e. coli dna ligase Thermo Fisher Search Results


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  • 99
    Thermo Fisher e coli dna ligase
    Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand <t>cDNA</t> synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear <t>DNA</t> was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).
    E Coli Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total rna
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
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    93
    Thermo Fisher competent escherichia coli
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
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    Thermo Fisher competent top10 e coli cells
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
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    Thermo Fisher competent e coli cells
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
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    Thermo Fisher e coli dna polymerase
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
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    Thermo Fisher competent escherichia coli top10
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
    Competent Escherichia Coli Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher e coli dh10b cells
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
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    Thermo Fisher e coli dh5α
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
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    Thermo Fisher e coli top10
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
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    Thermo Fisher shot top10 electrocomp e coli cells
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
    Shot Top10 Electrocomp E Coli Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher topo ta cloning kit
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
    Topo Ta Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53970 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna polymerase i
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
    Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2890 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher e coli expression vector ptrchis b
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
    E Coli Expression Vector Ptrchis B, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher e coli strain dh10b
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
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    Thermo Fisher escherichia coli strain dh5α cells
    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. <t>RNA</t> was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P
    Escherichia Coli Strain Dh5α Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher e coli rnase iii
    Target RNA conversion into a primer for RCA, the impact of E. coli <t>RNase</t> <t>III.</t> The reactions were carried out under the conditions described in Materials and Methods. The 5′-end-labeled ( A ) RNA2-PP1 (panel 1 ), ( B ) RNA2-PP2 (panel 1 ), and RNA1-PP1 (panel 2 ) hybrids were incubated with Phi29 DNA polymerase in the absence or presence of dNTP. ( A , panel 2 ) The experiments with E. coli RNase III were performed using RNA2-PP1 duplex. The RNA degradation and RCA products were analyzed by electrophoresis through denaturing 8% polyacrylamide gels. Different RNA-PP duplexes are shown above the gels. The radioactive label is indicated by the star. The contents of samples are shown above the gel lines. Reaction products are labeled as “ a ” for the 5′-end-labeled RNA1 hydrolysis product serving as an RNA primer for RCA and “ b ” for the 5′-end-labeled RCA product. ( C ) The reaction scheme represents the conversions of target RNA.
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    93
    Thermo Fisher e coli dh10b
    Target RNA conversion into a primer for RCA, the impact of E. coli <t>RNase</t> <t>III.</t> The reactions were carried out under the conditions described in Materials and Methods. The 5′-end-labeled ( A ) RNA2-PP1 (panel 1 ), ( B ) RNA2-PP2 (panel 1 ), and RNA1-PP1 (panel 2 ) hybrids were incubated with Phi29 DNA polymerase in the absence or presence of dNTP. ( A , panel 2 ) The experiments with E. coli RNase III were performed using RNA2-PP1 duplex. The RNA degradation and RCA products were analyzed by electrophoresis through denaturing 8% polyacrylamide gels. Different RNA-PP duplexes are shown above the gels. The radioactive label is indicated by the star. The contents of samples are shown above the gel lines. Reaction products are labeled as “ a ” for the 5′-end-labeled RNA1 hydrolysis product serving as an RNA primer for RCA and “ b ” for the 5′-end-labeled RCA product. ( C ) The reaction scheme represents the conversions of target RNA.
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    Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand cDNA synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear DNA was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).

    Journal: Nucleic Acids Research

    Article Title: A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae

    doi:

    Figure Lengend Snippet: Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand cDNA synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear DNA was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).

    Article Snippet: Total RNA (3 µg) was reverse transcribed and second strand cDNA synthesis was carried out with Escherichia coli DNA polymerase I, E.coli RNase H and E.coli DNA Ligase (SuperScript™ Choice System for cDNA Synthesis, Invitrogen).

    Techniques: Isolation, Agarose Gel Electrophoresis, Derivative Assay, Labeling, Generated, Polymerase Chain Reaction, Amplification, Clone Assay

    RNAse protection assay. The upper panel shows a schematic representation of part of the 5′ region of the human CD45 gene and the RNA probes generated in vitro . The lower panel shows the protected fragments in different cell lines: Jurkat (human T cell), Raji (human B cell), K562 (human erythroid), U937 (human myeloid), EL-4 (mouse T cell) and HFB-1 (human B cell). M is the Hin fI-digested φX174 DNA used as a marker. The size of the bands in bp is indicated at both sides.

    Journal: Immunology

    Article Title: Structural and functional analysis of the human CD45 gene (PTPRC) upstream region: evidence for a functional promoter within the first intron of the gene

    doi: 10.1046/j.1365-2567.2001.01177.x

    Figure Lengend Snippet: RNAse protection assay. The upper panel shows a schematic representation of part of the 5′ region of the human CD45 gene and the RNA probes generated in vitro . The lower panel shows the protected fragments in different cell lines: Jurkat (human T cell), Raji (human B cell), K562 (human erythroid), U937 (human myeloid), EL-4 (mouse T cell) and HFB-1 (human B cell). M is the Hin fI-digested φX174 DNA used as a marker. The size of the bands in bp is indicated at both sides.

