digoxigenin dig labeled β actin rna  (Roche)


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    Structured Review

    Roche digoxigenin dig labeled β actin rna
    Separation and purification of unlabeled and labeled <t>β-actin</t> <t>RNA</t> transcripts. <t>DIG-labeled</t> β-actin RNA transcript (1 µg) was analyzed by IP RP HPLC under elution condition 3. Unlabeled β-actin RNA elutes with a retention time of ∼4.7 min. DIG-labeled β-actin RNA elutes at the end of the gradient. Each peak was captured by manual collection.
    Digoxigenin Dig Labeled β Actin Rna, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "RNA analysis by ion-pair reversed-phase high performance liquid chromatography"

    Article Title: RNA analysis by ion-pair reversed-phase high performance liquid chromatography

    Journal: Nucleic Acids Research

    doi:

    Separation and purification of unlabeled and labeled β-actin RNA transcripts. DIG-labeled β-actin RNA transcript (1 µg) was analyzed by IP RP HPLC under elution condition 3. Unlabeled β-actin RNA elutes with a retention time of ∼4.7 min. DIG-labeled β-actin RNA elutes at the end of the gradient. Each peak was captured by manual collection.
    Figure Legend Snippet: Separation and purification of unlabeled and labeled β-actin RNA transcripts. DIG-labeled β-actin RNA transcript (1 µg) was analyzed by IP RP HPLC under elution condition 3. Unlabeled β-actin RNA elutes with a retention time of ∼4.7 min. DIG-labeled β-actin RNA elutes at the end of the gradient. Each peak was captured by manual collection.

    Techniques Used: Purification, Labeling, High Performance Liquid Chromatography

    2) Product Images from "Prevalence and Characterization of Murine Leukemia Virus Contamination in Human Cell Lines"

    Article Title: Prevalence and Characterization of Murine Leukemia Virus Contamination in Human Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125622

    Detection of RT activity in cell lines infected with MLV of contaminated cell lines by PERT assay. Cell culture supernatants of the infected cell lines NCI-H82 and VERO-B4 were harvested after 45 and 44 days after infection, respectively. MS2 phage RNA was reverse transcribed with an MS2-specific primer and the MLV derived RT of the samples in the presence of MnCl 2 . The cDNA was then amplified with a second MS2-specific primer applying PCR. The signals of the cell lines infected with LCL-HO supernatant may potentially be ascribed to the activity of SMRV derived RT. dpi: days post infection.
    Figure Legend Snippet: Detection of RT activity in cell lines infected with MLV of contaminated cell lines by PERT assay. Cell culture supernatants of the infected cell lines NCI-H82 and VERO-B4 were harvested after 45 and 44 days after infection, respectively. MS2 phage RNA was reverse transcribed with an MS2-specific primer and the MLV derived RT of the samples in the presence of MnCl 2 . The cDNA was then amplified with a second MS2-specific primer applying PCR. The signals of the cell lines infected with LCL-HO supernatant may potentially be ascribed to the activity of SMRV derived RT. dpi: days post infection.

    Techniques Used: Activity Assay, Infection, Cell Culture, Derivative Assay, Amplification, Polymerase Chain Reaction

    3) Product Images from "Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration"

    Article Title: Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21114089

    Optimization of microRNA acquirement from seminal plasma. MicroRNA yields were compared by qPCR between different RNA purification methods, with variation of the input volume of seminal plasma in the extraction procedure (25, 50, 100, 200 µL), with or without addition of an exosome enrichment kit, with or without incorporation of MS2 RNA carrier and proteinase K for the RNA extraction procedure, and with variation of the input volume of RNA in the cDNA reaction (1, 2, 4 µL). Data are represented as means ± SEM.
    Figure Legend Snippet: Optimization of microRNA acquirement from seminal plasma. MicroRNA yields were compared by qPCR between different RNA purification methods, with variation of the input volume of seminal plasma in the extraction procedure (25, 50, 100, 200 µL), with or without addition of an exosome enrichment kit, with or without incorporation of MS2 RNA carrier and proteinase K for the RNA extraction procedure, and with variation of the input volume of RNA in the cDNA reaction (1, 2, 4 µL). Data are represented as means ± SEM.

    Techniques Used: Real-time Polymerase Chain Reaction, Purification, RNA Extraction

    4) Product Images from "Detection of Differential Gene Expression in Biofilm-Forming versus Planktonic Populations of Staphylococcus aureus Using Micro-Representational-Difference Analysis"

    Article Title: Detection of Differential Gene Expression in Biofilm-Forming versus Planktonic Populations of Staphylococcus aureus Using Micro-Representational-Difference Analysis

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.67.7.2958-2965.2001

    Micro-RDA sensitivity test. Different amounts, ranging from 1 copy per cell up to 100 copies per cell, of a known RNA species (MS2 RNA) was mixed with total RNA of S. aureus . In the subsequent micro-RDA, this mixture was used as the tester and total S. aureus RNA was used as the driver. DP 3 was electrophoresed through a 2% agarose gel and stained with ethidium bromide. Fragments typical of the MS2 amplicon could be detected in the spike with a ratio of minimally 5 copies per cell. Lane 1, the S. aureus amplicon (MS2 RNA) at 100 copies/cell in RNA (3-h culture); lanes 2 to 6, DP 3 (MS2 RNA spike in RNA [3-h culture]) at 1 copy of MS2/cell and 5, 10, 50, and 100 copies of MS2/cell, respectively; lane 7, the MS 2 amplicon; lanes M, molecular size markers.
    Figure Legend Snippet: Micro-RDA sensitivity test. Different amounts, ranging from 1 copy per cell up to 100 copies per cell, of a known RNA species (MS2 RNA) was mixed with total RNA of S. aureus . In the subsequent micro-RDA, this mixture was used as the tester and total S. aureus RNA was used as the driver. DP 3 was electrophoresed through a 2% agarose gel and stained with ethidium bromide. Fragments typical of the MS2 amplicon could be detected in the spike with a ratio of minimally 5 copies per cell. Lane 1, the S. aureus amplicon (MS2 RNA) at 100 copies/cell in RNA (3-h culture); lanes 2 to 6, DP 3 (MS2 RNA spike in RNA [3-h culture]) at 1 copy of MS2/cell and 5, 10, 50, and 100 copies of MS2/cell, respectively; lane 7, the MS 2 amplicon; lanes M, molecular size markers.

    Techniques Used: Agarose Gel Electrophoresis, Staining, Amplification, Mass Spectrometry

    5) Product Images from "Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum"

    Article Title: Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00400-12

    Sequence-specific RNA cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.
    Figure Legend Snippet: Sequence-specific RNA cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.

    Techniques Used: Sequencing, Purification, SDS Page, Affinity Chromatography, In Vitro, Labeling, Incubation

    6) Product Images from "CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis"

    Article Title: CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1900176116

    CarD slows promoter escape from the Mtb rrnA P3 promoter. ( A ) Single-round in vitro promoter escape assay results with representative gel images showing the time-dependent increase in 32 P-labeled RNA transcripts formed by M. bovis RNAP from the Mtb rrnA P3 promoter construct in the presence and absence of CarD. Promoter escape rate is quantified by the time until 90% of the final transcript intensity is reached (t 90% ). The graph shows the mean t 90% ± SEM (−CarD n = 3; +CarD n = 7). Statistical significance was analyzed by Welch’s t test. * P
    Figure Legend Snippet: CarD slows promoter escape from the Mtb rrnA P3 promoter. ( A ) Single-round in vitro promoter escape assay results with representative gel images showing the time-dependent increase in 32 P-labeled RNA transcripts formed by M. bovis RNAP from the Mtb rrnA P3 promoter construct in the presence and absence of CarD. Promoter escape rate is quantified by the time until 90% of the final transcript intensity is reached (t 90% ). The graph shows the mean t 90% ± SEM (−CarD n = 3; +CarD n = 7). Statistical significance was analyzed by Welch’s t test. * P

    Techniques Used: In Vitro, Labeling, Construct

    CarD mutants with similar effects on RP o stability show similar gene expression profiles. ( A ) A principal component analysis of RNA sequencing samples based on read counts generated by mapping reads to a library of protein encoding sequences from the Mtb H37Rv genome. Each point represents one sequencing sample colored by genotype, and the distance between 2 points reflects the variance in gene expression between the 2 samples. The first 2 principle components, PC1 and PC2, define the x - and y -axes, respectively, and account for 70% and 11.4% of the variance, respectively. ( B ) Hierarchical clustering of RNAseq samples based on read count data in which each column and row represents one sample. The color of each cell represents the Euclidean distance, calculated according to relative expression of all Mtb protein encoding genes, between each sample pair. ( C and D ) Venn diagrams and scatter plots show the overlap in the lists of Mtb genes that were significantly differentially expressed ( P adj
    Figure Legend Snippet: CarD mutants with similar effects on RP o stability show similar gene expression profiles. ( A ) A principal component analysis of RNA sequencing samples based on read counts generated by mapping reads to a library of protein encoding sequences from the Mtb H37Rv genome. Each point represents one sequencing sample colored by genotype, and the distance between 2 points reflects the variance in gene expression between the 2 samples. The first 2 principle components, PC1 and PC2, define the x - and y -axes, respectively, and account for 70% and 11.4% of the variance, respectively. ( B ) Hierarchical clustering of RNAseq samples based on read count data in which each column and row represents one sample. The color of each cell represents the Euclidean distance, calculated according to relative expression of all Mtb protein encoding genes, between each sample pair. ( C and D ) Venn diagrams and scatter plots show the overlap in the lists of Mtb genes that were significantly differentially expressed ( P adj

    Techniques Used: Expressing, RNA Sequencing Assay, Generated, Sequencing

    7) Product Images from "Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum"

    Article Title: Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00400-12

    Sequence-specific RNA cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.
    Figure Legend Snippet: Sequence-specific RNA cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.

