t>RNA</t> Immunoprecipitation experiments of confirmed BC200 targets. ( A ) <t>RT-qPCR</t> analysis of BC200, GAPDH and 7SL enrichment by immunoprecipitation of the indicated proteins. RNA extracted from 10% of the input sample was used as a reference to calculate percent of input for each RNA that was bound to the immunoprecipitated protein. Data represents the mean of three independent replicates ± standard deviation. Dashed line represents the threshold value of 5% input. ( B ) Percent input values of BC200 and 7SL were compared to GAPDH to demonstrate the degree of specificity of the interactions analyzed in (A). Dashed line represents the threshold value of 2-fold enrichment. ( C ) Immunoprecipitation efficiency was monitored by performing western blot on 50 μg of PRE and POST IP samples as well as 2% of the IP.
    Ct One Step Rt Qpcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher one step real time quantitative pcr rt qpcr analysis
    The replication abilities of viruses with different NPs underexpression of eqMx1. ( A ) MDCK cells expressing HA-tagged eqMx1 were infected with wild type (H3N8 JL89 ) or mutant viruses (H3N8 JL89- H52N-NP) at an MOI of 0.001 and the supernatants were collected at 0, 12, 24, 36, and 48 hr post-infection. Total RNA from the collected supernatants was extracted using the RNeasy plus mini kit (Qiagen) and subjected to one-step real-time quantitative <t>PCR</t> <t>(qPCR)</t> analysis using the AgPath-ID™ One-Step RT-PCR reagents according to manufacturer’s protocol. Relative mRNA expression levels were determined using double-standard curve methods. All the experiments were performed three times and with three replicates with means ± SE shown. ( B ) MDCK cells expressing eqMx1 were infected with wild type (H3N8 JL89 ) or mutant viruses (H3N8 JL89 -H52N-NP) at an MOI of 0.001 and the supernatants were collected at 0, 12, 24, 36, and 48 hr post-infection. These supernatants were subsequently used to infect MDCK cells at different dilutions (10 −1 to 10 −11 ) with at least four repeats. 48 hr post-infection, immunofluorescence assays (IFA) were performed using specific antibodies against viral NP (from our lab) and FITC-labelled secondary antibody. Finally the viral titers were calculated using Reed and Munech methodology and results are shown as TCID 50 /mL. The results of a single experiment performed with four repeats are shown and results were subsequently confirmed in three separate experiments. Error bars indicate standard deviations (SD).
    One Step Real Time Quantitative Pcr Rt Qpcr Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    qRT-PCR analysis of miR-134 expression in relapsed/refractory AML patients and human leukemia AML multidrug-resistant cell lines. Notes: ( A ) Relative expression of miR-134 in 41 newly diagnosed AML patients, 25 relapsed/refractory AML patients, and 27 healthy controls. ( B ) Relative expression of miR-134 in six matched-pair bone marrow samples of AML patients at the diagnosis time prior to treatment and the relapsed/refractory state. ( C ) Relative expression of miR-134 in K562 cells and multidrug-resistant K562/A02 cells. ( D ) Relative expression of miR-134 in HL-60 cells and multidrug-resistant HL-60/ADM. * p

    Journal: OncoTargets and therapy

    Article Title: miR-134 increases the antitumor effects of cytarabine by targeting Mnks in acute myeloid leukemia cells

    doi: 10.2147/OTT.S143465

    Figure Lengend Snippet: qRT-PCR analysis of miR-134 expression in relapsed/refractory AML patients and human leukemia AML multidrug-resistant cell lines. Notes: ( A ) Relative expression of miR-134 in 41 newly diagnosed AML patients, 25 relapsed/refractory AML patients, and 27 healthy controls. ( B ) Relative expression of miR-134 in six matched-pair bone marrow samples of AML patients at the diagnosis time prior to treatment and the relapsed/refractory state. ( C ) Relative expression of miR-134 in K562 cells and multidrug-resistant K562/A02 cells. ( D ) Relative expression of miR-134 in HL-60 cells and multidrug-resistant HL-60/ADM. * p

    Article Snippet: The qRT-PCR assays were analyzed by the Step One Plus RT-PCR system (Thermo Fisher Scientific) using SYBR PrimeScript RT-PCR Kit (Takara Bio, Shiga, Japan).