    Article Snippet: Second strand was generated by incubating the first-strand product with DNA polymerase I in the presence of RNase H and DNA ligase (all from Life Technologies) for 2 hr at 16°.

    Techniques: Rnase Protection Assay, Generated, In Vitro, Marker

    Promotion of plasma cell differentiation and Ig production in lupus B cells by ALD-DNA. Splenic B cells from 16- to 20-week-old MRL/lpr mice and 12-week-old MRL +/+ mice were cultured in media alone, or in the presence of ALD-DNA (50 µg/ml), LPS (100 ng/ml), or both for different periods as indicated below. ( A ) After 72 h stimulation, viable cells were analyzed for B220 and CD138 surface expression by flow cytometry. Representative dot plots showed the frequencies of plasmablasts (B220 + CD138 hi , lower panels, MRL/lpr, n = 6) or plasma cells (B220 − CD138 + , upper panels, MRL +/+ , n = 6) generated after each treatment. ( B ) Expression of Blimp-1 and XBP1 mRNA from lupus B cells cultured for 96 h was examined by real-time PCR. Results are expressed as the fold induction of gene transcription relative to their untreated controls (means ±SEM, n = 6). ( C ) Ig production in cell culture supernatants harvested on day 6 was determined by ELISA. Bars represent the means ±SEM of three independent experiments (n = 6). * P

    Journal: PLoS ONE

    Article Title: Self DNA from Lymphocytes That Have Undergone Activation-Induced Cell Death Enhances Murine B Cell Proliferation and Antibody Production

    doi: 10.1371/journal.pone.0109095

    Figure Lengend Snippet: Promotion of plasma cell differentiation and Ig production in lupus B cells by ALD-DNA. Splenic B cells from 16- to 20-week-old MRL/lpr mice and 12-week-old MRL +/+ mice were cultured in media alone, or in the presence of ALD-DNA (50 µg/ml), LPS (100 ng/ml), or both for different periods as indicated below. ( A ) After 72 h stimulation, viable cells were analyzed for B220 and CD138 surface expression by flow cytometry. Representative dot plots showed the frequencies of plasmablasts (B220 + CD138 hi , lower panels, MRL/lpr, n = 6) or plasma cells (B220 − CD138 + , upper panels, MRL +/+ , n = 6) generated after each treatment. ( B ) Expression of Blimp-1 and XBP1 mRNA from lupus B cells cultured for 96 h was examined by real-time PCR. Results are expressed as the fold induction of gene transcription relative to their untreated controls (means ±SEM, n = 6). ( C ) Ig production in cell culture supernatants harvested on day 6 was determined by ELISA. Bars represent the means ±SEM of three independent experiments (n = 6). * P

    Article Snippet: CFSE-labeled B cells stimulated with ALD-DNA, E. coli DNA, LPS, anti-CD40, or different combinations of these stimuli for 3 days were stained with B220-APC (RA3-6B2, eBioscience) or CD138-APC (281-2, BD Biosciences).

    Techniques: Cell Differentiation, Mouse Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry, Generated, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Up-regulation of CD86 and MHC class II expression in naïve B cells by ALD-DNA stimulation. ( A ) Naïve B cells were incubated with ALD-DNA (50 µg/ml) in the presence or absence of LPS ranging from 0.01 µg/mL to 10 µg/ml for 72 h. The numbers of CD86-expressing B cells after each treatment were determined by flow cytometry. ( B ) Naive B cells were stimulated with ALD-DNA or E. coli single-stranded (ss) DNA (both at 50 µg/ml) for 72 h. CD86 and MHC class II expression was determined by performing flow cytometry. Histogram plots show the mean fluorescence intensity (MFI) of these surface molecules. The data are representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Self DNA from Lymphocytes That Have Undergone Activation-Induced Cell Death Enhances Murine B Cell Proliferation and Antibody Production

    doi: 10.1371/journal.pone.0109095

    Figure Lengend Snippet: Up-regulation of CD86 and MHC class II expression in naïve B cells by ALD-DNA stimulation. ( A ) Naïve B cells were incubated with ALD-DNA (50 µg/ml) in the presence or absence of LPS ranging from 0.01 µg/mL to 10 µg/ml for 72 h. The numbers of CD86-expressing B cells after each treatment were determined by flow cytometry. ( B ) Naive B cells were stimulated with ALD-DNA or E. coli single-stranded (ss) DNA (both at 50 µg/ml) for 72 h. CD86 and MHC class II expression was determined by performing flow cytometry. Histogram plots show the mean fluorescence intensity (MFI) of these surface molecules. The data are representative of three independent experiments.