    Techniques Used: Sequencing, Purification, SDS Page, Affinity Chromatography, In Vitro, Labeling, Incubation

    8) Product Images from "Detection of Differential Gene Expression in Biofilm-Forming versus Planktonic Populations of Staphylococcus aureus Using Micro-Representational-Difference Analysis"

    Article Title: Detection of Differential Gene Expression in Biofilm-Forming versus Planktonic Populations of Staphylococcus aureus Using Micro-Representational-Difference Analysis

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.67.7.2958-2965.2001

    Micro-RDA sensitivity test. Different amounts, ranging from 1 copy per cell up to 100 copies per cell, of a known RNA species (MS2 RNA) was mixed with total RNA of S. aureus . In the subsequent micro-RDA, this mixture was used as the tester and total S. aureus RNA was used as the driver. DP 3 was electrophoresed through a 2% agarose gel and stained with ethidium bromide. Fragments typical of the MS2 amplicon could be detected in the spike with a ratio of minimally 5 copies per cell. Lane 1, the S. aureus amplicon (MS2 RNA) at 100 copies/cell in RNA (3-h culture); lanes 2 to 6, DP 3 (MS2 RNA spike in RNA [3-h culture]) at 1 copy of MS2/cell and 5, 10, 50, and 100 copies of MS2/cell, respectively; lane 7, the MS 2 amplicon; lanes M, molecular size markers.
    Figure Legend Snippet: Micro-RDA sensitivity test. Different amounts, ranging from 1 copy per cell up to 100 copies per cell, of a known RNA species (MS2 RNA) was mixed with total RNA of S. aureus . In the subsequent micro-RDA, this mixture was used as the tester and total S. aureus RNA was used as the driver. DP 3 was electrophoresed through a 2% agarose gel and stained with ethidium bromide. Fragments typical of the MS2 amplicon could be detected in the spike with a ratio of minimally 5 copies per cell. Lane 1, the S. aureus amplicon (MS2 RNA) at 100 copies/cell in RNA (3-h culture); lanes 2 to 6, DP 3 (MS2 RNA spike in RNA [3-h culture]) at 1 copy of MS2/cell and 5, 10, 50, and 100 copies of MS2/cell, respectively; lane 7, the MS 2 amplicon; lanes M, molecular size markers.

    Techniques Used: Agarose Gel Electrophoresis, Staining, Amplification, Mass Spectrometry

    9) Product Images from "Growth and Translation Inhibition through Sequence-specific RNA Binding by Mycobacterium tuberculosis VapC Toxin"

    Article Title: Growth and Translation Inhibition through Sequence-specific RNA Binding by Mycobacterium tuberculosis VapC Toxin

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.340109

    VapC-mt4-mediated MS2 RNA cleavage. Equal molar amounts of His 6 -VapC-mt4 ( A ) or His 6 -MazF-mt1 ( B ) were added to MS2 RNA, and RNA cleavage was monitored over time. Preincubation o f His 6 -VapC-mt4 with His 6 -VapB-mt4 before the addition of the MS2 RNA prevented RNA cleavage. Data shown are representative of three independent experiments.
    Figure Legend Snippet: VapC-mt4-mediated MS2 RNA cleavage. Equal molar amounts of His 6 -VapC-mt4 ( A ) or His 6 -MazF-mt1 ( B ) were added to MS2 RNA, and RNA cleavage was monitored over time. Preincubation o f His 6 -VapC-mt4 with His 6 -VapB-mt4 before the addition of the MS2 RNA prevented RNA cleavage. Data shown are representative of three independent experiments.

    Techniques Used:

    VapC-mt4-mediated sequence-specific MS2 RNA cleavage. A–K , primer extension analysis for MS2 RNA identified 12 VapC-mt4 cleavage sites. Lanes labeled “−” represent control reactions to which no proteins were added. In lanes labeled “ B ,” purified His 6 -VapB-mt4 was incubated with the MS2 RNA. In lanes labeled “ C ,” the addition of His 6 -VapC-mt4 resulted in the cleavage of the MS2 RNA. In lanes labeled “ BC ,” preincubation of His 6 -VapC-mt4 with His 6 -VapB-mt4 prior to the addition of the MS2 RNA prevented RNA cleavage. Cleavage sites/products are indicated by black arrowheads on the right side of gels and in the relevant RNA sequences below each gel. Apparent secondary structure is indicated by white arrowheads on the right side of gels. Lanes G , A , T , and C correspond to DNA sequencing ladders prepared by reverse transcription using the same primers used in the primer extension reactions. Data shown are representative of two independent experiments. L , alignment of sequences containing VapC-mt4 cleavage sites. The ACGC consensus sequence is in bold .
    Figure Legend Snippet: VapC-mt4-mediated sequence-specific MS2 RNA cleavage. A–K , primer extension analysis for MS2 RNA identified 12 VapC-mt4 cleavage sites. Lanes labeled “−” represent control reactions to which no proteins were added. In lanes labeled “ B ,” purified His 6 -VapB-mt4 was incubated with the MS2 RNA. In lanes labeled “ C ,” the addition of His 6 -VapC-mt4 resulted in the cleavage of the MS2 RNA. In lanes labeled “ BC ,” preincubation of His 6 -VapC-mt4 with His 6 -VapB-mt4 prior to the addition of the MS2 RNA prevented RNA cleavage. Cleavage sites/products are indicated by black arrowheads on the right side of gels and in the relevant RNA sequences below each gel. Apparent secondary structure is indicated by white arrowheads on the right side of gels. Lanes G , A , T , and C correspond to DNA sequencing ladders prepared by reverse transcription using the same primers used in the primer extension reactions. Data shown are representative of two independent experiments. L , alignment of sequences containing VapC-mt4 cleavage sites. The ACGC consensus sequence is in bold .

    Techniques Used: Sequencing, Labeling, Purification, Incubation, DNA Sequencing

    10) Product Images from "CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis"

    Article Title: CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1900176116

    CarD slows promoter escape from the Mtb rrnA P3 promoter. ( A ) Single-round in vitro promoter escape assay results with representative gel images showing the time-dependent increase in 32 P-labeled RNA transcripts formed by M. bovis RNAP from the Mtb rrnA P3 promoter construct in the presence and absence of CarD. Promoter escape rate is quantified by the time until 90% of the final transcript intensity is reached (t 90% ). The graph shows the mean t 90% ± SEM (−CarD n = 3; +CarD n = 7). Statistical significance was analyzed by Welch’s t test. * P
    Figure Legend Snippet: CarD slows promoter escape from the Mtb rrnA P3 promoter. ( A ) Single-round in vitro promoter escape assay results with representative gel images showing the time-dependent increase in 32 P-labeled RNA transcripts formed by M. bovis RNAP from the Mtb rrnA P3 promoter construct in the presence and absence of CarD. Promoter escape rate is quantified by the time until 90% of the final transcript intensity is reached (t 90% ). The graph shows the mean t 90% ± SEM (−CarD n = 3; +CarD n = 7). Statistical significance was analyzed by Welch’s t test. * P

    Techniques Used: In Vitro, Labeling, Construct

    CarD mutants with similar effects on RP o stability show similar gene expression profiles. ( A ) A principal component analysis of RNA sequencing samples based on read counts generated by mapping reads to a library of protein encoding sequences from the Mtb H37Rv genome. Each point represents one sequencing sample colored by genotype, and the distance between 2 points reflects the variance in gene expression between the 2 samples. The first 2 principle components, PC1 and PC2, define the x - and y -axes, respectively, and account for 70% and 11.4% of the variance, respectively. ( B ) Hierarchical clustering of RNAseq samples based on read count data in which each column and row represents one sample. The color of each cell represents the Euclidean distance, calculated according to relative expression of all Mtb protein encoding genes, between each sample pair. ( C and D ) Venn diagrams and scatter plots show the overlap in the lists of Mtb genes that were significantly differentially expressed ( P adj
    Figure Legend Snippet: CarD mutants with similar effects on RP o stability show similar gene expression profiles. ( A ) A principal component analysis of RNA sequencing samples based on read counts generated by mapping reads to a library of protein encoding sequences from the Mtb H37Rv genome. Each point represents one sequencing sample colored by genotype, and the distance between 2 points reflects the variance in gene expression between the 2 samples. The first 2 principle components, PC1 and PC2, define the x - and y -axes, respectively, and account for 70% and 11.4% of the variance, respectively. ( B ) Hierarchical clustering of RNAseq samples based on read count data in which each column and row represents one sample. The color of each cell represents the Euclidean distance, calculated according to relative expression of all Mtb protein encoding genes, between each sample pair. ( C and D ) Venn diagrams and scatter plots show the overlap in the lists of Mtb genes that were significantly differentially expressed ( P adj

    Techniques Used: Expressing, RNA Sequencing Assay, Generated, Sequencing

    11) Product Images from "Domains within RbpA Serve Specific Functional Roles That Regulate the Expression of Distinct Mycobacterial Gene Subsets"

    Article Title: Domains within RbpA Serve Specific Functional Roles That Regulate the Expression of Distinct Mycobacterial Gene Subsets

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00690-17

    Multiple RbpA structural domains are important for mycobacterial growth and viability. (A) Diagram showing that M. tuberculosis RbpA is composed of an N-terminal tail (NTT) (amino acids 1 to 25), a core domain (CD) (amino acids 26 to 66), a basic linker (BL) (amino acids 67 to 80), and a σ-interaction domain (SID) (amino acids 81 to 111). (B) Table of M. tuberculosis and M. smegmatis strains engineered or determined to be nonviable with replacement of the RbpA Mtb WT expression cassette with a cassette expressing RbpA Mtb R79A , RbpA Mtb R88A , RbpA Mtb 1–71 , or RbpA Mtb 72–111 . An empty expression cassette was transformed as a negative control, while replacement of RbpA Mtb WT with RbpA Mtb WT was used as a positive control. (C) Growth curves of M. smegmatis expressing RbpA Mtb WT , RbpA Mtb R79A , RbpA Mtb R88A , or RbpA Mtb 72–111 , with nine replicates for each strain. (D) M. smegmatis doubling times calculated from the growth curves in panel C. Results are plotted as means ± standard deviations. Statistical significance was analyzed by analysis of variance (ANOVA) and Tukey's multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. All comparisons were included in the analysis, but only statistically significant differences are indicated in the figure. (E) Lysates from M. smegmatis Δ rbpA attB :: tet-rbpA expressing RbpA Mtb WT -FLAG (lane 1), RbpA Mtb R79A -FLAG (lane 2), RbpA Mtb R88A -FLAG (lane 3), or RbpA Mtb 72–111 -FLAG (lane 4), analyzed with monoclonal antibodies specific for either RNAP β or FLAG. (F) Graphical representation of RbpA stability as the ratio of RbpA molecules per RNAP β, showing the means ± standard errors of the means of three replicates. Statistical significance was analyzed by ANOVA and Tukey's multiple-comparison test. *, P ≤ 0.05; ns, not significant.
    Figure Legend Snippet: Multiple RbpA structural domains are important for mycobacterial growth and viability. (A) Diagram showing that M. tuberculosis RbpA is composed of an N-terminal tail (NTT) (amino acids 1 to 25), a core domain (CD) (amino acids 26 to 66), a basic linker (BL) (amino acids 67 to 80), and a σ-interaction domain (SID) (amino acids 81 to 111). (B) Table of M. tuberculosis and M. smegmatis strains engineered or determined to be nonviable with replacement of the RbpA Mtb WT expression cassette with a cassette expressing RbpA Mtb R79A , RbpA Mtb R88A , RbpA Mtb 1–71 , or RbpA Mtb 72–111 . An empty expression cassette was transformed as a negative control, while replacement of RbpA Mtb WT with RbpA Mtb WT was used as a positive control. (C) Growth curves of M. smegmatis expressing RbpA Mtb WT , RbpA Mtb R79A , RbpA Mtb R88A , or RbpA Mtb 72–111 , with nine replicates for each strain. (D) M. smegmatis doubling times calculated from the growth curves in panel C. Results are plotted as means ± standard deviations. Statistical significance was analyzed by analysis of variance (ANOVA) and Tukey's multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. All comparisons were included in the analysis, but only statistically significant differences are indicated in the figure. (E) Lysates from M. smegmatis Δ rbpA attB :: tet-rbpA expressing RbpA Mtb WT -FLAG (lane 1), RbpA Mtb R79A -FLAG (lane 2), RbpA Mtb R88A -FLAG (lane 3), or RbpA Mtb 72–111 -FLAG (lane 4), analyzed with monoclonal antibodies specific for either RNAP β or FLAG. (F) Graphical representation of RbpA stability as the ratio of RbpA molecules per RNAP β, showing the means ± standard errors of the means of three replicates. Statistical significance was analyzed by ANOVA and Tukey's multiple-comparison test. *, P ≤ 0.05; ns, not significant.