    Techniques: Quantitative RT-PCR, Expressing

    Transfusion of pregnant A129 mice with early-stage (high dose) ZIKV + plasma resulted in significant viremia and broad tissue tropism. Pregnant (E10–12) A129 mice were transfused with ZIKV + plasma from early, middle, and late stages, respectively, and plasma and tissues were collected 6 days post-transfusion for below tests. Plasma ZIKV RNA (A) and ZIKV titers (B) were detected by qRT-PCR and plaque assay, respectively. Tissue ZIKV RNA (C) and viral titers (D) were detected by qRT-PCR and plaque assay, respectively. ∗ , ∗∗ , and ∗∗∗ indicate significant differences between early stage (high dose) and early stage (low dose) post-transfusion with ZIKV + plasma. The data are presented as means ± s.e.m ( n = 6 mice/group). In (A) and (C) , the limit of detection, shown as the horizontal lines, was about 2.5 × 10 3 copies/ml (for A ) or 2.5 × 10 3 copies/g (for C ) of ZIKV RNA. This was determined based on a detection limit of 10 2 copies in a linear standard curve (at serial dilutions of 10 2 to 10 10 copies) of ZIKV RNA, as described in Materials and Methods. In (B) and (D) , the detection limit was about 1 PFU/ml (for B ) or 1 PFU/g (for D ). The experiments were repeated twice, and similar results were obtained. Normal plasma: control plasma from A129 mice transfused with normal plasma without ZIKV.

    Journal: Frontiers in Microbiology

    Article Title: Transfusion-Transmitted Zika Virus Infection in Pregnant Mice Leads to Broad Tissue Tropism With Severe Placental Damage and Fetal Demise

    doi: 10.3389/fmicb.2019.00029

    Figure Lengend Snippet: Transfusion of pregnant A129 mice with early-stage (high dose) ZIKV + plasma resulted in significant viremia and broad tissue tropism. Pregnant (E10–12) A129 mice were transfused with ZIKV + plasma from early, middle, and late stages, respectively, and plasma and tissues were collected 6 days post-transfusion for below tests. Plasma ZIKV RNA (A) and ZIKV titers (B) were detected by qRT-PCR and plaque assay, respectively. Tissue ZIKV RNA (C) and viral titers (D) were detected by qRT-PCR and plaque assay, respectively. ∗ , ∗∗ , and ∗∗∗ indicate significant differences between early stage (high dose) and early stage (low dose) post-transfusion with ZIKV + plasma. The data are presented as means ± s.e.m ( n = 6 mice/group). In (A) and (C) , the limit of detection, shown as the horizontal lines, was about 2.5 × 10 3 copies/ml (for A ) or 2.5 × 10 3 copies/g (for C ) of ZIKV RNA. This was determined based on a detection limit of 10 2 copies in a linear standard curve (at serial dilutions of 10 2 to 10 10 copies) of ZIKV RNA, as described in Materials and Methods. In (B) and (D) , the detection limit was about 1 PFU/ml (for B ) or 1 PFU/g (for D ). The experiments were repeated twice, and similar results were obtained. Normal plasma: control plasma from A129 mice transfused with normal plasma without ZIKV.

    Article Snippet: Briefly, RNA was extracted using QIAamp MinElute Virus Spin Kit (for plasma and amniotic fluid) (Qiagen) and RNeasy Mini Kit (for tissues) (Qiagen), and quantified using one-step qRT-PCR in the presence of Power SYBR Green PCR Master Mix, MultiScribe Reverse Transcriptase, and AmbionTM RNase Inhibitor (Thermo Fisher Scientific) in ViiA 7 Master Cycler PCR System (Thermo Fisher Scientific).