    Article Snippet: CFSE-labeled B cells stimulated with ALD-DNA, E. coli DNA, LPS, anti-CD40, or different combinations of these stimuli for 3 days were stained with B220-APC (RA3-6B2, eBioscience) or CD138-APC (281-2, BD Biosciences).

    Techniques: Expressing, Incubation, Flow Cytometry, Cytometry, Fluorescence

    Enhancement of LPS-stimulated B cell survival and division by ALD-DNA. ( A ) CFSE-labeled B cells were stimulated with ALD-DNA (50 µg/ml) in the presence or absence of LPS (100 ng/ml) for 72 h. Division progression of B cells was determined by dilution of CFSE. Representative histogram plots show the percentage of proliferating (CFSE-low) B cells after each treatment (mean values ±SEM, n = 6). Shaded areas show the cell division peaks predicted by the ModFit software. ( B ) Flow cytometry dot plots showing the gate used to select living cells and eliminate cell debris, and showing the viability of B cells (mean values ±SEM, n = 11) under different culture conditions as described above for 72 h. ( C ) Nude mice were administered one intravenous injection of indicated stimuli at day 1 and an additional intraperitoneal injection of BrdU 24 h before sacrifice. After 72 h, spleens were collected, and B220 + B cells with incorporated BrdU combined with total DNA content (with 7-AAD) were analyzed by FACS. The region gates applied to the 7-AAD versus BrdU dot plot indicate the cell subsets that were resided in the S phase of the cell cycle. Results represent 6 mice/experimental group from two separate experiments.

    Journal: PLoS ONE

    Article Title: Self DNA from Lymphocytes That Have Undergone Activation-Induced Cell Death Enhances Murine B Cell Proliferation and Antibody Production

    doi: 10.1371/journal.pone.0109095

    Figure Lengend Snippet: Enhancement of LPS-stimulated B cell survival and division by ALD-DNA. ( A ) CFSE-labeled B cells were stimulated with ALD-DNA (50 µg/ml) in the presence or absence of LPS (100 ng/ml) for 72 h. Division progression of B cells was determined by dilution of CFSE. Representative histogram plots show the percentage of proliferating (CFSE-low) B cells after each treatment (mean values ±SEM, n = 6). Shaded areas show the cell division peaks predicted by the ModFit software. ( B ) Flow cytometry dot plots showing the gate used to select living cells and eliminate cell debris, and showing the viability of B cells (mean values ±SEM, n = 11) under different culture conditions as described above for 72 h. ( C ) Nude mice were administered one intravenous injection of indicated stimuli at day 1 and an additional intraperitoneal injection of BrdU 24 h before sacrifice. After 72 h, spleens were collected, and B220 + B cells with incorporated BrdU combined with total DNA content (with 7-AAD) were analyzed by FACS. The region gates applied to the 7-AAD versus BrdU dot plot indicate the cell subsets that were resided in the S phase of the cell cycle. Results represent 6 mice/experimental group from two separate experiments.

    Article Snippet: CFSE-labeled B cells stimulated with ALD-DNA, E. coli DNA, LPS, anti-CD40, or different combinations of these stimuli for 3 days were stained with B220-APC (RA3-6B2, eBioscience) or CD138-APC (281-2, BD Biosciences).

    Techniques: Labeling, Software, Flow Cytometry, Cytometry, Mouse Assay, Injection, FACS

    Enhancement of CD40-stimulated naïve B cell proliferation by ALD-DNA. ( A ) Naïve B cells were pre-incubated for 20 min with polymyxin B (PMB; 20 µg/ml) before the addition of ALD-DNA (50 µg/ml), LPS (100 ng/ml), anti-CD40 (1 µg/ml), LPS (100 ng/ml) plus ALD-DNA (50 µg/ml), or anti-CD40 (1 µg/ml) plus ALD-DNA (50 µg/ml). After 72 h stimulation, cell proliferation was detected by performing BrdU incorporation assay using ELISA. Data are presented as the means ±SEM of three independent experiments. ( B ) CFSE-labeled naïve B cells were cultured in media containing ALD-DNA, anti-CD40, or both (at the concentrations as described above) for 72 h. Cell division was monitored by measuring the dilution of CFSE. Shaded areas show the cell division peaks predicted by the ModFit software. Results from one representative experiment of three are shown. ** P