    Techniques Used: Expressing, Transformation Assay, Negative Control, Positive Control

    12) Product Images from "Structural and biophysical characterization of Staphylococcus aureus SaMazF shows conservation of functional dynamics"

    Article Title: Structural and biophysical characterization of Staphylococcus aureus SaMazF shows conservation of functional dynamics

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku266

    Purification of Sa MazF. ( A ) Ni-NTA purification of Sa MazE and Sa MazF. After loading, Sa MazE is eluted using a gradient of guanidinium hydrochloride while Sa MazF remains bound to the column. Sa MazF is eluted using an immidazole gradient and subsequently dialyzed to remove the guanidinium. ( B ) SDS-PAGE showing the progress of expression and purification. Lane 1: molecular weight marker (Fermentas PageRuler). Lane 2: E. coli extract prior to induction. Lane 3: E. coli 2 h post induction. Lane 4: E. coli extract after overnight induction. Lane 5: Sa MazE eluted from the Ni-NTA column. Lane 6: fractions in between the Sa MazE and Sa MazF peaks. Lane 7: Sa MazF eluted from the Ni-NTA column. Lanes 8 and 9: Sa MazF after further purification on SEC. ( C ) RNase activity of Sa MazF. The figure shows the ribonuclease activity of Sa MazF against bacteriophage MS2 genomic RNA. Lane 1: New England Biolabs Inc. low range ssRNA ladder (50, 80, 150, 300, 500 and 1000 bases). Lane 2: intact RNA control, excluding any nonspecific RNase contamination. Lanes 3 and 4: cleaved RNA by an active Sa MazF at 1 μM and 2 μM, respectively. Lane 5: RNA degradation inhibition of Sa MazF by the presence of Sa MazE. Lane 6: Sa MazE sample incubated with RNA.
    Figure Legend Snippet: Purification of Sa MazF. ( A ) Ni-NTA purification of Sa MazE and Sa MazF. After loading, Sa MazE is eluted using a gradient of guanidinium hydrochloride while Sa MazF remains bound to the column. Sa MazF is eluted using an immidazole gradient and subsequently dialyzed to remove the guanidinium. ( B ) SDS-PAGE showing the progress of expression and purification. Lane 1: molecular weight marker (Fermentas PageRuler). Lane 2: E. coli extract prior to induction. Lane 3: E. coli 2 h post induction. Lane 4: E. coli extract after overnight induction. Lane 5: Sa MazE eluted from the Ni-NTA column. Lane 6: fractions in between the Sa MazE and Sa MazF peaks. Lane 7: Sa MazF eluted from the Ni-NTA column. Lanes 8 and 9: Sa MazF after further purification on SEC. ( C ) RNase activity of Sa MazF. The figure shows the ribonuclease activity of Sa MazF against bacteriophage MS2 genomic RNA. Lane 1: New England Biolabs Inc. low range ssRNA ladder (50, 80, 150, 300, 500 and 1000 bases). Lane 2: intact RNA control, excluding any nonspecific RNase contamination. Lanes 3 and 4: cleaved RNA by an active Sa MazF at 1 μM and 2 μM, respectively. Lane 5: RNA degradation inhibition of Sa MazF by the presence of Sa MazE. Lane 6: Sa MazE sample incubated with RNA.

    Techniques Used: Purification, SDS Page, Expressing, Molecular Weight, Marker, Size-exclusion Chromatography, Activity Assay, Inhibition, Incubation

    13) Product Images from "Changes of micro-RNAs in asymptomatic subjects sensitized to Japanese cedar pollen after prophylactic sublingual immunotherapy"

    Article Title: Changes of micro-RNAs in asymptomatic subjects sensitized to Japanese cedar pollen after prophylactic sublingual immunotherapy

    Journal: Allergy & Rhinology

    doi: 10.2500/ar.2015.6.0107

    Comparison of expression levels of let-7b between preseason and postseason among the (A) placebo and (B) sublingual immunotherapy (SLIT) groups. The value of micro-RNA (miRNA) in preseason samples was designated as 1 for each subject, and the fold changes in miRNA values in postseason samples were then determined. Statistical significance was determined with a Student's paired t-test using ΔCt value adjusted with the reference value. Placebo group, closed circles and solid lines; SLIT group, open circles and dotted lines.
    Figure Legend Snippet: Comparison of expression levels of let-7b between preseason and postseason among the (A) placebo and (B) sublingual immunotherapy (SLIT) groups. The value of micro-RNA (miRNA) in preseason samples was designated as 1 for each subject, and the fold changes in miRNA values in postseason samples were then determined. Statistical significance was determined with a Student's paired t-test using ΔCt value adjusted with the reference value. Placebo group, closed circles and solid lines; SLIT group, open circles and dotted lines.

    Techniques Used: Expressing

    Change in serum miR-223 expression for each participant. The value of micro-RNA (miRNA) in preseason samples was designated as 1 for each subject, and the fold changes in miRNA value in postseason samples were then determined. Statistical significance was determined with a Student's paired t-test using ΔCt values adjusted with the reference value. Placebo group, closed circles and solid lines; sublingual immunotherapy (SLIT) group, open circles and dotted lines.
    Figure Legend Snippet: Change in serum miR-223 expression for each participant. The value of micro-RNA (miRNA) in preseason samples was designated as 1 for each subject, and the fold changes in miRNA value in postseason samples were then determined. Statistical significance was determined with a Student's paired t-test using ΔCt values adjusted with the reference value. Placebo group, closed circles and solid lines; sublingual immunotherapy (SLIT) group, open circles and dotted lines.

    Techniques Used: Expressing

    14) Product Images from "Inhibition of LINE-1 retrotransposon-encoded reverse transcriptase modulates the expression of cell differentiation genes in breast cancer cells"

    Article Title: Inhibition of LINE-1 retrotransposon-encoded reverse transcriptase modulates the expression of cell differentiation genes in breast cancer cells

    Journal: Breast Cancer Research and Treatment

    doi: 10.1007/s10549-013-2812-7

    L1-encoded RT activity in breast cancer cells. a Endogenous RT activity was detected after incubation of synthetic MS2 phage RNA with cell extracts from a panel of breast cancer cells. Control reactions were set up by omitting cell extract ( negative control ) or adding cell extracts from NTera.2D1 human embryonic carcinoma cells ( positive control ). The PCR product of 110 bp (corresponding to the reverse-transcribed MS2 cDNA) is shown. Marker, 1 kb-plus DNA marker. b The L1-encoded ORF2p, which contains the RT enzyme, was detected by western blotting of whole-cell lysates from normal and a panel of breast cancer cells. NTera.2D1 were used as positive controls. For protein normalization, α-tubulin was used as a loading control. c The L1 ORF2 mRNAs derived from L1 expression were quantified by qRT-PCR with primer specific for the ORF2 sequence. The data are shown as fold change in each breast cancer panel cells compared to normal MCF10A after normalization to the HPRT1 housekeeping gene. Each point represents the average of three independent experiments, with each experiment performed in triplicate. Error bars indicate SD ( n = 3)
    Figure Legend Snippet: L1-encoded RT activity in breast cancer cells. a Endogenous RT activity was detected after incubation of synthetic MS2 phage RNA with cell extracts from a panel of breast cancer cells. Control reactions were set up by omitting cell extract ( negative control ) or adding cell extracts from NTera.2D1 human embryonic carcinoma cells ( positive control ). The PCR product of 110 bp (corresponding to the reverse-transcribed MS2 cDNA) is shown. Marker, 1 kb-plus DNA marker. b The L1-encoded ORF2p, which contains the RT enzyme, was detected by western blotting of whole-cell lysates from normal and a panel of breast cancer cells. NTera.2D1 were used as positive controls. For protein normalization, α-tubulin was used as a loading control. c The L1 ORF2 mRNAs derived from L1 expression were quantified by qRT-PCR with primer specific for the ORF2 sequence. The data are shown as fold change in each breast cancer panel cells compared to normal MCF10A after normalization to the HPRT1 housekeeping gene. Each point represents the average of three independent experiments, with each experiment performed in triplicate. Error bars indicate SD ( n = 3)

    Techniques Used: Activity Assay, Incubation, Negative Control, Positive Control, Polymerase Chain Reaction, Marker, Western Blot, Derivative Assay, Expressing, Quantitative RT-PCR, Sequencing

    15) Product Images from "Effect of Rickettsial Toxin VapC on Its Eukaryotic Host"