    Techniques: Mouse Assay, Quantitative RT-PCR, Plaque Assay

    Transfusion of pregnant A129 mice with early-stage (high dose) ZIKV + plasma led to significant placental infection. Pregnant (E10–12) A129 mice were transfused with ZIKV + plasma from early, middle, and late stages, respectively, and placentas were collected 6 days after transfusion for below tests. Detection of ZIKV RNA by qRT-PCR ( A , top) and ZIKV titers by plaque assay ( A , bottom). The data are presented as means ± s.e.m ( n = 6 mice/group). ∗ indicates significant differences between early stage (high dose) and early or middle stages (low dose). The detection limit, shown as the horizontal lines, for qRT-PCR and plaque assay was about 2.5 × 10 3 RNA copies/g and 1 PFU/g, respectively. (B) Immunofluorescence staining of placental tissues of pregnant mice transfused with early-stage (high dose) ZIKV + plasma. ZIKV (green) was stained using ZIKV EDIII-specific mAb ZV-64. Nuclei were stained with DAPI, and are shown in blue. Representative images are listed. Magnification: 63X; Scale bar: 10 μm. (C) Quantification of ZIKV staining in (B) was calculated by ImageJ software for the relative intensity (particle analysis) of ZIKV staining with fluorescent signals. The data are presented as mean fluorescence intensity (e.g., ZIKV + staining) in each field ± s.e.m ( n = 6, “ n ” indicates numbers of image from different placentas). ∗ indicates significant differences between early stage (high dose) and normal plasma groups. (D) TEM analysis of placental tissues collected from the mice transfused with early-stage (high dose) ZIKV + plasma. D1–D2, microphotographs show ZIKV particles in the placentas of ZIKV + plasma-transfused mice. Infected cells were highly vacuolated and abundant with lipid droplets. Short arrows indicate ZIKV particles. D3, placental tissues collected from normal plasma-transfused mice. Vac, vacuole; LD, lipid droplet; M, mitochondria; N, nuclei. Scale bar: 500 nm for D1, D1”, D2, and D3, and 100 nm for D1’ insertion, D2’, and D2”. Normal plasma: control plasma from A129 mice transfused with normal plasma without ZIKV.

    Journal: Frontiers in Microbiology

    Article Title: Transfusion-Transmitted Zika Virus Infection in Pregnant Mice Leads to Broad Tissue Tropism With Severe Placental Damage and Fetal Demise

    doi: 10.3389/fmicb.2019.00029

    Figure Lengend Snippet: Transfusion of pregnant A129 mice with early-stage (high dose) ZIKV + plasma led to significant placental infection. Pregnant (E10–12) A129 mice were transfused with ZIKV + plasma from early, middle, and late stages, respectively, and placentas were collected 6 days after transfusion for below tests. Detection of ZIKV RNA by qRT-PCR ( A , top) and ZIKV titers by plaque assay ( A , bottom). The data are presented as means ± s.e.m ( n = 6 mice/group). ∗ indicates significant differences between early stage (high dose) and early or middle stages (low dose). The detection limit, shown as the horizontal lines, for qRT-PCR and plaque assay was about 2.5 × 10 3 RNA copies/g and 1 PFU/g, respectively. (B) Immunofluorescence staining of placental tissues of pregnant mice transfused with early-stage (high dose) ZIKV + plasma. ZIKV (green) was stained using ZIKV EDIII-specific mAb ZV-64. Nuclei were stained with DAPI, and are shown in blue. Representative images are listed. Magnification: 63X; Scale bar: 10 μm. (C) Quantification of ZIKV staining in (B) was calculated by ImageJ software for the relative intensity (particle analysis) of ZIKV staining with fluorescent signals. The data are presented as mean fluorescence intensity (e.g., ZIKV + staining) in each field ± s.e.m ( n = 6, “ n ” indicates numbers of image from different placentas). ∗ indicates significant differences between early stage (high dose) and normal plasma groups. (D) TEM analysis of placental tissues collected from the mice transfused with early-stage (high dose) ZIKV + plasma. D1–D2, microphotographs show ZIKV particles in the placentas of ZIKV + plasma-transfused mice. Infected cells were highly vacuolated and abundant with lipid droplets. Short arrows indicate ZIKV particles. D3, placental tissues collected from normal plasma-transfused mice. Vac, vacuole; LD, lipid droplet; M, mitochondria; N, nuclei. Scale bar: 500 nm for D1, D1”, D2, and D3, and 100 nm for D1’ insertion, D2’, and D2”. Normal plasma: control plasma from A129 mice transfused with normal plasma without ZIKV.