    Journal: PLoS ONE

    Article Title: Self DNA from Lymphocytes That Have Undergone Activation-Induced Cell Death Enhances Murine B Cell Proliferation and Antibody Production

    doi: 10.1371/journal.pone.0109095

    Figure Lengend Snippet: Enhancement of CD40-stimulated naïve B cell proliferation by ALD-DNA. ( A ) Naïve B cells were pre-incubated for 20 min with polymyxin B (PMB; 20 µg/ml) before the addition of ALD-DNA (50 µg/ml), LPS (100 ng/ml), anti-CD40 (1 µg/ml), LPS (100 ng/ml) plus ALD-DNA (50 µg/ml), or anti-CD40 (1 µg/ml) plus ALD-DNA (50 µg/ml). After 72 h stimulation, cell proliferation was detected by performing BrdU incorporation assay using ELISA. Data are presented as the means ±SEM of three independent experiments. ( B ) CFSE-labeled naïve B cells were cultured in media containing ALD-DNA, anti-CD40, or both (at the concentrations as described above) for 72 h. Cell division was monitored by measuring the dilution of CFSE. Shaded areas show the cell division peaks predicted by the ModFit software. Results from one representative experiment of three are shown. ** P

    Article Snippet: CFSE-labeled B cells stimulated with ALD-DNA, E. coli DNA, LPS, anti-CD40, or different combinations of these stimuli for 3 days were stained with B220-APC (RA3-6B2, eBioscience) or CD138-APC (281-2, BD Biosciences).

    Techniques: Incubation, BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay, Labeling, Cell Culture, Software

    Synergistic enhancement of LPS-driven IgG production in naive B cells by ALD-DNA stimulation. Naive B cells were cultured for 6 days in media containing ALD-DNA (50 µg/ml) with or without LPS (100 ng/ml). Cell culture supernatants were collected, and the levels of IgM and IgG were measured by ELISA. Bars represent the means ±SEM of three independent experiments (n = 5). ND (not detected). ** P

    Journal: PLoS ONE

    Article Title: Self DNA from Lymphocytes That Have Undergone Activation-Induced Cell Death Enhances Murine B Cell Proliferation and Antibody Production

    doi: 10.1371/journal.pone.0109095

    Figure Lengend Snippet: Synergistic enhancement of LPS-driven IgG production in naive B cells by ALD-DNA stimulation. Naive B cells were cultured for 6 days in media containing ALD-DNA (50 µg/ml) with or without LPS (100 ng/ml). Cell culture supernatants were collected, and the levels of IgM and IgG were measured by ELISA. Bars represent the means ±SEM of three independent experiments (n = 5). ND (not detected). ** P

    Article Snippet: CFSE-labeled B cells stimulated with ALD-DNA, E. coli DNA, LPS, anti-CD40, or different combinations of these stimuli for 3 days were stained with B220-APC (RA3-6B2, eBioscience) or CD138-APC (281-2, BD Biosciences).

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

    Effect of ALD-DNA on CD138, Blimp-1, XBP1, and IL-6 expression in naïve B cells. Naïve B cells were cultured in media containing ALD-DNA (50 µg/ml), LPS (100 ng/ml), or both for different periods as described below. ( A ) After 72 h stimulation, Cells were analyzed for B220 and CD138 surface expression by flow cytometry. The number indicates the percentage of B220 − CD138 + cells in the gate. Similar results were obtained in three independent experiments. ( B ) Expression of Blimp-1 and XBP1 mRNA from B cells cultured for 96 hours was examined by real-time PCR. Fold induction relative to their untreated controls is shown (means ±SEM, n = 5). ( C ) Expression of IL-6 mRNA in B cells after 6 h stimulation was examined by real-time PCR. Fold induction relative to their untreated controls is shown (means ±SEM, n = 5); The amounts of IL-6 protein in the culture supernatants of B cells after 72 h stimulation were measured by ELISA (n = 6, a line linked two dots represents a pair observation). Up-regulation of IL-6 protein was undetectable in all samples after ALD-DNA treatment. * P

    Journal: PLoS ONE

    Article Title: Self DNA from Lymphocytes That Have Undergone Activation-Induced Cell Death Enhances Murine B Cell Proliferation and Antibody Production

    doi: 10.1371/journal.pone.0109095

    Figure Lengend Snippet: Effect of ALD-DNA on CD138, Blimp-1, XBP1, and IL-6 expression in naïve B cells. Naïve B cells were cultured in media containing ALD-DNA (50 µg/ml), LPS (100 ng/ml), or both for different periods as described below. ( A ) After 72 h stimulation, Cells were analyzed for B220 and CD138 surface expression by flow cytometry. The number indicates the percentage of B220 − CD138 + cells in the gate. Similar results were obtained in three independent experiments. ( B ) Expression of Blimp-1 and XBP1 mRNA from B cells cultured for 96 hours was examined by real-time PCR. Fold induction relative to their untreated controls is shown (means ±SEM, n = 5). ( C ) Expression of IL-6 mRNA in B cells after 6 h stimulation was examined by real-time PCR. Fold induction relative to their untreated controls is shown (means ±SEM, n = 5); The amounts of IL-6 protein in the culture supernatants of B cells after 72 h stimulation were measured by ELISA (n = 6, a line linked two dots represents a pair observation). Up-regulation of IL-6 protein was undetectable in all samples after ALD-DNA treatment. * P

    Article Snippet: CFSE-labeled B cells stimulated with ALD-DNA, E. coli DNA, LPS, anti-CD40, or different combinations of these stimuli for 3 days were stained with B220-APC (RA3-6B2, eBioscience) or CD138-APC (281-2, BD Biosciences).