    Article Title: Effect of Rickettsial Toxin VapC on Its Eukaryotic Host

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026528

    Heterologous host growth inhibition by R. felis VapC toxin is controlled by antitoxin VapB. a. Growth curves of E. coli cultures expressing the vapB-1 , vapC-1 or vapBC-1 operon. b. Growth curves of E. coli cultures expressing the vapB-2 , vapC-2 or vapBC-2 operon. The error bars represent the mean of triplicates ± standard deviation. c. Growth curves of yeast cells in Sgal induction media expressing R. felis vapB-2 , vapC-2 , VapB2-VapC2 and/or control pBNS. d. Yeast toxicity assays of S. cerevisiae harboring plasmids with indicated genes or empty vector, under inducing (Sgal) or noninducing conditions (SD). e. In vitro RNase activity of free R. felis and R. bellii VapC-1 proteins: The MS2 phage RNA profiles after incubation reactions with irrelevant protein (lane 1 and 2), R. felis VapC-1 (lane 3), R. felis VapB/C-1 complex (lane 4), R.felis antitoxin VapB-1 (lane 5), R. bellii VapC-1 (lane 6), R. bellii VapB/C-1 complex (lane 7), R. bellii antitoxin VapB-1 (lane 8), RNase A as positive control (lane 9), without protein (lane 10).
    Figure Legend Snippet: Heterologous host growth inhibition by R. felis VapC toxin is controlled by antitoxin VapB. a. Growth curves of E. coli cultures expressing the vapB-1 , vapC-1 or vapBC-1 operon. b. Growth curves of E. coli cultures expressing the vapB-2 , vapC-2 or vapBC-2 operon. The error bars represent the mean of triplicates ± standard deviation. c. Growth curves of yeast cells in Sgal induction media expressing R. felis vapB-2 , vapC-2 , VapB2-VapC2 and/or control pBNS. d. Yeast toxicity assays of S. cerevisiae harboring plasmids with indicated genes or empty vector, under inducing (Sgal) or noninducing conditions (SD). e. In vitro RNase activity of free R. felis and R. bellii VapC-1 proteins: The MS2 phage RNA profiles after incubation reactions with irrelevant protein (lane 1 and 2), R. felis VapC-1 (lane 3), R. felis VapB/C-1 complex (lane 4), R.felis antitoxin VapB-1 (lane 5), R. bellii VapC-1 (lane 6), R. bellii VapB/C-1 complex (lane 7), R. bellii antitoxin VapB-1 (lane 8), RNase A as positive control (lane 9), without protein (lane 10).

    Techniques Used: Inhibition, Expressing, Standard Deviation, Plasmid Preparation, In Vitro, Activity Assay, Incubation, Positive Control

    16) Product Images from "Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library"

    Article Title: Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37233-y

    Evolved KlenTaq DNA polymerase with reverse transcriptase activity. ( A ) Depiction of novel mutations contributing to reverse transcriptase activity marked in red. ( B ) Reverse transcriptase activity studied by primer extension experiments for WT and Mut_RT. Reactions carried out for the indicated time points under identical conditions. P = primer, C = Control reaction that was carried out with the corresponding DNA template. Full-length gel is presented in Supplementary Fig. S16 . ( C ) Reverse transcriptase activity of Mut_RT in real time PCR assay employed with varying amounts of RNA template (left) and their corresponding melting peak analysis (right). ( D ) Employability of Mut_RT in reverse transcription from total RNA extract (100 ng) using the HPRT mRNA target (left) and the agarose gel analysis of amplified product of HRPT transcript (lane 1) and the no template control (lane2) Full-length gel is presented in Supplementary Fig. S17 .
    Figure Legend Snippet: Evolved KlenTaq DNA polymerase with reverse transcriptase activity. ( A ) Depiction of novel mutations contributing to reverse transcriptase activity marked in red. ( B ) Reverse transcriptase activity studied by primer extension experiments for WT and Mut_RT. Reactions carried out for the indicated time points under identical conditions. P = primer, C = Control reaction that was carried out with the corresponding DNA template. Full-length gel is presented in Supplementary Fig. S16 . ( C ) Reverse transcriptase activity of Mut_RT in real time PCR assay employed with varying amounts of RNA template (left) and their corresponding melting peak analysis (right). ( D ) Employability of Mut_RT in reverse transcription from total RNA extract (100 ng) using the HPRT mRNA target (left) and the agarose gel analysis of amplified product of HRPT transcript (lane 1) and the no template control (lane2) Full-length gel is presented in Supplementary Fig. S17 .

    Techniques Used: Activity Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification

    17) Product Images from "RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements"

    Article Title: RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements

    Journal: bioRxiv

    doi: 10.1101/2020.07.01.183574

    Application of RanDeL-seq to map Zika Virus (ZIKV) cis elements. A. pMR766(+), a Zika virus molecular clone. The MR766 Zika virus genome is encoded as a cDNA driven by the CMV IE2 promoter. At the 3’ end of the genome, a self-cleaving Hepatitis Delta ribozyme allows for creation of an authentic 3’ end post-transcription. An intron sequence is present within NS1 to allow maintenance in bacteria but is spliced out during transcription in host cells. B. Restriction enzyme characterization of completed ZIKV deletion libraries compared to insertion libraries (“ins.”). (+) and (-) designate the template ZIKV plasmid. “S” and “L” designate the chewback length for deletion libraries. Undigested, completed deletion libraries (Lanes 1-4) were run next to undigested insertion libraries (Lanes 5-6). Insertion libraries (Lanes 7-10) treated with I-SceI or I-CeuI to excise transposon (∼1.4 kb). Deletion libraries linearized by unique ZIKV cutter KpnI (Lanes 11-14). C. Deletion depth profile of the pMR766(+)L library. The ZIKV genome is well-represented in the pMR766(+)L library, with some bias. Each base of the ZIKV genome is covered by several hundred different deletion mutants. D. Detection and quantification of ZIKV barcode cassettes by RT-qPCR. Genomic percentage of barcoded mutants to total ZIKV genomes at each day in passage 1 of the high MOI screen, and a wild-type ZIKV control (WT). RT-qPCR data was normalized to a MS2 RNA spike-in. E. Deletion Depth Profile of intracellular RNA of 293T co-transfected with the wild-type ZIKV plasmid and the pooled deletion libraries. F. Deletion Depth Profile of pMR766(+)L after Passage 1. Only deletions in Pr–NS1 can be trans-complemented by wild-type ZIKV. H. Final map of ZIKV cis- and trans-acting elements after Passage 2. The two cis-acting regions are highlighted in blue and do not tolerate deletion (i.e., must be present for efficient transmission to occur). The trans-acting region is highlighted in green and can be complemented in trans (i.e., if deleted, transmission occurs by complementation from wild-type virus).
    Figure Legend Snippet: Application of RanDeL-seq to map Zika Virus (ZIKV) cis elements. A. pMR766(+), a Zika virus molecular clone. The MR766 Zika virus genome is encoded as a cDNA driven by the CMV IE2 promoter. At the 3’ end of the genome, a self-cleaving Hepatitis Delta ribozyme allows for creation of an authentic 3’ end post-transcription. An intron sequence is present within NS1 to allow maintenance in bacteria but is spliced out during transcription in host cells. B. Restriction enzyme characterization of completed ZIKV deletion libraries compared to insertion libraries (“ins.”). (+) and (-) designate the template ZIKV plasmid. “S” and “L” designate the chewback length for deletion libraries. Undigested, completed deletion libraries (Lanes 1-4) were run next to undigested insertion libraries (Lanes 5-6). Insertion libraries (Lanes 7-10) treated with I-SceI or I-CeuI to excise transposon (∼1.4 kb). Deletion libraries linearized by unique ZIKV cutter KpnI (Lanes 11-14). C. Deletion depth profile of the pMR766(+)L library. The ZIKV genome is well-represented in the pMR766(+)L library, with some bias. Each base of the ZIKV genome is covered by several hundred different deletion mutants. D. Detection and quantification of ZIKV barcode cassettes by RT-qPCR. Genomic percentage of barcoded mutants to total ZIKV genomes at each day in passage 1 of the high MOI screen, and a wild-type ZIKV control (WT). RT-qPCR data was normalized to a MS2 RNA spike-in. E. Deletion Depth Profile of intracellular RNA of 293T co-transfected with the wild-type ZIKV plasmid and the pooled deletion libraries. F. Deletion Depth Profile of pMR766(+)L after Passage 1. Only deletions in Pr–NS1 can be trans-complemented by wild-type ZIKV. H. Final map of ZIKV cis- and trans-acting elements after Passage 2. The two cis-acting regions are highlighted in blue and do not tolerate deletion (i.e., must be present for efficient transmission to occur). The trans-acting region is highlighted in green and can be complemented in trans (i.e., if deleted, transmission occurs by complementation from wild-type virus).

    Techniques Used: Sequencing, Plasmid Preparation, Quantitative RT-PCR, Transfection, Transmission Assay

    Genetic Screen of random deletion library to map viral cis- and trans- elements A. Block design of high-MOI passage. Wild-type NL4-3 and deletion library plasmids co-transfected 293T cells. Virus-containing supernatant infected MT-4 cells in triplicate (K, L, M) at high MOI. Infections were passaged at the end of every week, for 4 weeks. At the same time, flasks with only NL4-3 wild-type virus were passaged identically (A, B, C not shown). B. Passage details of high MOI screen. MT-4 cells (blue double discs) are infected at high MOI with a pool of virus (HIV-1) containing both wild-type (red stars) and deletion mutants (blue stars). At days 1 and 2, additional naïve MT-4 were added and the culture volume expanded. On day 3, cell-free supernatant was harvested, and virus purified by ultracentrifugation for transfer or analysis. One passage corresponds to 3 rounds of replication. C. Detection and quantification of barcode cassettes by RT-qPCR. Genomic percentage of barcoded mutants to total HIV genomes in transfection (293T), each stage of high MOI passage (P3-P12), and a wild-type HIV control (WT). RT-qPCR data was normalized to a MS2 RNA spike-in. Error bars are standard deviation from averaging each flask (K, L, M) per passage. D. Representative deletion variant trajectories during high MOI passage. The slope in prevalence versus passage number was determined by linear regression and classified deletions as adaptive, neutral or deleterious. Data points correspond to the triplicate flasks (K, L, M) at each passage. Prevalence is in reference to the total barcode cassette pool (tagged mutants). E. Distribution of fitness in deletion variants that are not extinct by passage 12. The dashed vertical line marks the neutral fitness boundary (slope of 0). 1390 of 4390 persistent mutants were adaptive. F. The deletion depth profile of poly(A) RNA from transfected 293T cells, representing mutants able to be transcribed. G. The deletion depth profile built from the virus-containing supernatant of transfected 293T representing mutants able to be transcribed, encapsulated, and egressed. H. Deletion depth profile from virus-containing supernatant after 3 passages. I. Deletion depth profile after 6 passages. J. A model of HIV-1 cis- and trans-acting elements after 12 passages. The HIV-1 genome is composed of 4 cis-acting elements, CAE1–CAE4, (highlighted in blue) and 3 trans-acting elements, TAE1–TAE3, (highlighted in green).
    Figure Legend Snippet: Genetic Screen of random deletion library to map viral cis- and trans- elements A. Block design of high-MOI passage. Wild-type NL4-3 and deletion library plasmids co-transfected 293T cells. Virus-containing supernatant infected MT-4 cells in triplicate (K, L, M) at high MOI. Infections were passaged at the end of every week, for 4 weeks. At the same time, flasks with only NL4-3 wild-type virus were passaged identically (A, B, C not shown). B. Passage details of high MOI screen. MT-4 cells (blue double discs) are infected at high MOI with a pool of virus (HIV-1) containing both wild-type (red stars) and deletion mutants (blue stars). At days 1 and 2, additional naïve MT-4 were added and the culture volume expanded. On day 3, cell-free supernatant was harvested, and virus purified by ultracentrifugation for transfer or analysis. One passage corresponds to 3 rounds of replication. C. Detection and quantification of barcode cassettes by RT-qPCR. Genomic percentage of barcoded mutants to total HIV genomes in transfection (293T), each stage of high MOI passage (P3-P12), and a wild-type HIV control (WT). RT-qPCR data was normalized to a MS2 RNA spike-in. Error bars are standard deviation from averaging each flask (K, L, M) per passage. D. Representative deletion variant trajectories during high MOI passage. The slope in prevalence versus passage number was determined by linear regression and classified deletions as adaptive, neutral or deleterious. Data points correspond to the triplicate flasks (K, L, M) at each passage. Prevalence is in reference to the total barcode cassette pool (tagged mutants). E. Distribution of fitness in deletion variants that are not extinct by passage 12. The dashed vertical line marks the neutral fitness boundary (slope of 0). 1390 of 4390 persistent mutants were adaptive. F. The deletion depth profile of poly(A) RNA from transfected 293T cells, representing mutants able to be transcribed. G. The deletion depth profile built from the virus-containing supernatant of transfected 293T representing mutants able to be transcribed, encapsulated, and egressed. H. Deletion depth profile from virus-containing supernatant after 3 passages. I. Deletion depth profile after 6 passages. J. A model of HIV-1 cis- and trans-acting elements after 12 passages. The HIV-1 genome is composed of 4 cis-acting elements, CAE1–CAE4, (highlighted in blue) and 3 trans-acting elements, TAE1–TAE3, (highlighted in green).