    Article Snippet: Briefly, RNA was extracted using QIAamp MinElute Virus Spin Kit (for plasma and amniotic fluid) (Qiagen) and RNeasy Mini Kit (for tissues) (Qiagen), and quantified using one-step qRT-PCR in the presence of Power SYBR Green PCR Master Mix, MultiScribe Reverse Transcriptase, and AmbionTM RNase Inhibitor (Thermo Fisher Scientific) in ViiA 7 Master Cycler PCR System (Thermo Fisher Scientific).

    Techniques: Mouse Assay, Infection, Quantitative RT-PCR, Plaque Assay, Immunofluorescence, Staining, Software, Fluorescence, Transmission Electron Microscopy

    Transfusion of pregnant A129 mice with early-stage (high dose) ZIKV + plasma led to significant fetal infection and fetal and pup death. Pregnant (E10–12) A129 mice were transfused with ZIKV + plasma from early, middle, and late stages, respectively. ZIKV RNA and ZIKV titers were detected by qRT-PCR (A) and plaque assay (B) in amniotic fluid and embryonic brain collected 6 days post-transfusion. The detection limit, shown as the horizontal lines, was about 2.5 × 10 3 RNA copies/ml and 1 PFU/ml (for amniotic fluid), or 2.5 × 10 3 RNA copies/g and 1 PFU/g (for embryonic brain), respectively, for qRT-PCR and plaque assay. ∗ and ∗∗ indicate significant differences between early stage (high dose) and early or middle stages (low dose), or between early and middle stages (low dose). The data are presented as means ± s.e.m ( n = 6 mice/group). (C) Morphology of mouse uteri and placentas and conditions of fetuses 6 days after transfusion of early-stage (high dose) ZIKV + plasma. (a) E13–15 uteri from pregnant mice transfused at E7–9. (b) Representative images of fetuses in (a) . (c) E16–18 placentas from pregnant mice transfused at E10–12. (d) Representative images of E16–18 fetuses in (c) . Scale bar: 1 cm. Placental diameter in (c) and fetal size in (d) are shown. ∗ and ∗∗ indicate significant differences between early stage (high dose) and normal plasma groups. The data are presented as means ± s.e.m ( n = 6 mice/group). (D) Total born and surviving pups 24 h after birth from pregnant (E10–12) mice transfused with early-stage (high dose) ZIKV + plasma. Normal plasma: control plasma from A129 mice transfused with normal plasma without ZIKV.

    Journal: Frontiers in Microbiology

    Article Title: Transfusion-Transmitted Zika Virus Infection in Pregnant Mice Leads to Broad Tissue Tropism With Severe Placental Damage and Fetal Demise

    doi: 10.3389/fmicb.2019.00029

    Figure Lengend Snippet: Transfusion of pregnant A129 mice with early-stage (high dose) ZIKV + plasma led to significant fetal infection and fetal and pup death. Pregnant (E10–12) A129 mice were transfused with ZIKV + plasma from early, middle, and late stages, respectively. ZIKV RNA and ZIKV titers were detected by qRT-PCR (A) and plaque assay (B) in amniotic fluid and embryonic brain collected 6 days post-transfusion. The detection limit, shown as the horizontal lines, was about 2.5 × 10 3 RNA copies/ml and 1 PFU/ml (for amniotic fluid), or 2.5 × 10 3 RNA copies/g and 1 PFU/g (for embryonic brain), respectively, for qRT-PCR and plaque assay. ∗ and ∗∗ indicate significant differences between early stage (high dose) and early or middle stages (low dose), or between early and middle stages (low dose). The data are presented as means ± s.e.m ( n = 6 mice/group). (C) Morphology of mouse uteri and placentas and conditions of fetuses 6 days after transfusion of early-stage (high dose) ZIKV + plasma. (a) E13–15 uteri from pregnant mice transfused at E7–9. (b) Representative images of fetuses in (a) . (c) E16–18 placentas from pregnant mice transfused at E10–12. (d) Representative images of E16–18 fetuses in (c) . Scale bar: 1 cm. Placental diameter in (c) and fetal size in (d) are shown. ∗ and ∗∗ indicate significant differences between early stage (high dose) and normal plasma groups. The data are presented as means ± s.e.m ( n = 6 mice/group). (D) Total born and surviving pups 24 h after birth from pregnant (E10–12) mice transfused with early-stage (high dose) ZIKV + plasma. Normal plasma: control plasma from A129 mice transfused with normal plasma without ZIKV.