    Techniques: Expressing, Cell Culture, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. RNA was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P

    Journal: Endocrinology

    Article Title: mir-374-5p, mir-379-5p, and mir-503-5p Regulate Proliferation and Hypertrophic Differentiation of Growth Plate Chondrocytes in Male Rats

    doi: 10.1210/en.2017-00780

    Figure Lengend Snippet: mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p show greater expression of growth plate cartilage in the PZ than in the HZ. Growth plate cartilage from 4-day-old rats was separated into specific zones by microdissection. RNA was purified, and miRNA levels were assessed by solution hybridization (Nanostring, mean ± standard error of the mean of four biological replicates each from an individual animal). * P

    Article Snippet: After 6 hours of treatment, cells were collected, and total RNA was extracted using the mirVana kit. miRNAs were reverse transcribed and amplified with the following steps according to the manufacturer’s instructions (TaqManAdvanced miRNA Assays; Thermo Fisher Scientific): (1) Poly(A) tail reaction, (2) adaptor ligation, (3) reverse transcription, and (4) miRNA amplification reaction.

    Techniques: Expressing, Laser Capture Microdissection, Purification, Hybridization

    Combined treatment of transfected primary chondrocytes with miRNA inhibitors and PTH 1-34. Primary chondrocytes were transfected with a scrambled RNA inhibitor or one of three miRNA inhibitors (mir-374-5p, mir-379-5p, or mir-503-5p). After 42 hours, the cells were treated with PTH 1-34 or vehicle for 6 hours, and mRNA levels of (a) Ihh and (b) Col10a1 were measured by real-time PCR. Six biological replicates were performed. * P

    Journal: Endocrinology

    Article Title: mir-374-5p, mir-379-5p, and mir-503-5p Regulate Proliferation and Hypertrophic Differentiation of Growth Plate Chondrocytes in Male Rats

    doi: 10.1210/en.2017-00780

    Figure Lengend Snippet: Combined treatment of transfected primary chondrocytes with miRNA inhibitors and PTH 1-34. Primary chondrocytes were transfected with a scrambled RNA inhibitor or one of three miRNA inhibitors (mir-374-5p, mir-379-5p, or mir-503-5p). After 42 hours, the cells were treated with PTH 1-34 or vehicle for 6 hours, and mRNA levels of (a) Ihh and (b) Col10a1 were measured by real-time PCR. Six biological replicates were performed. * P

    Article Snippet: After 6 hours of treatment, cells were collected, and total RNA was extracted using the mirVana kit. miRNAs were reverse transcribed and amplified with the following steps according to the manufacturer’s instructions (TaqManAdvanced miRNA Assays; Thermo Fisher Scientific): (1) Poly(A) tail reaction, (2) adaptor ligation, (3) reverse transcription, and (4) miRNA amplification reaction.

    Techniques: Transfection, Real-time Polymerase Chain Reaction

    Inhibitors of mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p decrease growth plate chondrocyte proliferation and stimulate hypertrophic differentiation. Primary chondrocytes from 4-day-old rat growth plates were transfected with inhibitors for mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p or with a scrambled miRNA inhibitor. (a) Proliferation rates (mean ± standard error of the mean) were measured by 3 H-thymidine incorporation beginning 48 hours after transfection. (b–e) In separate experiments, RNA was isolated from the primary chondrocytes 48 hours after transfection with inhibitors for (b) mir-369-3p, (c) mir-374-5p, (d) mir-379-5p, or (e) mir-503-5. Real-time RT-PCR was used to measure mRNA levels (mean ± standard error of the mean) for specific hypertrophy-associated genes. The scrambled miRNA inhibitor served as the control. * P

    Journal: Endocrinology

    Article Title: mir-374-5p, mir-379-5p, and mir-503-5p Regulate Proliferation and Hypertrophic Differentiation of Growth Plate Chondrocytes in Male Rats

    doi: 10.1210/en.2017-00780

    Figure Lengend Snippet: Inhibitors of mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p decrease growth plate chondrocyte proliferation and stimulate hypertrophic differentiation. Primary chondrocytes from 4-day-old rat growth plates were transfected with inhibitors for mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p or with a scrambled miRNA inhibitor. (a) Proliferation rates (mean ± standard error of the mean) were measured by 3 H-thymidine incorporation beginning 48 hours after transfection. (b–e) In separate experiments, RNA was isolated from the primary chondrocytes 48 hours after transfection with inhibitors for (b) mir-369-3p, (c) mir-374-5p, (d) mir-379-5p, or (e) mir-503-5. Real-time RT-PCR was used to measure mRNA levels (mean ± standard error of the mean) for specific hypertrophy-associated genes. The scrambled miRNA inhibitor served as the control. * P

    Article Snippet: After 6 hours of treatment, cells were collected, and total RNA was extracted using the mirVana kit. miRNAs were reverse transcribed and amplified with the following steps according to the manufacturer’s instructions (TaqManAdvanced miRNA Assays; Thermo Fisher Scientific): (1) Poly(A) tail reaction, (2) adaptor ligation, (3) reverse transcription, and (4) miRNA amplification reaction.