    Techniques Used: Blocking Assay, Transfection, Infection, Purification, Quantitative RT-PCR, Standard Deviation, Variant Assay

    18) Product Images from "Effect of Rickettsial Toxin VapC on Its Eukaryotic Host"

    Article Title: Effect of Rickettsial Toxin VapC on Its Eukaryotic Host

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026528

    Heterologous host growth inhibition by R. felis VapC toxin is controlled by antitoxin VapB. a. Growth curves of E. coli cultures expressing the vapB-1 , vapC-1 or vapBC-1 operon. b. Growth curves of E. coli cultures expressing the vapB-2 , vapC-2 or vapBC-2 operon. The error bars represent the mean of triplicates ± standard deviation. c. Growth curves of yeast cells in Sgal induction media expressing R. felis vapB-2 , vapC-2 , VapB2-VapC2 and/or control pBNS. d. Yeast toxicity assays of S. cerevisiae harboring plasmids with indicated genes or empty vector, under inducing (Sgal) or noninducing conditions (SD). e. In vitro RNase activity of free R. felis and R. bellii VapC-1 proteins: The MS2 phage RNA profiles after incubation reactions with irrelevant protein (lane 1 and 2), R. felis VapC-1 (lane 3), R. felis VapB/C-1 complex (lane 4), R.felis antitoxin VapB-1 (lane 5), R. bellii VapC-1 (lane 6), R. bellii VapB/C-1 complex (lane 7), R. bellii antitoxin VapB-1 (lane 8), RNase A as positive control (lane 9), without protein (lane 10).
    Figure Legend Snippet: Heterologous host growth inhibition by R. felis VapC toxin is controlled by antitoxin VapB. a. Growth curves of E. coli cultures expressing the vapB-1 , vapC-1 or vapBC-1 operon. b. Growth curves of E. coli cultures expressing the vapB-2 , vapC-2 or vapBC-2 operon. The error bars represent the mean of triplicates ± standard deviation. c. Growth curves of yeast cells in Sgal induction media expressing R. felis vapB-2 , vapC-2 , VapB2-VapC2 and/or control pBNS. d. Yeast toxicity assays of S. cerevisiae harboring plasmids with indicated genes or empty vector, under inducing (Sgal) or noninducing conditions (SD). e. In vitro RNase activity of free R. felis and R. bellii VapC-1 proteins: The MS2 phage RNA profiles after incubation reactions with irrelevant protein (lane 1 and 2), R. felis VapC-1 (lane 3), R. felis VapB/C-1 complex (lane 4), R.felis antitoxin VapB-1 (lane 5), R. bellii VapC-1 (lane 6), R. bellii VapB/C-1 complex (lane 7), R. bellii antitoxin VapB-1 (lane 8), RNase A as positive control (lane 9), without protein (lane 10).

    Techniques Used: Inhibition, Expressing, Standard Deviation, Plasmid Preparation, In Vitro, Activity Assay, Incubation, Positive Control

    Dexamethasone treatment inhibited apoptosis induction in R. conorii -infected cells or in a model of cells microinjected with R. felis VapC -2 protein. a. The mean ± standard deviation (sd) of the lytic plaque sizes from infected cells that were treated with chloramphenicol (red square), pre-incubated with dexamethasone and treated with chloramphenicol (green arrowhead) or mock treated with PBS (blue circle) at 0, 2, 4 and 6 hours after treatment. b. Microinjected L929 cells with dextran-FITC associated with VapC-2 protein (T) or VapB-2 (AT). From left to right, all panels show fluorescence microscopy images using DAPI, dextran-FITC and Anx-V -Cy3 staining and the overlay channel. In the lower panels (T+dexa), the L929 cells were pre-incubated for 24 hours in MEM supplemented with 1 µM dexamethasone before the toxin microinjection. Scale bars represent 40 µm (see the experimental procedures section).
    Figure Legend Snippet: Dexamethasone treatment inhibited apoptosis induction in R. conorii -infected cells or in a model of cells microinjected with R. felis VapC -2 protein. a. The mean ± standard deviation (sd) of the lytic plaque sizes from infected cells that were treated with chloramphenicol (red square), pre-incubated with dexamethasone and treated with chloramphenicol (green arrowhead) or mock treated with PBS (blue circle) at 0, 2, 4 and 6 hours after treatment. b. Microinjected L929 cells with dextran-FITC associated with VapC-2 protein (T) or VapB-2 (AT). From left to right, all panels show fluorescence microscopy images using DAPI, dextran-FITC and Anx-V -Cy3 staining and the overlay channel. In the lower panels (T+dexa), the L929 cells were pre-incubated for 24 hours in MEM supplemented with 1 µM dexamethasone before the toxin microinjection. Scale bars represent 40 µm (see the experimental procedures section).

    Techniques Used: Infection, Standard Deviation, Incubation, Fluorescence, Microscopy, Staining

    19) Product Images from "Droplet digital polymerase chain reaction-based quantification of circulating microRNAs using small RNA concentration normalization"

    Article Title: Droplet digital polymerase chain reaction-based quantification of circulating microRNAs using small RNA concentration normalization

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-66072-z

    Concentration-normalization in setup phase revealed similar miR-23a-3p levels in the 50-μl and 100-μl groups with TaqMan chemistry. (A) With un-normalized samples, mean miR-23a-3p Cq decreased with corresponding increase in starting plasma volumes. For the normalized samples, mean miR-23a-3p Cq was similar between the 50-μl and 100-μl groups. The 200-μl group still had a lower mean Cq than either of the other groups. As a result of normalization, the mean miR-23a-3p Cq for each of the normalized plasma volume groups was higher than the corresponding un-normalized counterpart. (B) With ddPCR, the expression pattern was similar to that of RT-qPCR. The mean miR-23a-3p copy number per 20 μl of PCR reaction volume did not differ between the unnormalized and normalized 50 μl group. (C) Addition of the MS2 carrier RNA did not improve the miR-23a-3p yield. Statistical significances: *p
    Figure Legend Snippet: Concentration-normalization in setup phase revealed similar miR-23a-3p levels in the 50-μl and 100-μl groups with TaqMan chemistry. (A) With un-normalized samples, mean miR-23a-3p Cq decreased with corresponding increase in starting plasma volumes. For the normalized samples, mean miR-23a-3p Cq was similar between the 50-μl and 100-μl groups. The 200-μl group still had a lower mean Cq than either of the other groups. As a result of normalization, the mean miR-23a-3p Cq for each of the normalized plasma volume groups was higher than the corresponding un-normalized counterpart. (B) With ddPCR, the expression pattern was similar to that of RT-qPCR. The mean miR-23a-3p copy number per 20 μl of PCR reaction volume did not differ between the unnormalized and normalized 50 μl group. (C) Addition of the MS2 carrier RNA did not improve the miR-23a-3p yield. Statistical significances: *p

    Techniques Used: Concentration Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

    Concentration-normalization in validation phase revealed similar miR-103a-3p levels in the 50-μl and 100-μl groups, and similar miR-451a levels in all the three plasma volume groups. (A,D) As expected with un-normalized samples, mean miR-103a-3p and miR-451a Cq decreased with increase in starting plasma volumes. Unlike miR-23a-3p in the setup phase, miRCURY RT-qPCR from the normalized samples revealed similar miR-103a-3p and miR-451a levels in all the three groups. (B,E) With miRCURY ddPCR setup 6, un-normalized samples revealed increased miR-103a-3p and miR-451a copy numbers with increase in starting plasma volumes. For miR-103a-3p, the normalized 50-μl and 100-μl groups had similar levels, but the 200-μl group still showed higher miR-103a-3p levels than the 100-μl group. On the other hand, all the three groups showed normalized miR-451a copy number. (C,F) Addition of the MS2 carrier RNA improved both miR-103a-3p and miR-451a yield (miR-103a-3p: 2.38-fold, p
    Figure Legend Snippet: Concentration-normalization in validation phase revealed similar miR-103a-3p levels in the 50-μl and 100-μl groups, and similar miR-451a levels in all the three plasma volume groups. (A,D) As expected with un-normalized samples, mean miR-103a-3p and miR-451a Cq decreased with increase in starting plasma volumes. Unlike miR-23a-3p in the setup phase, miRCURY RT-qPCR from the normalized samples revealed similar miR-103a-3p and miR-451a levels in all the three groups. (B,E) With miRCURY ddPCR setup 6, un-normalized samples revealed increased miR-103a-3p and miR-451a copy numbers with increase in starting plasma volumes. For miR-103a-3p, the normalized 50-μl and 100-μl groups had similar levels, but the 200-μl group still showed higher miR-103a-3p levels than the 100-μl group. On the other hand, all the three groups showed normalized miR-451a copy number. (C,F) Addition of the MS2 carrier RNA improved both miR-103a-3p and miR-451a yield (miR-103a-3p: 2.38-fold, p

    Techniques Used: Concentration Assay, Quantitative RT-PCR

    Concentration-normalization in setup phase revealed similar miR-23a-3p levels in the 50-μl and 100-μl groups with miRCURY chemistry. (A) With un-normalized samples, mean miR-23a-3p Cq was lower in the 100-μl group than in the 50-μl group. The 200-μl group had a lower mean Cq compared with the 50-μl, but not the 100-μl group. For the normalized samples, mean Cq was similar between the 50-μl and 100-μl groups, but the 200-μl group had a lower mean Cq compared with both other groups. (B) RT-qPCR from the spike-in UniSp6 revealed a similar RT efficiency for all samples. (C) Addition of the MS2 carrier RNA did not improve the miR-23a-3p yield. (D) With ddPCR setup 3, the 200-μl group did not have a higher mean miR-23a-3p copy number than either the 50-μl or 100-μl group. For the normalized samples, however, the expression pattern was similar to that of RT-qPCR. (E) Setup 6 in ddPCR performed better with the un-normalized samples, as it showed a higher mean miR-23a-3p copy number in the 200-μl group than in the 50-μl group, as with RT-qPCR. Further, it also performed better with the normalized samples, since it reduced the difference between the 200-μl and 50-μl groups to 1.86-fold (p
    Figure Legend Snippet: Concentration-normalization in setup phase revealed similar miR-23a-3p levels in the 50-μl and 100-μl groups with miRCURY chemistry. (A) With un-normalized samples, mean miR-23a-3p Cq was lower in the 100-μl group than in the 50-μl group. The 200-μl group had a lower mean Cq compared with the 50-μl, but not the 100-μl group. For the normalized samples, mean Cq was similar between the 50-μl and 100-μl groups, but the 200-μl group had a lower mean Cq compared with both other groups. (B) RT-qPCR from the spike-in UniSp6 revealed a similar RT efficiency for all samples. (C) Addition of the MS2 carrier RNA did not improve the miR-23a-3p yield. (D) With ddPCR setup 3, the 200-μl group did not have a higher mean miR-23a-3p copy number than either the 50-μl or 100-μl group. For the normalized samples, however, the expression pattern was similar to that of RT-qPCR. (E) Setup 6 in ddPCR performed better with the un-normalized samples, as it showed a higher mean miR-23a-3p copy number in the 200-μl group than in the 50-μl group, as with RT-qPCR. Further, it also performed better with the normalized samples, since it reduced the difference between the 200-μl and 50-μl groups to 1.86-fold (p

    Techniques Used: Concentration Assay, Quantitative RT-PCR, Expressing

    20) Product Images from "CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis"

    Article Title: CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1900176116

    CarD slows promoter escape from the Mtb rrnA P3 promoter. ( A ) Single-round in vitro promoter escape assay results with representative gel images showing the time-dependent increase in 32 P-labeled RNA transcripts formed by M. bovis RNAP from the Mtb rrnA P3 promoter construct in the presence and absence of CarD. Promoter escape rate is quantified by the time until 90% of the final transcript intensity is reached (t 90% ). The graph shows the mean t 90% ± SEM (−CarD n = 3; +CarD n = 7). Statistical significance was analyzed by Welch’s t test. * P
    Figure Legend Snippet: CarD slows promoter escape from the Mtb rrnA P3 promoter. ( A ) Single-round in vitro promoter escape assay results with representative gel images showing the time-dependent increase in 32 P-labeled RNA transcripts formed by M. bovis RNAP from the Mtb rrnA P3 promoter construct in the presence and absence of CarD. Promoter escape rate is quantified by the time until 90% of the final transcript intensity is reached (t 90% ). The graph shows the mean t 90% ± SEM (−CarD n = 3; +CarD n = 7). Statistical significance was analyzed by Welch’s t test. * P

    Techniques Used: In Vitro, Labeling, Construct

    CarD mutants with similar effects on RP o stability show similar gene expression profiles. ( A ) A principal component analysis of RNA sequencing samples based on read counts generated by mapping reads to a library of protein encoding sequences from the Mtb H37Rv genome. Each point represents one sequencing sample colored by genotype, and the distance between 2 points reflects the variance in gene expression between the 2 samples. The first 2 principle components, PC1 and PC2, define the x - and y -axes, respectively, and account for 70% and 11.4% of the variance, respectively. ( B ) Hierarchical clustering of RNAseq samples based on read count data in which each column and row represents one sample. The color of each cell represents the Euclidean distance, calculated according to relative expression of all Mtb protein encoding genes, between each sample pair. ( C and D ) Venn diagrams and scatter plots show the overlap in the lists of Mtb genes that were significantly differentially expressed ( P adj
    Figure Legend Snippet: CarD mutants with similar effects on RP o stability show similar gene expression profiles. ( A ) A principal component analysis of RNA sequencing samples based on read counts generated by mapping reads to a library of protein encoding sequences from the Mtb H37Rv genome. Each point represents one sequencing sample colored by genotype, and the distance between 2 points reflects the variance in gene expression between the 2 samples. The first 2 principle components, PC1 and PC2, define the x - and y -axes, respectively, and account for 70% and 11.4% of the variance, respectively. ( B ) Hierarchical clustering of RNAseq samples based on read count data in which each column and row represents one sample. The color of each cell represents the Euclidean distance, calculated according to relative expression of all Mtb protein encoding genes, between each sample pair. ( C and D ) Venn diagrams and scatter plots show the overlap in the lists of Mtb genes that were significantly differentially expressed ( P adj

    Techniques Used: Expressing, RNA Sequencing Assay, Generated, Sequencing

    21) Product Images from "Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library"

    Article Title: Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37233-y

    Evolved KlenTaq DNA polymerase with reverse transcriptase activity. ( A ) Depiction of novel mutations contributing to reverse transcriptase activity marked in red. ( B ) Reverse transcriptase activity studied by primer extension experiments for WT and Mut_RT. Reactions carried out for the indicated time points under identical conditions. P = primer, C = Control reaction that was carried out with the corresponding DNA template. Full-length gel is presented in Supplementary Fig. S16 . ( C ) Reverse transcriptase activity of Mut_RT in real time PCR assay employed with varying amounts of RNA template (left) and their corresponding melting peak analysis (right). ( D ) Employability of Mut_RT in reverse transcription from total RNA extract (100 ng) using the HPRT mRNA target (left) and the agarose gel analysis of amplified product of HRPT transcript (lane 1) and the no template control (lane2) Full-length gel is presented in Supplementary Fig. S17 .
    Figure Legend Snippet: Evolved KlenTaq DNA polymerase with reverse transcriptase activity. ( A ) Depiction of novel mutations contributing to reverse transcriptase activity marked in red. ( B ) Reverse transcriptase activity studied by primer extension experiments for WT and Mut_RT. Reactions carried out for the indicated time points under identical conditions. P = primer, C = Control reaction that was carried out with the corresponding DNA template. Full-length gel is presented in Supplementary Fig. S16 . ( C ) Reverse transcriptase activity of Mut_RT in real time PCR assay employed with varying amounts of RNA template (left) and their corresponding melting peak analysis (right). ( D ) Employability of Mut_RT in reverse transcription from total RNA extract (100 ng) using the HPRT mRNA target (left) and the agarose gel analysis of amplified product of HRPT transcript (lane 1) and the no template control (lane2) Full-length gel is presented in Supplementary Fig. S17 .

    Techniques Used: Activity Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification

    22) Product Images from "Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase"

    Article Title: Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase

    Journal: FEBS letters

    doi: 10.1016/j.febslet.2011.07.008

    Primer extension analysis of MazF-bs cleavage sites in MS2 RNA. CspA was added in all reactions (A to F) except for lanes 6 and 8 in A and B. Cleavage sites indicated by arrows on the RNA sequences were determined using the RNA ladder shown on the left.
    Figure Legend Snippet: Primer extension analysis of MazF-bs cleavage sites in MS2 RNA. CspA was added in all reactions (A to F) except for lanes 6 and 8 in A and B. Cleavage sites indicated by arrows on the RNA sequences were determined using the RNA ladder shown on the left.

    Techniques Used:

    23) Product Images from "Replacement of All Arginine Residues with Canavanine in MazF-bs mRNA Interferase Changes Its Specificity *"

    Article Title: Replacement of All Arginine Residues with Canavanine in MazF-bs mRNA Interferase Changes Its Specificity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.434969

    Acquirement of a higher RNA cleavage specificity in MazF-bs( can ). A , analysis of cleavage sites in MS2 phage RNA by MazF-bs( arg ) or MazF-bs( can ). Lane C represents a control reaction in which no protein was added; MS2 RNA was incubated with MazF-bs( arg
    Figure Legend Snippet: Acquirement of a higher RNA cleavage specificity in MazF-bs( can ). A , analysis of cleavage sites in MS2 phage RNA by MazF-bs( arg ) or MazF-bs( can ). Lane C represents a control reaction in which no protein was added; MS2 RNA was incubated with MazF-bs( arg

    Techniques Used: Incubation

    24) Product Images from "Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration"

    Article Title: Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21114089

    Optimization of microRNA acquirement from seminal plasma. MicroRNA yields were compared by qPCR between different RNA purification methods, with variation of the input volume of seminal plasma in the extraction procedure (25, 50, 100, 200 µL), with or without addition of an exosome enrichment kit, with or without incorporation of MS2 RNA carrier and proteinase K for the RNA extraction procedure, and with variation of the input volume of RNA in the cDNA reaction (1, 2, 4 µL). Data are represented as means ± SEM.
    Figure Legend Snippet: Optimization of microRNA acquirement from seminal plasma. MicroRNA yields were compared by qPCR between different RNA purification methods, with variation of the input volume of seminal plasma in the extraction procedure (25, 50, 100, 200 µL), with or without addition of an exosome enrichment kit, with or without incorporation of MS2 RNA carrier and proteinase K for the RNA extraction procedure, and with variation of the input volume of RNA in the cDNA reaction (1, 2, 4 µL). Data are represented as means ± SEM.

    Techniques Used: Real-time Polymerase Chain Reaction, Purification, RNA Extraction

    25) Product Images from "Effect of Rickettsial Toxin VapC on Its Eukaryotic Host"

    Article Title: Effect of Rickettsial Toxin VapC on Its Eukaryotic Host

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026528

    Heterologous host growth inhibition by R. felis VapC toxin is controlled by antitoxin VapB. a. Growth curves of E. coli cultures expressing the vapB-1 , vapC-1 or vapBC-1 operon. b. Growth curves of E. coli cultures expressing the vapB-2 , vapC-2 or vapBC-2 operon. The error bars represent the mean of triplicates ± standard deviation. c. Growth curves of yeast cells in Sgal induction media expressing R. felis vapB-2 , vapC-2 , VapB2-VapC2 and/or control pBNS. d. Yeast toxicity assays of S. cerevisiae harboring plasmids with indicated genes or empty vector, under inducing (Sgal) or noninducing conditions (SD). e. In vitro RNase activity of free R. felis and R. bellii VapC-1 proteins: The MS2 phage RNA profiles after incubation reactions with irrelevant protein (lane 1 and 2), R. felis VapC-1 (lane 3), R. felis VapB/C-1 complex (lane 4), R.felis antitoxin VapB-1 (lane 5), R. bellii VapC-1 (lane 6), R. bellii VapB/C-1 complex (lane 7), R. bellii antitoxin VapB-1 (lane 8), RNase A as positive control (lane 9), without protein (lane 10).
    Figure Legend Snippet: Heterologous host growth inhibition by R. felis VapC toxin is controlled by antitoxin VapB. a. Growth curves of E. coli cultures expressing the vapB-1 , vapC-1 or vapBC-1 operon. b. Growth curves of E. coli cultures expressing the vapB-2 , vapC-2 or vapBC-2 operon. The error bars represent the mean of triplicates ± standard deviation. c. Growth curves of yeast cells in Sgal induction media expressing R. felis vapB-2 , vapC-2 , VapB2-VapC2 and/or control pBNS. d. Yeast toxicity assays of S. cerevisiae harboring plasmids with indicated genes or empty vector, under inducing (Sgal) or noninducing conditions (SD). e. In vitro RNase activity of free R. felis and R. bellii VapC-1 proteins: The MS2 phage RNA profiles after incubation reactions with irrelevant protein (lane 1 and 2), R. felis VapC-1 (lane 3), R. felis VapB/C-1 complex (lane 4), R.felis antitoxin VapB-1 (lane 5), R. bellii VapC-1 (lane 6), R. bellii VapB/C-1 complex (lane 7), R. bellii antitoxin VapB-1 (lane 8), RNase A as positive control (lane 9), without protein (lane 10).

    Techniques Used: Inhibition, Expressing, Standard Deviation, Plasmid Preparation, In Vitro, Activity Assay, Incubation, Positive Control

    26) Product Images from "Increased expression and copy number amplification of LINE-1 and SINE B1 retrotransposable elements in murine mammary carcinoma progression"

    Article Title: Increased expression and copy number amplification of LINE-1 and SINE B1 retrotransposable elements in murine mammary carcinoma progression

    Journal: Oncotarget

    doi:

    RT activity in murine breast cancer tissues A. Western blot analysis of ORF2p (upper panel) and alpha-tubulin (lower panel) in tissue extracts from normal breast (NB) and breast cancer (stages 1+2, 3+4, and 5+6 were pooled). B. RT activity functional assay after incubation of MS2 phage RNA with extracts from breast carcinoma tissues. Histograms represent the retrotranscribed cDNA yield from each reaction; means and SD values from three independent assays are expressed in arbitrary units.
    Figure Legend Snippet: RT activity in murine breast cancer tissues A. Western blot analysis of ORF2p (upper panel) and alpha-tubulin (lower panel) in tissue extracts from normal breast (NB) and breast cancer (stages 1+2, 3+4, and 5+6 were pooled). B. RT activity functional assay after incubation of MS2 phage RNA with extracts from breast carcinoma tissues. Histograms represent the retrotranscribed cDNA yield from each reaction; means and SD values from three independent assays are expressed in arbitrary units.

    Techniques Used: Activity Assay, Western Blot, Functional Assay, Incubation

    27) Product Images from "Development of RT-qPCR and semi-nested RT-PCR assays for molecular diagnosis of hantavirus pulmonary syndrome"

    Article Title: Development of RT-qPCR and semi-nested RT-PCR assays for molecular diagnosis of hantavirus pulmonary syndrome

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007884

    Comparison between amplification efficiencies of hantavirus RT-qPCR in singleplex and multiplex with MS2 EIC formats. Amplification plot and Standard Curve for each assay format are depicted. Threshold was fixed where the amplification efficiency was higher, inside the exponential phase of each amplification plot in logarithmic scale, with 0.3481 ΔRn in singleplex (A) and 0.4492 ΔRn in multiplex (B). Ct variation between singleplex and multiplex was within 1 Ct. Standard curves were constructed with seven 1:10 dilutions ranging from 10 7 copies to 10 1 copies of in vitro RNA. Amplification efficiency was 97.6% for singleplex (R 2 :0.999) (A) and 97.7% for multiplex (R 2 :0.998) (B). Ct: Cycle threshold, HTN M: hantavirus RT-qPCR multiplex format, HTN S: hantavirus RT-qPCR singleplex format.
    Figure Legend Snippet: Comparison between amplification efficiencies of hantavirus RT-qPCR in singleplex and multiplex with MS2 EIC formats. Amplification plot and Standard Curve for each assay format are depicted. Threshold was fixed where the amplification efficiency was higher, inside the exponential phase of each amplification plot in logarithmic scale, with 0.3481 ΔRn in singleplex (A) and 0.4492 ΔRn in multiplex (B). Ct variation between singleplex and multiplex was within 1 Ct. Standard curves were constructed with seven 1:10 dilutions ranging from 10 7 copies to 10 1 copies of in vitro RNA. Amplification efficiency was 97.6% for singleplex (R 2 :0.999) (A) and 97.7% for multiplex (R 2 :0.998) (B). Ct: Cycle threshold, HTN M: hantavirus RT-qPCR multiplex format, HTN S: hantavirus RT-qPCR singleplex format.

    Techniques Used: Amplification, Quantitative RT-PCR, Multiplex Assay, Construct, In Vitro

    28) Product Images from "Effect of Rickettsial Toxin VapC on Its Eukaryotic Host"

    Article Title: Effect of Rickettsial Toxin VapC on Its Eukaryotic Host

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026528

    Heterologous host growth inhibition by R. felis VapC toxin is controlled by antitoxin VapB. a. Growth curves of E. coli cultures expressing the vapB-1 , vapC-1 or vapBC-1 operon. b. Growth curves of E. coli cultures expressing the vapB-2 , vapC-2 or vapBC-2 operon. The error bars represent the mean of triplicates ± standard deviation. c. Growth curves of yeast cells in Sgal induction media expressing R. felis vapB-2 , vapC-2 , VapB2-VapC2 and/or control pBNS. d. Yeast toxicity assays of S. cerevisiae harboring plasmids with indicated genes or empty vector, under inducing (Sgal) or noninducing conditions (SD). e. In vitro RNase activity of free R. felis and R. bellii VapC-1 proteins: The MS2 phage RNA profiles after incubation reactions with irrelevant protein (lane 1 and 2), R. felis VapC-1 (lane 3), R. felis VapB/C-1 complex (lane 4), R.felis antitoxin VapB-1 (lane 5), R. bellii VapC-1 (lane 6), R. bellii VapB/C-1 complex (lane 7), R. bellii antitoxin VapB-1 (lane 8), RNase A as positive control (lane 9), without protein (lane 10).
    Figure Legend Snippet: Heterologous host growth inhibition by R. felis VapC toxin is controlled by antitoxin VapB. a. Growth curves of E. coli cultures expressing the vapB-1 , vapC-1 or vapBC-1 operon. b. Growth curves of E. coli cultures expressing the vapB-2 , vapC-2 or vapBC-2 operon. The error bars represent the mean of triplicates ± standard deviation. c. Growth curves of yeast cells in Sgal induction media expressing R. felis vapB-2 , vapC-2 , VapB2-VapC2 and/or control pBNS. d. Yeast toxicity assays of S. cerevisiae harboring plasmids with indicated genes or empty vector, under inducing (Sgal) or noninducing conditions (SD). e. In vitro RNase activity of free R. felis and R. bellii VapC-1 proteins: The MS2 phage RNA profiles after incubation reactions with irrelevant protein (lane 1 and 2), R. felis VapC-1 (lane 3), R. felis VapB/C-1 complex (lane 4), R.felis antitoxin VapB-1 (lane 5), R. bellii VapC-1 (lane 6), R. bellii VapB/C-1 complex (lane 7), R. bellii antitoxin VapB-1 (lane 8), RNase A as positive control (lane 9), without protein (lane 10).

    Techniques Used: Inhibition, Expressing, Standard Deviation, Plasmid Preparation, In Vitro, Activity Assay, Incubation, Positive Control

    29) Product Images from "A high rate of polymerization during synthesis of mouse mammary tumor virus DNA alleviates hypermutation by APOBEC3 proteins"

    Article Title: A high rate of polymerization during synthesis of mouse mammary tumor virus DNA alleviates hypermutation by APOBEC3 proteins

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007533

    MMTV RT is more processive and faster than HIV-1 RT. RT enzymes were extracted from preparations containing equal virion levels (quantified by RTqPCR and verified by physical particle counting (EM, S3 Fig ). (A) To determine RTs processivity an MS2 cDNA was synthesized in the presence of an excess of the template and a trap to limit re-association of RTs with cDNAs. The cDNA products were A-tailed and amplified using anchor- and MS2-specific primers. A schematic diagram of the method is shown in the upper panel. The length distribution of the cDNA products was analyzed on 1.5% agarose gels (lower panel); lane 1: MassRuler DNA ladder (Fermentas), lane 2: extension terminated after 0 min, lane 3: extension terminated after 15 min. The assay was repeated three times with similar outcome. (B) Kinetics of MS2 cDNA synthesis was assayed in the absence of the trap and with a limited amount of the MS2 RNA/primer template. The cDNA polymerization was terminated after the various amount of time and the presence of 0.4 kb- or 1.4 kb-long cDNA determined by PCR with MS2-specific primers. PCR products were analyzed on agarose gels (marker: 2-Log DNA ladder (NEB)). The Fig shows a representative example of three assays.
    Figure Legend Snippet: MMTV RT is more processive and faster than HIV-1 RT. RT enzymes were extracted from preparations containing equal virion levels (quantified by RTqPCR and verified by physical particle counting (EM, S3 Fig ). (A) To determine RTs processivity an MS2 cDNA was synthesized in the presence of an excess of the template and a trap to limit re-association of RTs with cDNAs. The cDNA products were A-tailed and amplified using anchor- and MS2-specific primers. A schematic diagram of the method is shown in the upper panel. The length distribution of the cDNA products was analyzed on 1.5% agarose gels (lower panel); lane 1: MassRuler DNA ladder (Fermentas), lane 2: extension terminated after 0 min, lane 3: extension terminated after 15 min. The assay was repeated three times with similar outcome. (B) Kinetics of MS2 cDNA synthesis was assayed in the absence of the trap and with a limited amount of the MS2 RNA/primer template. The cDNA polymerization was terminated after the various amount of time and the presence of 0.4 kb- or 1.4 kb-long cDNA determined by PCR with MS2-specific primers. PCR products were analyzed on agarose gels (marker: 2-Log DNA ladder (NEB)). The Fig shows a representative example of three assays.

    Techniques Used: Synthesized, Amplification, Polymerase Chain Reaction, Marker

    30) Product Images from "CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis"

    Article Title: CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1900176116

    CarD slows promoter escape from the Mtb rrnA P3 promoter. ( A ) Single-round in vitro promoter escape assay results with representative gel images showing the time-dependent increase in 32 P-labeled RNA transcripts formed by M. bovis RNAP from the Mtb rrnA P3 promoter construct in the presence and absence of CarD. Promoter escape rate is quantified by the time until 90% of the final transcript intensity is reached (t 90% ). The graph shows the mean t 90% ± SEM (−CarD n = 3; +CarD n = 7). Statistical significance was analyzed by Welch’s t test. * P
    Figure Legend Snippet: CarD slows promoter escape from the Mtb rrnA P3 promoter. ( A ) Single-round in vitro promoter escape assay results with representative gel images showing the time-dependent increase in 32 P-labeled RNA transcripts formed by M. bovis RNAP from the Mtb rrnA P3 promoter construct in the presence and absence of CarD. Promoter escape rate is quantified by the time until 90% of the final transcript intensity is reached (t 90% ). The graph shows the mean t 90% ± SEM (−CarD n = 3; +CarD n = 7). Statistical significance was analyzed by Welch’s t test. * P

    Techniques Used: In Vitro, Labeling, Construct

    CarD mutants with similar effects on RP o stability show similar gene expression profiles. ( A ) A principal component analysis of RNA sequencing samples based on read counts generated by mapping reads to a library of protein encoding sequences from the Mtb H37Rv genome. Each point represents one sequencing sample colored by genotype, and the distance between 2 points reflects the variance in gene expression between the 2 samples. The first 2 principle components, PC1 and PC2, define the x - and y -axes, respectively, and account for 70% and 11.4% of the variance, respectively. ( B ) Hierarchical clustering of RNAseq samples based on read count data in which each column and row represents one sample. The color of each cell represents the Euclidean distance, calculated according to relative expression of all Mtb protein encoding genes, between each sample pair. ( C and D ) Venn diagrams and scatter plots show the overlap in the lists of Mtb genes that were significantly differentially expressed ( P adj
    Figure Legend Snippet: CarD mutants with similar effects on RP o stability show similar gene expression profiles. ( A ) A principal component analysis of RNA sequencing samples based on read counts generated by mapping reads to a library of protein encoding sequences from the Mtb H37Rv genome. Each point represents one sequencing sample colored by genotype, and the distance between 2 points reflects the variance in gene expression between the 2 samples. The first 2 principle components, PC1 and PC2, define the x - and y -axes, respectively, and account for 70% and 11.4% of the variance, respectively. ( B ) Hierarchical clustering of RNAseq samples based on read count data in which each column and row represents one sample. The color of each cell represents the Euclidean distance, calculated according to relative expression of all Mtb protein encoding genes, between each sample pair. ( C and D ) Venn diagrams and scatter plots show the overlap in the lists of Mtb genes that were significantly differentially expressed ( P adj

    Techniques Used: Expressing, RNA Sequencing Assay, Generated, Sequencing

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  • 89
    Roche bacteriophage ms2 rna
    Detection of RT activity in cell lines infected with MLV of contaminated cell lines by PERT assay. Cell culture supernatants of the infected cell lines NCI-H82 and VERO-B4 were harvested after 45 and 44 days after infection, respectively. <t>MS2</t> phage <t>RNA</t> was reverse transcribed with an MS2-specific primer and the MLV derived RT of the samples in the presence of MnCl 2 . The cDNA was then amplified with a second MS2-specific primer applying PCR. The signals of the cell lines infected with LCL-HO supernatant may potentially be ascribed to the activity of SMRV derived RT. dpi: days post infection.
    Bacteriophage Ms2 Rna, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bacteriophage ms2 rna/product/Roche
    Average 89 stars, based on 15 article reviews
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    bacteriophage ms2 rna - by Bioz Stars, 2020-09
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    88
    Roche ms2 phage rna
    Sequence-specific <t>RNA</t> cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in <t>MS2</t> RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.
    Ms2 Phage Rna, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Detection of RT activity in cell lines infected with MLV of contaminated cell lines by PERT assay. Cell culture supernatants of the infected cell lines NCI-H82 and VERO-B4 were harvested after 45 and 44 days after infection, respectively. MS2 phage RNA was reverse transcribed with an MS2-specific primer and the MLV derived RT of the samples in the presence of MnCl 2 . The cDNA was then amplified with a second MS2-specific primer applying PCR. The signals of the cell lines infected with LCL-HO supernatant may potentially be ascribed to the activity of SMRV derived RT. dpi: days post infection.

    Journal: PLoS ONE

    Article Title: Prevalence and Characterization of Murine Leukemia Virus Contamination in Human Cell Lines

    doi: 10.1371/journal.pone.0125622

    Figure Lengend Snippet: Detection of RT activity in cell lines infected with MLV of contaminated cell lines by PERT assay. Cell culture supernatants of the infected cell lines NCI-H82 and VERO-B4 were harvested after 45 and 44 days after infection, respectively. MS2 phage RNA was reverse transcribed with an MS2-specific primer and the MLV derived RT of the samples in the presence of MnCl 2 . The cDNA was then amplified with a second MS2-specific primer applying PCR. The signals of the cell lines infected with LCL-HO supernatant may potentially be ascribed to the activity of SMRV derived RT. dpi: days post infection.

    Article Snippet: Primer template annealing One μl of PERT-RT1R primer at 10 mM was mixed with 0.4 μl bacteriophage MS2 RNA at 0.8 μg/μl (Roche Diagnostics, Mannheim, Germany) per reaction, denatured at 95°C for 5 min and subsequently incubated for 30 min at 37°C for annealing of the primer to the template.

    Techniques: Activity Assay, Infection, Cell Culture, Derivative Assay, Amplification, Polymerase Chain Reaction

    Optimization of microRNA acquirement from seminal plasma. MicroRNA yields were compared by qPCR between different RNA purification methods, with variation of the input volume of seminal plasma in the extraction procedure (25, 50, 100, 200 µL), with or without addition of an exosome enrichment kit, with or without incorporation of MS2 RNA carrier and proteinase K for the RNA extraction procedure, and with variation of the input volume of RNA in the cDNA reaction (1, 2, 4 µL). Data are represented as means ± SEM.

    Journal: International Journal of Molecular Sciences

    Article Title: Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration

    doi: 10.3390/ijms21114089

    Figure Lengend Snippet: Optimization of microRNA acquirement from seminal plasma. MicroRNA yields were compared by qPCR between different RNA purification methods, with variation of the input volume of seminal plasma in the extraction procedure (25, 50, 100, 200 µL), with or without addition of an exosome enrichment kit, with or without incorporation of MS2 RNA carrier and proteinase K for the RNA extraction procedure, and with variation of the input volume of RNA in the cDNA reaction (1, 2, 4 µL). Data are represented as means ± SEM.

    Article Snippet: Protocols, including the incorporation of proteinase K and MS2 bacteriophage RNA (#10165948001; Roche) to the Biofluid kit method, were carried out according to the supplier’s manuals and a previous paper [ ].

    Techniques: Real-time Polymerase Chain Reaction, Purification, RNA Extraction

    Micro-RDA sensitivity test. Different amounts, ranging from 1 copy per cell up to 100 copies per cell, of a known RNA species (MS2 RNA) was mixed with total RNA of S. aureus . In the subsequent micro-RDA, this mixture was used as the tester and total S. aureus RNA was used as the driver. DP 3 was electrophoresed through a 2% agarose gel and stained with ethidium bromide. Fragments typical of the MS2 amplicon could be detected in the spike with a ratio of minimally 5 copies per cell. Lane 1, the S. aureus amplicon (MS2 RNA) at 100 copies/cell in RNA (3-h culture); lanes 2 to 6, DP 3 (MS2 RNA spike in RNA [3-h culture]) at 1 copy of MS2/cell and 5, 10, 50, and 100 copies of MS2/cell, respectively; lane 7, the MS 2 amplicon; lanes M, molecular size markers.

    Journal: Applied and Environmental Microbiology

    Article Title: Detection of Differential Gene Expression in Biofilm-Forming versus Planktonic Populations of Staphylococcus aureus Using Micro-Representational-Difference Analysis

    doi: 10.1128/AEM.67.7.2958-2965.2001

    Figure Lengend Snippet: Micro-RDA sensitivity test. Different amounts, ranging from 1 copy per cell up to 100 copies per cell, of a known RNA species (MS2 RNA) was mixed with total RNA of S. aureus . In the subsequent micro-RDA, this mixture was used as the tester and total S. aureus RNA was used as the driver. DP 3 was electrophoresed through a 2% agarose gel and stained with ethidium bromide. Fragments typical of the MS2 amplicon could be detected in the spike with a ratio of minimally 5 copies per cell. Lane 1, the S. aureus amplicon (MS2 RNA) at 100 copies/cell in RNA (3-h culture); lanes 2 to 6, DP 3 (MS2 RNA spike in RNA [3-h culture]) at 1 copy of MS2/cell and 5, 10, 50, and 100 copies of MS2/cell, respectively; lane 7, the MS 2 amplicon; lanes M, molecular size markers.

    Article Snippet: To evaluate the sensitivity of micro-RDA, a series of spiked controls containing various amounts of MS2 bacteriophage RNA (Roche Diagnostics) added to a fixed amount of S. aureus driver RNA was used as the tester fraction against driver RNA in the micro-RDA procedure.

    Techniques: Agarose Gel Electrophoresis, Staining, Amplification, Mass Spectrometry

    Sequence-specific RNA cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.

    Journal: Journal of Bacteriology

    Article Title: Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum

    doi: 10.1128/JB.00400-12

    Figure Lengend Snippet: Sequence-specific RNA cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.

    Article Snippet: Avian myeloblastosis virus reverse transcriptase (AMV RT), MS2 phage RNA, and RNase inhibitor for primer extension were purchased from Roche (Indianapolis, IN).

    Techniques: Sequencing, Purification, SDS Page, Affinity Chromatography, In Vitro, Labeling, Incubation