    Article Snippet: Briefly, RNA was extracted using QIAamp MinElute Virus Spin Kit (for plasma and amniotic fluid) (Qiagen) and RNeasy Mini Kit (for tissues) (Qiagen), and quantified using one-step qRT-PCR in the presence of Power SYBR Green PCR Master Mix, MultiScribe Reverse Transcriptase, and AmbionTM RNase Inhibitor (Thermo Fisher Scientific) in ViiA 7 Master Cycler PCR System (Thermo Fisher Scientific).

    Techniques: Mouse Assay, Infection, Quantitative RT-PCR, Plaque Assay

    RNA Immunoprecipitation experiments of confirmed BC200 targets. ( A ) RT-qPCR analysis of BC200, GAPDH and 7SL enrichment by immunoprecipitation of the indicated proteins. RNA extracted from 10% of the input sample was used as a reference to calculate percent of input for each RNA that was bound to the immunoprecipitated protein. Data represents the mean of three independent replicates ± standard deviation. Dashed line represents the threshold value of 5% input. ( B ) Percent input values of BC200 and 7SL were compared to GAPDH to demonstrate the degree of specificity of the interactions analyzed in (A). Dashed line represents the threshold value of 2-fold enrichment. ( C ) Immunoprecipitation efficiency was monitored by performing western blot on 50 μg of PRE and POST IP samples as well as 2% of the IP.

    Journal: Nucleic Acids Research

    Article Title: Comprehensive analysis of the BC200 ribonucleoprotein reveals a reciprocal regulatory function with CSDE1/UNR

    doi: 10.1093/nar/gky860

    Figure Lengend Snippet: RNA Immunoprecipitation experiments of confirmed BC200 targets. ( A ) RT-qPCR analysis of BC200, GAPDH and 7SL enrichment by immunoprecipitation of the indicated proteins. RNA extracted from 10% of the input sample was used as a reference to calculate percent of input for each RNA that was bound to the immunoprecipitated protein. Data represents the mean of three independent replicates ± standard deviation. Dashed line represents the threshold value of 5% input. ( B ) Percent input values of BC200 and 7SL were compared to GAPDH to demonstrate the degree of specificity of the interactions analyzed in (A). Dashed line represents the threshold value of 2-fold enrichment. ( C ) Immunoprecipitation efficiency was monitored by performing western blot on 50 μg of PRE and POST IP samples as well as 2% of the IP.

    Article Snippet: For both native and crosslinking immunoprecipitations, RT-qPCR analysis was performed using an Applied Biosystems StepOnePlus instrument with the RNA to Ct One-step RT-qPCR kit (Thermo-Fisher Scientific).