    Techniques: Transfection, Isolation, Quantitative RT-PCR

    5’ RACE. PCR products from wildtype and DrrS 5’ variants separated on 3% agarose. Total RNA was treated with RNA pyrophosphohydrolase (PPH) before linker ligation, cDNA synthesis and PCR amplification.

    Journal: PLoS ONE

    Article Title: Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

    doi: 10.1371/journal.pone.0174079

    Figure Lengend Snippet: 5’ RACE. PCR products from wildtype and DrrS 5’ variants separated on 3% agarose. Total RNA was treated with RNA pyrophosphohydrolase (PPH) before linker ligation, cDNA synthesis and PCR amplification.

    Article Snippet: 3’ RACE was performed by poly(A) tailing total RNA using E . coli poly-A-polymerase (Ambion), following manufacturer’s instructions. cDNA was synthesised using the oligo(d)T primer, and PCRs were done using Red-Taq DNA polymerase (Sigma) with primers shown in .

    Techniques: Polymerase Chain Reaction, Ligation, Amplification

    The pre-EJC regulates the precise splicing of dlg1 . ( A ) Aberrant dlg1 transcripts (indicated by asterisks) were detected by RT-PCR in RNA samples prepared from fly larvae expressing tsu RNAi driven by da -Gal4. ( B ) Shown is the genomic structure of dlg1 that contains 23 coding exons (blue boxes). The positions of primer pairs used to amplify RB, RH and RL splicing isoforms are indicated by red arrows. ( C ) Shown are RB, RH, RL splicing isoforms that are amplified by the above indicated primer pair. ( D – H ) Aberrant isoforms indicated by asterisks in panel A. were excised and purified from the agarose gel, cloned into a pGEM-T vector and subsequently subject to sequencing. After comparing the mRNA sequences deduced from sequencing results with wildype RB, RH and RL isoforms, three classes of aberrant splicing events were detected. The first class represents a previously reported exon skipping event, which was observed in all splicing forms ( D – G ). The second class represents exon inclusion, which contains the exons that are normally efficiently spliced in wildtype isoforms (green boxes). The third class includes exons that are generated by previously unidentified splice sites (SS; red boxes). These three classes are not mutually exclusive as classes 1 and 2 were found in aberrant isoforms shown in panel E, classes 1 and 3 in panel F, and classes 1–3 in panel G. Δ indicates missing exons. 1’ indicates a 5’ SS in exon 1. 16’’ indicates a 3’SS sites in exon 16. 7’’’ and 23’’’ indicate 5’ and 3’ SS present in the exon 7 and 23, respectively. Dashed lines indicate novel ligation of exons. Details on usage of previously unidentified splice sites are shown ( H ). DOI: http://dx.doi.org/10.7554/eLife.17200.013

    Journal: eLife

    Article Title: The exon junction complex regulates the splicing of cell polarity gene dlg1 to control Wingless signaling in development

    doi: 10.7554/eLife.17200

    Figure Lengend Snippet: The pre-EJC regulates the precise splicing of dlg1 . ( A ) Aberrant dlg1 transcripts (indicated by asterisks) were detected by RT-PCR in RNA samples prepared from fly larvae expressing tsu RNAi driven by da -Gal4. ( B ) Shown is the genomic structure of dlg1 that contains 23 coding exons (blue boxes). The positions of primer pairs used to amplify RB, RH and RL splicing isoforms are indicated by red arrows. ( C ) Shown are RB, RH, RL splicing isoforms that are amplified by the above indicated primer pair. ( D – H ) Aberrant isoforms indicated by asterisks in panel A. were excised and purified from the agarose gel, cloned into a pGEM-T vector and subsequently subject to sequencing. After comparing the mRNA sequences deduced from sequencing results with wildype RB, RH and RL isoforms, three classes of aberrant splicing events were detected. The first class represents a previously reported exon skipping event, which was observed in all splicing forms ( D – G ). The second class represents exon inclusion, which contains the exons that are normally efficiently spliced in wildtype isoforms (green boxes). The third class includes exons that are generated by previously unidentified splice sites (SS; red boxes). These three classes are not mutually exclusive as classes 1 and 2 were found in aberrant isoforms shown in panel E, classes 1 and 3 in panel F, and classes 1–3 in panel G. Δ indicates missing exons. 1’ indicates a 5’ SS in exon 1. 16’’ indicates a 3’SS sites in exon 16. 7’’’ and 23’’’ indicate 5’ and 3’ SS present in the exon 7 and 23, respectively. Dashed lines indicate novel ligation of exons. Details on usage of previously unidentified splice sites are shown ( H ). DOI: http://dx.doi.org/10.7554/eLife.17200.013

    Article Snippet: RNA isolation, quantitative real-time PCR and RT-PCR Total RNA of pooled third instar larvae or imaginal wing discs was extracted using TRIzol reagent (Invitrogen).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Purification, Agarose Gel Electrophoresis, Clone Assay, Plasmid Preparation, Sequencing, Generated, Ligation

    Target RNA conversion into a primer for RCA, the impact of E. coli RNase III. The reactions were carried out under the conditions described in Materials and Methods. The 5′-end-labeled ( A ) RNA2-PP1 (panel 1 ), ( B ) RNA2-PP2 (panel 1 ), and RNA1-PP1 (panel 2 ) hybrids were incubated with Phi29 DNA polymerase in the absence or presence of dNTP. ( A , panel 2 ) The experiments with E. coli RNase III were performed using RNA2-PP1 duplex. The RNA degradation and RCA products were analyzed by electrophoresis through denaturing 8% polyacrylamide gels. Different RNA-PP duplexes are shown above the gels. The radioactive label is indicated by the star. The contents of samples are shown above the gel lines. Reaction products are labeled as “ a ” for the 5′-end-labeled RNA1 hydrolysis product serving as an RNA primer for RCA and “ b ” for the 5′-end-labeled RCA product. ( C ) The reaction scheme represents the conversions of target RNA.

    Journal: RNA

    Article Title: Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3?-end

    doi: 10.1261/rna.2068510

    Figure Lengend Snippet: Target RNA conversion into a primer for RCA, the impact of E. coli RNase III. The reactions were carried out under the conditions described in Materials and Methods. The 5′-end-labeled ( A ) RNA2-PP1 (panel 1 ), ( B ) RNA2-PP2 (panel 1 ), and RNA1-PP1 (panel 2 ) hybrids were incubated with Phi29 DNA polymerase in the absence or presence of dNTP. ( A , panel 2 ) The experiments with E. coli RNase III were performed using RNA2-PP1 duplex. The RNA degradation and RCA products were analyzed by electrophoresis through denaturing 8% polyacrylamide gels. Different RNA-PP duplexes are shown above the gels. The radioactive label is indicated by the star. The contents of samples are shown above the gel lines. Reaction products are labeled as “ a ” for the 5′-end-labeled RNA1 hydrolysis product serving as an RNA primer for RCA and “ b ” for the 5′-end-labeled RCA product. ( C ) The reaction scheme represents the conversions of target RNA.

    Article Snippet: Some RCA reactions were performed in the presence of 0.5 U of E. coli RNase III (Ambion).

    Techniques: Labeling, Incubation, Electrophoresis

    Detection of individual mouse GAPDH and human ACTB and PPIA transcripts in situ, the impact of E. coli RNase III. The reactions in situ were carried out with target transcript–padlock probe duplexes ( A , duplexes 1–3) under the conditions described in Materials and Methods. The oligonucleotides ( B , D ) PP9 (complementary to the hybridization region in the middle of mGAPDH transcript), ( F , H ) PP11 (complementary to the hybridization region in the middle of hACTB transcript), and ( J , L ) PP13* (complementary to the hybridization region at the 5′-end PPIA transcript) were used as specific tools for target transcript detection in situ. ( C , E , G , I , K , M ) The oligonucleotides PP10*, PP12, and PP14 (in the ligation junction regions slightly differing from PP9*, PP11, and PP13, respectively) were used as controls for reaction specificity. The RCA reactions were carried in the absence ( B , C , F , G , J , K ) or presence ( D , E , H , I , L , M ) of E. coli RNase III. The reaction products were hybridized with the oligonucleotides containing FITC or Alexa555 labels, and the slides were analyzed using the fluorescence microscope Olympus IX70 with 20× objectives. The images are presented as the overlay of DAPI and FITC ( B–I ) or Alexa555 ( J–M ) projections. DAPI stains nuclei blue, FITC or Alexa555 labels specific RCA products green or red, respectively. Scale bars, 25 μm.

    Journal: RNA

    Article Title: Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3?-end

    doi: 10.1261/rna.2068510

    Figure Lengend Snippet: Detection of individual mouse GAPDH and human ACTB and PPIA transcripts in situ, the impact of E. coli RNase III. The reactions in situ were carried out with target transcript–padlock probe duplexes ( A , duplexes 1–3) under the conditions described in Materials and Methods. The oligonucleotides ( B , D ) PP9 (complementary to the hybridization region in the middle of mGAPDH transcript), ( F , H ) PP11 (complementary to the hybridization region in the middle of hACTB transcript), and ( J , L ) PP13* (complementary to the hybridization region at the 5′-end PPIA transcript) were used as specific tools for target transcript detection in situ. ( C , E , G , I , K , M ) The oligonucleotides PP10*, PP12, and PP14 (in the ligation junction regions slightly differing from PP9*, PP11, and PP13, respectively) were used as controls for reaction specificity. The RCA reactions were carried in the absence ( B , C , F , G , J , K ) or presence ( D , E , H , I , L , M ) of E. coli RNase III. The reaction products were hybridized with the oligonucleotides containing FITC or Alexa555 labels, and the slides were analyzed using the fluorescence microscope Olympus IX70 with 20× objectives. The images are presented as the overlay of DAPI and FITC ( B–I ) or Alexa555 ( J–M ) projections. DAPI stains nuclei blue, FITC or Alexa555 labels specific RCA products green or red, respectively. Scale bars, 25 μm.

    Article Snippet: Some RCA reactions were performed in the presence of 0.5 U of E. coli RNase III (Ambion).

    Techniques: In Situ, Hybridization, Ligation, Fluorescence, Microscopy

    Detection of GAPDH transcripts in vitro, the impact of E. coli RNase III. The reactions were carried out with GAPDH-padlock probe duplexes ( A , duplexes 1–3) under the conditions described in Materials and Methods. ( B ) The oligonucleotides PP5 (panel 1 ) and PP10* (panel 2 ) were used as specific tools for the hGAPDH transcript detection in the human total RNA isolated from HeLa cells. The oligonucleotides PP6 (panel 1 ) and PP9* (panel 2 ) were used as controls for reaction specificity. The oligonucleotides PP9* and PP10* (panels 3.1 , 3.2 ) were used as probes for mGAPDH transcript detection and controls for reaction specificity, respectively, when testing mouse total RNAs isolated from the tongue (panel 3.1 ) and liver (panel 3.2 ) tissues. The RCA reactions were carried out in the absence or presence of E. coli RNase III (panels 1–3 ). The reaction products were monomerized by cleaving them with Mva1269I (panel 1 ) or LguI (panels 2 , 3.1 , 3.2 ) REases and analyzed by electrophoresis through a denaturing 8% polyacrylamide gel. The contents of samples are shown above the gel lines. The reaction products are labeled as “ a ,” “ b ,” and “ c ” for the 70-nt-long labeled monomers of RCA products obtained using transcript-padlock probe duplexes 1, 2, and 3, respectively ( A ).

    Journal: RNA

    Article Title: Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3?-end

    doi: 10.1261/rna.2068510

    Figure Lengend Snippet: Detection of GAPDH transcripts in vitro, the impact of E. coli RNase III. The reactions were carried out with GAPDH-padlock probe duplexes ( A , duplexes 1–3) under the conditions described in Materials and Methods. ( B ) The oligonucleotides PP5 (panel 1 ) and PP10* (panel 2 ) were used as specific tools for the hGAPDH transcript detection in the human total RNA isolated from HeLa cells. The oligonucleotides PP6 (panel 1 ) and PP9* (panel 2 ) were used as controls for reaction specificity. The oligonucleotides PP9* and PP10* (panels 3.1 , 3.2 ) were used as probes for mGAPDH transcript detection and controls for reaction specificity, respectively, when testing mouse total RNAs isolated from the tongue (panel 3.1 ) and liver (panel 3.2 ) tissues. The RCA reactions were carried out in the absence or presence of E. coli RNase III (panels 1–3 ). The reaction products were monomerized by cleaving them with Mva1269I (panel 1 ) or LguI (panels 2 , 3.1 , 3.2 ) REases and analyzed by electrophoresis through a denaturing 8% polyacrylamide gel. The contents of samples are shown above the gel lines. The reaction products are labeled as “ a ,” “ b ,” and “ c ” for the 70-nt-long labeled monomers of RCA products obtained using transcript-padlock probe duplexes 1, 2, and 3, respectively ( A ).

    Article Snippet: Some RCA reactions were performed in the presence of 0.5 U of E. coli RNase III (Ambion).

    Techniques: In Vitro, Isolation, Electrophoresis, Labeling

    The impact of double-stranded RNA-specific E. coli RNase III on the detection of target transcripts in situ

    Journal: RNA

    Article Title: Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3?-end

    doi: 10.1261/rna.2068510

    Figure Lengend Snippet: The impact of double-stranded RNA-specific E. coli RNase III on the detection of target transcripts in situ

    Article Snippet: Some RCA reactions were performed in the presence of 0.5 U of E. coli RNase III (Ambion).

    Techniques: In Situ