    Techniques: Immunoprecipitation, Quantitative RT-PCR, Standard Deviation, Western Blot

    CSDE1 knock-down decreases stability of the BC200 RNA. ( A ) RT-qPCR analysis of BC200 expression following 72-hour CSDE1 or control knock-down and Actinomycin D treatment at T = 0. Indicated half-lives were calculated by fitting the data to a one-phase decay equation with GraphPad Prism software. Data represents the mean of three independent biological replicates measured in duplicate. ( B ) Relative total RNA quantities purified at the indicated time points from an equal number of cells to monitor total RNA decay. Data represents the mean of three independent replicates ± standard deviation. ( C ) MCF-7 cells were reverse transfected with control or CSDE1 siRNA and following 24 h were forward transfected with the plasmids containing BC200 under control of either the U6 snRNA promoter or endogenous BC200 promoter. Absolute expression levels of RNA from plasmid was calculated by RT-qPCR in parallel to a standard curve generated with purified BC200 RNA. Data represents the mean of three independent replicates ± standard deviation. ( D ) Relative CSDE1 mRNA expression was monitored by RT-qPCR analysis of the same RNA samples as used in (C).

    Journal: Nucleic Acids Research

    Article Title: Comprehensive analysis of the BC200 ribonucleoprotein reveals a reciprocal regulatory function with CSDE1/UNR

    doi: 10.1093/nar/gky860

    Figure Lengend Snippet: CSDE1 knock-down decreases stability of the BC200 RNA. ( A ) RT-qPCR analysis of BC200 expression following 72-hour CSDE1 or control knock-down and Actinomycin D treatment at T = 0. Indicated half-lives were calculated by fitting the data to a one-phase decay equation with GraphPad Prism software. Data represents the mean of three independent biological replicates measured in duplicate. ( B ) Relative total RNA quantities purified at the indicated time points from an equal number of cells to monitor total RNA decay. Data represents the mean of three independent replicates ± standard deviation. ( C ) MCF-7 cells were reverse transfected with control or CSDE1 siRNA and following 24 h were forward transfected with the plasmids containing BC200 under control of either the U6 snRNA promoter or endogenous BC200 promoter. Absolute expression levels of RNA from plasmid was calculated by RT-qPCR in parallel to a standard curve generated with purified BC200 RNA. Data represents the mean of three independent replicates ± standard deviation. ( D ) Relative CSDE1 mRNA expression was monitored by RT-qPCR analysis of the same RNA samples as used in (C).

    Article Snippet: For both native and crosslinking immunoprecipitations, RT-qPCR analysis was performed using an Applied Biosystems StepOnePlus instrument with the RNA to Ct One-step RT-qPCR kit (Thermo-Fisher Scientific).

    Techniques: Quantitative RT-PCR, Expressing, Software, Purification, Standard Deviation, Transfection, Plasmid Preparation, Generated

    BC200 and CSDE1 expression are mutually codependent. ( A ) RT-qPCR analysis of BC200 RNA expression following transfection with the indicated RNA interference oligonucleotides. Data represents the mean of three independent replicates ± standard deviation. ( B ) RT-qPCR analysis of CSDE1 mRNA expression following transfection with the indicated RNA interference oligonucleotides. Data represents the mean of three independent replicates ± standard deviation. ( C ) Western blot analysis of protein samples from a 72-hour knock-down time-course with the indicated RNA interference oligonucleotides and antibodies. Data is representative of three independent replicates. ( D ) Densitometry measurements of CSDE1 protein expression from (C) as well as replicate experiments. Data represents the mean of three independent replicates ± standard deviation

    Journal: Nucleic Acids Research

    Article Title: Comprehensive analysis of the BC200 ribonucleoprotein reveals a reciprocal regulatory function with CSDE1/UNR

    doi: 10.1093/nar/gky860

    Figure Lengend Snippet: BC200 and CSDE1 expression are mutually codependent. ( A ) RT-qPCR analysis of BC200 RNA expression following transfection with the indicated RNA interference oligonucleotides. Data represents the mean of three independent replicates ± standard deviation. ( B ) RT-qPCR analysis of CSDE1 mRNA expression following transfection with the indicated RNA interference oligonucleotides. Data represents the mean of three independent replicates ± standard deviation. ( C ) Western blot analysis of protein samples from a 72-hour knock-down time-course with the indicated RNA interference oligonucleotides and antibodies. Data is representative of three independent replicates. ( D ) Densitometry measurements of CSDE1 protein expression from (C) as well as replicate experiments. Data represents the mean of three independent replicates ± standard deviation

    Article Snippet: For both native and crosslinking immunoprecipitations, RT-qPCR analysis was performed using an Applied Biosystems StepOnePlus instrument with the RNA to Ct One-step RT-qPCR kit (Thermo-Fisher Scientific).

    Techniques: Expressing, Quantitative RT-PCR, RNA Expression, Transfection, Standard Deviation, Western Blot

    The replication abilities of viruses with different NPs underexpression of eqMx1. ( A ) MDCK cells expressing HA-tagged eqMx1 were infected with wild type (H3N8 JL89 ) or mutant viruses (H3N8 JL89- H52N-NP) at an MOI of 0.001 and the supernatants were collected at 0, 12, 24, 36, and 48 hr post-infection. Total RNA from the collected supernatants was extracted using the RNeasy plus mini kit (Qiagen) and subjected to one-step real-time quantitative PCR (qPCR) analysis using the AgPath-ID™ One-Step RT-PCR reagents according to manufacturer’s protocol. Relative mRNA expression levels were determined using double-standard curve methods. All the experiments were performed three times and with three replicates with means ± SE shown. ( B ) MDCK cells expressing eqMx1 were infected with wild type (H3N8 JL89 ) or mutant viruses (H3N8 JL89 -H52N-NP) at an MOI of 0.001 and the supernatants were collected at 0, 12, 24, 36, and 48 hr post-infection. These supernatants were subsequently used to infect MDCK cells at different dilutions (10 −1 to 10 −11 ) with at least four repeats. 48 hr post-infection, immunofluorescence assays (IFA) were performed using specific antibodies against viral NP (from our lab) and FITC-labelled secondary antibody. Finally the viral titers were calculated using Reed and Munech methodology and results are shown as TCID 50 /mL. The results of a single experiment performed with four repeats are shown and results were subsequently confirmed in three separate experiments. Error bars indicate standard deviations (SD).

    Journal: Viruses

    Article Title: Equine Mx1 Restricts Influenza A Virus Replication by Targeting at Distinct Site of its Nucleoprotein

    doi: 10.3390/v11121114

    Figure Lengend Snippet: The replication abilities of viruses with different NPs underexpression of eqMx1. ( A ) MDCK cells expressing HA-tagged eqMx1 were infected with wild type (H3N8 JL89 ) or mutant viruses (H3N8 JL89- H52N-NP) at an MOI of 0.001 and the supernatants were collected at 0, 12, 24, 36, and 48 hr post-infection. Total RNA from the collected supernatants was extracted using the RNeasy plus mini kit (Qiagen) and subjected to one-step real-time quantitative PCR (qPCR) analysis using the AgPath-ID™ One-Step RT-PCR reagents according to manufacturer’s protocol. Relative mRNA expression levels were determined using double-standard curve methods. All the experiments were performed three times and with three replicates with means ± SE shown. ( B ) MDCK cells expressing eqMx1 were infected with wild type (H3N8 JL89 ) or mutant viruses (H3N8 JL89 -H52N-NP) at an MOI of 0.001 and the supernatants were collected at 0, 12, 24, 36, and 48 hr post-infection. These supernatants were subsequently used to infect MDCK cells at different dilutions (10 −1 to 10 −11 ) with at least four repeats. 48 hr post-infection, immunofluorescence assays (IFA) were performed using specific antibodies against viral NP (from our lab) and FITC-labelled secondary antibody. Finally the viral titers were calculated using Reed and Munech methodology and results are shown as TCID 50 /mL. The results of a single experiment performed with four repeats are shown and results were subsequently confirmed in three separate experiments. Error bars indicate standard deviations (SD).

    Article Snippet: Measurement of Gene Expression Using RT-qPCR Total RNA from the collected supernatants was extracted using the RNeasy plus Minikit (Qiagen, Venlo, Netherland) and were subjected to a one-step real-time quantitative PCR (RT-qPCR) analysis using the AgPath-ID™ One-Step RT-PCR reagents (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s protocol.

    Techniques: Expressing, Infection, Mutagenesis, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence