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  • 99
    Zymo Research rna isolation
    Rna Isolation, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 1638 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna isolation
    Rna Isolation, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 75697 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega sv total rna isolation system
    <t>SroC</t> induces GcvB degradation by a base-pairing mechanism GcvB-SroC base-pairing regions. The compensatory base pair changes in GcvB and SroC are indicated in red. Salmonella Δ gcvB Δ sroC (JVS-5822) strain was cotransformed with pP L -GcvB and pBAD-SroC derivatives (see Supplementary Table S4). Each transformant was grown to OD 600 of 0.5 (0 min) and incubated for another 10 min in the presence of 0.2% l -arabinose. Total <t>RNA</t> was prepared from the cells and subjected to Northern blot analysis. The fold repression of GcvB was determined from three independent replicates; standard deviation is shown. The chromosomally modified wild-type sroC strain (JVS-10108), sroC ** mutant (JVS-10111) and sroC deletion mutant (JVS-5821) were grown to early stationary phase in LB medium (OD 600 of 2.0) prior to the addition of rifampicin. Cultures were harvested at the indicated time points after rifampicin treatment. RNA was isolated and analyzed by Northern blot as in Fig 3B . Source data are available online for this figure.
    Sv Total Rna Isolation System, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 13726 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rna isolation
    <t>SroC</t> induces GcvB degradation by a base-pairing mechanism GcvB-SroC base-pairing regions. The compensatory base pair changes in GcvB and SroC are indicated in red. Salmonella Δ gcvB Δ sroC (JVS-5822) strain was cotransformed with pP L -GcvB and pBAD-SroC derivatives (see Supplementary Table S4). Each transformant was grown to OD 600 of 0.5 (0 min) and incubated for another 10 min in the presence of 0.2% l -arabinose. Total <t>RNA</t> was prepared from the cells and subjected to Northern blot analysis. The fold repression of GcvB was determined from three independent replicates; standard deviation is shown. The chromosomally modified wild-type sroC strain (JVS-10108), sroC ** mutant (JVS-10111) and sroC deletion mutant (JVS-5821) were grown to early stationary phase in LB medium (OD 600 of 2.0) prior to the addition of rifampicin. Cultures were harvested at the indicated time points after rifampicin treatment. RNA was isolated and analyzed by Northern blot as in Fig 3B . Source data are available online for this figure.
    Rna Isolation, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7571 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnaqueous kit
    <t>SroC</t> induces GcvB degradation by a base-pairing mechanism GcvB-SroC base-pairing regions. The compensatory base pair changes in GcvB and SroC are indicated in red. Salmonella Δ gcvB Δ sroC (JVS-5822) strain was cotransformed with pP L -GcvB and pBAD-SroC derivatives (see Supplementary Table S4). Each transformant was grown to OD 600 of 0.5 (0 min) and incubated for another 10 min in the presence of 0.2% l -arabinose. Total <t>RNA</t> was prepared from the cells and subjected to Northern blot analysis. The fold repression of GcvB was determined from three independent replicates; standard deviation is shown. The chromosomally modified wild-type sroC strain (JVS-10108), sroC ** mutant (JVS-10111) and sroC deletion mutant (JVS-5821) were grown to early stationary phase in LB medium (OD 600 of 2.0) prior to the addition of rifampicin. Cultures were harvested at the indicated time points after rifampicin treatment. RNA was isolated and analyzed by Northern blot as in Fig 3B . Source data are available online for this figure.
    Rnaqueous Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnaqueous micro kit
    Capillary electrophoresis analysis of pig LV biopsy RNA. ( A) Bioanalyzer nano chip traces of all tested samples. Inspection of electropherograms provides additional information to assess RNA quality and protocol performance. (B) Representative traces from each protocol with respective RIN values. Numbers 1–4 correspond to numbered boxes in A. Note that trace 3 has a high RIN despite a large smear in the gel. (C) Summary of RIN values of all the samples tested (mean±SEM). The <t>RNAqueous</t> protocol had undetectable RINs. The mir Vana and miRCURY tissue protocols gave consistently high RIN values, while despite high average RIN, the samples from the miRNeasy micro protocol are more variable. Numbers in brackets indicate the number of samples in each group. (D) Bioanalyzer small RNA chip traces for each kit. Although the RIN of samples marked * were similar (8.6–8.7), the spectrum of small RNAs recovered is highly heterogeneous.
    Rnaqueous Micro Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5953 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen rna isolation kit
    Capillary electrophoresis analysis of pig LV biopsy RNA. ( A) Bioanalyzer nano chip traces of all tested samples. Inspection of electropherograms provides additional information to assess RNA quality and protocol performance. (B) Representative traces from each protocol with respective RIN values. Numbers 1–4 correspond to numbered boxes in A. Note that trace 3 has a high RIN despite a large smear in the gel. (C) Summary of RIN values of all the samples tested (mean±SEM). The <t>RNAqueous</t> protocol had undetectable RINs. The mir Vana and miRCURY tissue protocols gave consistently high RIN values, while despite high average RIN, the samples from the miRNeasy micro protocol are more variable. Numbers in brackets indicate the number of samples in each group. (D) Bioanalyzer small RNA chip traces for each kit. Although the RIN of samples marked * were similar (8.6–8.7), the spectrum of small RNAs recovered is highly heterogeneous.
    Rna Isolation Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 4567 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher picopure rna isolation kit
    Capillary electrophoresis analysis of pig LV biopsy RNA. ( A) Bioanalyzer nano chip traces of all tested samples. Inspection of electropherograms provides additional information to assess RNA quality and protocol performance. (B) Representative traces from each protocol with respective RIN values. Numbers 1–4 correspond to numbered boxes in A. Note that trace 3 has a high RIN despite a large smear in the gel. (C) Summary of RIN values of all the samples tested (mean±SEM). The <t>RNAqueous</t> protocol had undetectable RINs. The mir Vana and miRCURY tissue protocols gave consistently high RIN values, while despite high average RIN, the samples from the miRNeasy micro protocol are more variable. Numbers in brackets indicate the number of samples in each group. (D) Bioanalyzer small RNA chip traces for each kit. Although the RIN of samples marked * were similar (8.6–8.7), the spectrum of small RNAs recovered is highly heterogeneous.
    Picopure Rna Isolation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen rneasy rna isolation kit
    Capillary electrophoresis analysis of pig LV biopsy RNA. ( A) Bioanalyzer nano chip traces of all tested samples. Inspection of electropherograms provides additional information to assess RNA quality and protocol performance. (B) Representative traces from each protocol with respective RIN values. Numbers 1–4 correspond to numbered boxes in A. Note that trace 3 has a high RIN despite a large smear in the gel. (C) Summary of RIN values of all the samples tested (mean±SEM). The <t>RNAqueous</t> protocol had undetectable RINs. The mir Vana and miRCURY tissue protocols gave consistently high RIN values, while despite high average RIN, the samples from the miRNeasy micro protocol are more variable. Numbers in brackets indicate the number of samples in each group. (D) Bioanalyzer small RNA chip traces for each kit. Although the RIN of samples marked * were similar (8.6–8.7), the spectrum of small RNAs recovered is highly heterogeneous.
    Rneasy Rna Isolation Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3696 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnaqueous 4pcr kit
    Capillary electrophoresis analysis of pig LV biopsy RNA. ( A) Bioanalyzer nano chip traces of all tested samples. Inspection of electropherograms provides additional information to assess RNA quality and protocol performance. (B) Representative traces from each protocol with respective RIN values. Numbers 1–4 correspond to numbered boxes in A. Note that trace 3 has a high RIN despite a large smear in the gel. (C) Summary of RIN values of all the samples tested (mean±SEM). The <t>RNAqueous</t> protocol had undetectable RINs. The mir Vana and miRCURY tissue protocols gave consistently high RIN values, while despite high average RIN, the samples from the miRNeasy micro protocol are more variable. Numbers in brackets indicate the number of samples in each group. (D) Bioanalyzer small RNA chip traces for each kit. Although the RIN of samples marked * were similar (8.6–8.7), the spectrum of small RNAs recovered is highly heterogeneous.
    Rnaqueous 4pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega sv total rna isolation kit
    The UME6 5′ UTR functions to inhibit translational efficiency. The indicated strains were grown overnight in YEPD medium at 30°C and diluted into prewarmed YEPD at 30°C (non-filament-inducing conditions) or YEPD + 10% serum at 37°C (filament-inducing conditions). At 30 minutes following serum and temperature induction, cells were treated with 0.1 mg mL −1 cycloheximide, lysed (in the presence or absence of 25 mM EDTA for cells grown in YEPD + 10% serum at 37°C), and subjected to sucrose gradient centrifugation for polysome isolation. (A) Polysome profiles across sucrose gradients for the indicated strains grown in YEPD at 30°C or YEPD + 10% serum at 37°C (+/− EDTA treatment) are shown; please note that profiles for each strain are offset. (B) <t>RNA</t> was extracted from each fraction of the indicated sucrose gradients to determine the abundance of UME6 transcript by <t>qRT-PCR</t> analysis. Data shown represents normalized mean UME6 transcript levels based on two independent experiments (± SEM). Please note that in the presence of serum at 37°C there is a statistically significant increase in UME6 transcript abundance in UME6 5′ utrΔ/Δ vs. wild-type (WT) strains for the polysome fractions 7,10 and 11 (p ≤ 0.01 as determined by Student’s t test).
    Sv Total Rna Isolation Kit, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 3160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna isolation kit
    The UME6 5′ UTR functions to inhibit translational efficiency. The indicated strains were grown overnight in YEPD medium at 30°C and diluted into prewarmed YEPD at 30°C (non-filament-inducing conditions) or YEPD + 10% serum at 37°C (filament-inducing conditions). At 30 minutes following serum and temperature induction, cells were treated with 0.1 mg mL −1 cycloheximide, lysed (in the presence or absence of 25 mM EDTA for cells grown in YEPD + 10% serum at 37°C), and subjected to sucrose gradient centrifugation for polysome isolation. (A) Polysome profiles across sucrose gradients for the indicated strains grown in YEPD at 30°C or YEPD + 10% serum at 37°C (+/− EDTA treatment) are shown; please note that profiles for each strain are offset. (B) <t>RNA</t> was extracted from each fraction of the indicated sucrose gradients to determine the abundance of UME6 transcript by <t>qRT-PCR</t> analysis. Data shown represents normalized mean UME6 transcript levels based on two independent experiments (± SEM). Please note that in the presence of serum at 37°C there is a statistically significant increase in UME6 transcript abundance in UME6 5′ utrΔ/Δ vs. wild-type (WT) strains for the polysome fractions 7,10 and 11 (p ≤ 0.01 as determined by Student’s t test).
    Rna Isolation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2701 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mirvana rna isolation kit
    The UME6 5′ UTR functions to inhibit translational efficiency. The indicated strains were grown overnight in YEPD medium at 30°C and diluted into prewarmed YEPD at 30°C (non-filament-inducing conditions) or YEPD + 10% serum at 37°C (filament-inducing conditions). At 30 minutes following serum and temperature induction, cells were treated with 0.1 mg mL −1 cycloheximide, lysed (in the presence or absence of 25 mM EDTA for cells grown in YEPD + 10% serum at 37°C), and subjected to sucrose gradient centrifugation for polysome isolation. (A) Polysome profiles across sucrose gradients for the indicated strains grown in YEPD at 30°C or YEPD + 10% serum at 37°C (+/− EDTA treatment) are shown; please note that profiles for each strain are offset. (B) <t>RNA</t> was extracted from each fraction of the indicated sucrose gradients to determine the abundance of UME6 transcript by <t>qRT-PCR</t> analysis. Data shown represents normalized mean UME6 transcript levels based on two independent experiments (± SEM). Please note that in the presence of serum at 37°C there is a statistically significant increase in UME6 transcript abundance in UME6 5′ utrΔ/Δ vs. wild-type (WT) strains for the polysome fractions 7,10 and 11 (p ≤ 0.01 as determined by Student’s t test).
    Mirvana Rna Isolation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL rna isolation
    Dual exon skipping of myostatin and dystrophin in DL589.2 DMD patient cells . DL589.2 myotubes were transfected with 200 nM of myostatin AON and h50AON1 DMD AON. <t>RNA</t> was isolated two days post-transfection and analyzed for myostatin and dystrophin skips by <t>RT-PCR.</t>
    Rna Isolation, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 94/100, based on 2806 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen mircury rna isolation kit
    Dual exon skipping of myostatin and dystrophin in DL589.2 DMD patient cells . DL589.2 myotubes were transfected with 200 nM of myostatin AON and h50AON1 DMD AON. <t>RNA</t> was isolated two days post-transfection and analyzed for myostatin and dystrophin skips by <t>RT-PCR.</t>
    Mircury Rna Isolation Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2227 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc rna isolation
    Global cDNA read count distribution and Hfq levels in Y. pseudotuberculosis during growth under environmental and infection-relevant conditions. (A) Percentage of uniquely mapped reads of the virulence plasmid pYV obtained by <t>RNA-seq</t> of Y . pseudotuberculosis YPIII and YPIIIΔ crp cDNA libraries. Libraries were generated from TAP-treated (+TAP) <t>rRNA</t> depleted RNA obtained from cultures grown to exponential (E) or stationary (S) growth phase at 25°C (25) and 37°C (37) (for detailed statistics see S1 Dataset ). The data represent the mean ± SEM from three independent biological replicates and were analyzed with Student’s t-test. **: P
    Rna Isolation, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 2472 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher plant rna isolation aid
    Global cDNA read count distribution and Hfq levels in Y. pseudotuberculosis during growth under environmental and infection-relevant conditions. (A) Percentage of uniquely mapped reads of the virulence plasmid pYV obtained by <t>RNA-seq</t> of Y . pseudotuberculosis YPIII and YPIIIΔ crp cDNA libraries. Libraries were generated from TAP-treated (+TAP) <t>rRNA</t> depleted RNA obtained from cultures grown to exponential (E) or stationary (S) growth phase at 25°C (25) and 37°C (37) (for detailed statistics see S1 Dataset ). The data represent the mean ± SEM from three independent biological replicates and were analyzed with Student’s t-test. **: P
    Plant Rna Isolation Aid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 890 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co rna isolation
    Silencing of NbCUL1 enhances CLCuMuV <t>DNA</t> accumulation and results in typical disease symptoms. (A1 and A2) Six- to seven-week-old N . benthamiana plants were agroinoculated with CLCuMuV (CA) and βM2- CUL1 F1 (A1), βM2- CUL1 F2 (A2) or βM2- βC1 F (as the control). (B1 and B2) Silencing of NbCUL1 enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1 and C2) Real-time RT-PCR confirmed silencing of NbCUL1 . Total <t>RNA</t> was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify NbCUL1 mRNA level. Actin was used as the internal reference. The raw data of (B1 and B2) and (C1 and C2) were analysed by two-sample t -test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1 and D2) 100% plants infected with CA+βM2- CUL1 F1 (D1) or CA+βM2- CUL1 F2 (D2) show severe symptoms at 21 dpi.
    Rna Isolation, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 1655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen rneasy total rna isolation kit
    Silencing of NbCUL1 enhances CLCuMuV <t>DNA</t> accumulation and results in typical disease symptoms. (A1 and A2) Six- to seven-week-old N . benthamiana plants were agroinoculated with CLCuMuV (CA) and βM2- CUL1 F1 (A1), βM2- CUL1 F2 (A2) or βM2- βC1 F (as the control). (B1 and B2) Silencing of NbCUL1 enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1 and C2) Real-time RT-PCR confirmed silencing of NbCUL1 . Total <t>RNA</t> was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify NbCUL1 mRNA level. Actin was used as the internal reference. The raw data of (B1 and B2) and (C1 and C2) were analysed by two-sample t -test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1 and D2) 100% plants infected with CA+βM2- CUL1 F1 (D1) or CA+βM2- CUL1 F2 (D2) show severe symptoms at 21 dpi.
    Rneasy Total Rna Isolation Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1512 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Molecular Research Center inc rna isolation
    Association of nELAVs with the APP pre-mRNA. ( A ) Localization of primers used for the detection of APP/App pre-mRNAs ( arrows ; APPI/AppI : Forward I and Reverse I; APPII/AppII : Forward II and Reverse II). Boxes indicate exons and bold lines introns. ( B – D ) Nuclear extracts prepared from human SK-N-SH ( B ), human SH-SY5Y ( C ) and mouse Neuro-2a ( D ) cells were immunoprecipitated with a mouse anti-ELAVL antibody or mouse IgGs as a control. <t>RNA</t> was isolated from immunoprecipitates, as well as their supernatants and analyzed by semi-quantitative and quantitative <t>RT-PCR,</t> respectively, using specific primers against human or mouse APP pre-mRNA ( arrows ) and intronic GAPDH. Minus RT lanes are included as controls. Note that APP pre-mRNA was detectable in the immunoprecipitate only when lysates from cells expressing nELAVLs were used. Bars in graphs depict mean ± standard deviation of three independent experiments (*P
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    5 PRIME isol rna lysis reagent
    Association of nELAVs with the APP pre-mRNA. ( A ) Localization of primers used for the detection of APP/App pre-mRNAs ( arrows ; APPI/AppI : Forward I and Reverse I; APPII/AppII : Forward II and Reverse II). Boxes indicate exons and bold lines introns. ( B – D ) Nuclear extracts prepared from human SK-N-SH ( B ), human SH-SY5Y ( C ) and mouse Neuro-2a ( D ) cells were immunoprecipitated with a mouse anti-ELAVL antibody or mouse IgGs as a control. <t>RNA</t> was isolated from immunoprecipitates, as well as their supernatants and analyzed by semi-quantitative and quantitative <t>RT-PCR,</t> respectively, using specific primers against human or mouse APP pre-mRNA ( arrows ) and intronic GAPDH. Minus RT lanes are included as controls. Note that APP pre-mRNA was detectable in the immunoprecipitate only when lysates from cells expressing nELAVLs were used. Bars in graphs depict mean ± standard deviation of three independent experiments (*P
    Isol Rna Lysis Reagent, supplied by 5 PRIME, used in various techniques. Bioz Stars score: 92/100, based on 1381 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SroC induces GcvB degradation by a base-pairing mechanism GcvB-SroC base-pairing regions. The compensatory base pair changes in GcvB and SroC are indicated in red. Salmonella Δ gcvB Δ sroC (JVS-5822) strain was cotransformed with pP L -GcvB and pBAD-SroC derivatives (see Supplementary Table S4). Each transformant was grown to OD 600 of 0.5 (0 min) and incubated for another 10 min in the presence of 0.2% l -arabinose. Total RNA was prepared from the cells and subjected to Northern blot analysis. The fold repression of GcvB was determined from three independent replicates; standard deviation is shown. The chromosomally modified wild-type sroC strain (JVS-10108), sroC ** mutant (JVS-10111) and sroC deletion mutant (JVS-5821) were grown to early stationary phase in LB medium (OD 600 of 2.0) prior to the addition of rifampicin. Cultures were harvested at the indicated time points after rifampicin treatment. RNA was isolated and analyzed by Northern blot as in Fig 3B . Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

    doi: 10.15252/embj.201490546

    Figure Lengend Snippet: SroC induces GcvB degradation by a base-pairing mechanism GcvB-SroC base-pairing regions. The compensatory base pair changes in GcvB and SroC are indicated in red. Salmonella Δ gcvB Δ sroC (JVS-5822) strain was cotransformed with pP L -GcvB and pBAD-SroC derivatives (see Supplementary Table S4). Each transformant was grown to OD 600 of 0.5 (0 min) and incubated for another 10 min in the presence of 0.2% l -arabinose. Total RNA was prepared from the cells and subjected to Northern blot analysis. The fold repression of GcvB was determined from three independent replicates; standard deviation is shown. The chromosomally modified wild-type sroC strain (JVS-10108), sroC ** mutant (JVS-10111) and sroC deletion mutant (JVS-5821) were grown to early stationary phase in LB medium (OD 600 of 2.0) prior to the addition of rifampicin. Cultures were harvested at the indicated time points after rifampicin treatment. RNA was isolated and analyzed by Northern blot as in Fig 3B . Source data are available online for this figure.

    Article Snippet: At optical density (OD600 ) of 1.5, l -arabinose was added to cultures at a final concentration of 0.2% to induced SroC expression for 10 min, and total RNA was prepared with SV total RNA isolation system (Promega).

    Techniques: Incubation, Northern Blot, Standard Deviation, Modification, Mutagenesis, Isolation

    Direct binding between GcvB and SroC sRNAs 20 nM of 5′-end-labeled GcvB and SroC were subjected to RNase T1 and lead(II) cleavage in the absence and presence of 100 nM cold sRNAs, SroC, and GcvB, respectively. Lane C: untreated RNA; lane T1: RNase T1 ladder of denatured RNA; lane OH: alkaline ladder. The position of G residues cleaved by RNase T1 is indicated at the left of picture. Secondary structures of GcvB and SroC, paired or alone. RNase T1 cleavage sites are indicated by arrowheads; color indicates those that appeared (red) or disappeared (blue) upon base-pairing. The base pairing sites BS1 and BS2 are shadowed. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

    doi: 10.15252/embj.201490546

    Figure Lengend Snippet: Direct binding between GcvB and SroC sRNAs 20 nM of 5′-end-labeled GcvB and SroC were subjected to RNase T1 and lead(II) cleavage in the absence and presence of 100 nM cold sRNAs, SroC, and GcvB, respectively. Lane C: untreated RNA; lane T1: RNase T1 ladder of denatured RNA; lane OH: alkaline ladder. The position of G residues cleaved by RNase T1 is indicated at the left of picture. Secondary structures of GcvB and SroC, paired or alone. RNase T1 cleavage sites are indicated by arrowheads; color indicates those that appeared (red) or disappeared (blue) upon base-pairing. The base pairing sites BS1 and BS2 are shadowed. Source data are available online for this figure.

    Article Snippet: At optical density (OD600 ) of 1.5, l -arabinose was added to cultures at a final concentration of 0.2% to induced SroC expression for 10 min, and total RNA was prepared with SV total RNA isolation system (Promega).

    Techniques: Binding Assay, Labeling

    RNase E mediates SroC processing and GcvB degradation in distinct pathways Salmonella Δ gltIJKL rne + (JVS-9257) and Δ gltIJKL rne3071 (JVS-9258) strains transformed with pBAD- gltI or pBAD-SroC were grown to OD 600 of 0.3 at 28°C and further incubated at 44°C for 30 min (OD 600 of ˜0.5, indicated as time point 0). SroC expression was then induced for 10 min in the presence of 0.2% of l -arabinose. Total RNA was analyzed by Northern blots. 100 nM of 5′PPP or 5′P in vitro -transcribed preSroC was incubated with 100 nM of purified RNase E (1–529) at 30°C for the indicated time in the presence (lanes 10–17) or absence (lanes 2–9) of 100 nM Hfq. The same amount of preSroC was loaded in lane 1 as a control. SroC transcripts were detected using 5′-end-labeled oligonucleotide JVO-2907. Salmonella Δ gltIJKL rne + (JVS-9257) and Δ gltIJKL rneR169K (JVS-11001) strains transformed with pBAD- gltI or pBAD-SroC were grown to OD 600 of 0.5 (0 min) at 37°C and was further incubated for 10 min in the presence of 0.2% l -arabinose. The asterisk indicates transcriptional read-through to the rrnB terminator located downstream on the plasmid. Total RNA was prepared from the Salmonella strains and subjected to Northern blot analysis. 9S rRNA accumulated in the rneR169K strain. Estimated size from pUC8 marker is indicated on the right. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

    doi: 10.15252/embj.201490546

    Figure Lengend Snippet: RNase E mediates SroC processing and GcvB degradation in distinct pathways Salmonella Δ gltIJKL rne + (JVS-9257) and Δ gltIJKL rne3071 (JVS-9258) strains transformed with pBAD- gltI or pBAD-SroC were grown to OD 600 of 0.3 at 28°C and further incubated at 44°C for 30 min (OD 600 of ˜0.5, indicated as time point 0). SroC expression was then induced for 10 min in the presence of 0.2% of l -arabinose. Total RNA was analyzed by Northern blots. 100 nM of 5′PPP or 5′P in vitro -transcribed preSroC was incubated with 100 nM of purified RNase E (1–529) at 30°C for the indicated time in the presence (lanes 10–17) or absence (lanes 2–9) of 100 nM Hfq. The same amount of preSroC was loaded in lane 1 as a control. SroC transcripts were detected using 5′-end-labeled oligonucleotide JVO-2907. Salmonella Δ gltIJKL rne + (JVS-9257) and Δ gltIJKL rneR169K (JVS-11001) strains transformed with pBAD- gltI or pBAD-SroC were grown to OD 600 of 0.5 (0 min) at 37°C and was further incubated for 10 min in the presence of 0.2% l -arabinose. The asterisk indicates transcriptional read-through to the rrnB terminator located downstream on the plasmid. Total RNA was prepared from the Salmonella strains and subjected to Northern blot analysis. 9S rRNA accumulated in the rneR169K strain. Estimated size from pUC8 marker is indicated on the right. Source data are available online for this figure.

    Article Snippet: At optical density (OD600 ) of 1.5, l -arabinose was added to cultures at a final concentration of 0.2% to induced SroC expression for 10 min, and total RNA was prepared with SV total RNA isolation system (Promega).

    Techniques: Transformation Assay, Incubation, Expressing, Northern Blot, In Vitro, Purification, Labeling, Plasmid Preparation, Marker

    Posttranscriptional regulation of GcvB by SroC Salmonella Δ sroC (JVS-5821), Δ gcvB Δ sroC (JVS-5822), and Δ sroC Δ hfq (JVS-9031) strains were transformed with pBAD-ctrl (pKP8-35) or pBAD-SroC (pYC6-4). Δ gcvB Δ sroC was cotransformed with pP L -GcvB (pMM03). Each strain was grown to OD 600 of 0.5 (0 min) and was further incubated for 10 min in the presence of 0.2% l -arabinose. Salmonella WT (JVS-1574), Δ gltIJKL (JVS-5823), ΔCA (JVS-10741), and Δ sroC (JVS-5821) strains were grown to OD 600 of 2.0 prior to the addition of rifampicin. Total RNA was analyzed by Northern blot to determine decay rates of GcvB. The half-lives were determined from three independent experiments; the standard deviation is indicated. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

    doi: 10.15252/embj.201490546

    Figure Lengend Snippet: Posttranscriptional regulation of GcvB by SroC Salmonella Δ sroC (JVS-5821), Δ gcvB Δ sroC (JVS-5822), and Δ sroC Δ hfq (JVS-9031) strains were transformed with pBAD-ctrl (pKP8-35) or pBAD-SroC (pYC6-4). Δ gcvB Δ sroC was cotransformed with pP L -GcvB (pMM03). Each strain was grown to OD 600 of 0.5 (0 min) and was further incubated for 10 min in the presence of 0.2% l -arabinose. Salmonella WT (JVS-1574), Δ gltIJKL (JVS-5823), ΔCA (JVS-10741), and Δ sroC (JVS-5821) strains were grown to OD 600 of 2.0 prior to the addition of rifampicin. Total RNA was analyzed by Northern blot to determine decay rates of GcvB. The half-lives were determined from three independent experiments; the standard deviation is indicated. Source data are available online for this figure.

    Article Snippet: At optical density (OD600 ) of 1.5, l -arabinose was added to cultures at a final concentration of 0.2% to induced SroC expression for 10 min, and total RNA was prepared with SV total RNA isolation system (Promega).

    Techniques: Transformation Assay, Incubation, Northern Blot, Standard Deviation

    SroC is the effector molecule for GcvB repression Expression changes of OppA and GcvB upon deletion of gltIJKL locus. WT (JVS-1574), Δ gltIJKL (JVS-5823), Δ gltJKL (JVS-10795), Δ sroC (JVS-5821), Δ gcvB Δ sroC (JVS-5822), and Δ gcvB (JVS-0236) strains were grown to early stationary phase (OD 600 of 2.0) in LB medium. Expression changes of OppA and GcvB by overexpression of gltI - sroC . Δ gltIJKL strain (JVS-5823) harboring pBAD-ctrl (lane 1), pBAD- gltI (pMM36) derivatives (lanes 2-5), or pBAD-SroC (lane 6) was grown to early stationary phase (OD 600 of 2.0) in LB medium supplemented with 0.02% l -arabinose. Data information: Upper two panels: Total protein was analyzed by Western blot to quantify OppA expression. GroEL served as a loading control. Lower three panels: Total RNA was analyzed by Northern blot, and expression of GcvB and SroC was monitored. 5S rRNA served as a loading control. Estimated size from pUC8 marker is indicated on the right. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

    doi: 10.15252/embj.201490546

    Figure Lengend Snippet: SroC is the effector molecule for GcvB repression Expression changes of OppA and GcvB upon deletion of gltIJKL locus. WT (JVS-1574), Δ gltIJKL (JVS-5823), Δ gltJKL (JVS-10795), Δ sroC (JVS-5821), Δ gcvB Δ sroC (JVS-5822), and Δ gcvB (JVS-0236) strains were grown to early stationary phase (OD 600 of 2.0) in LB medium. Expression changes of OppA and GcvB by overexpression of gltI - sroC . Δ gltIJKL strain (JVS-5823) harboring pBAD-ctrl (lane 1), pBAD- gltI (pMM36) derivatives (lanes 2-5), or pBAD-SroC (lane 6) was grown to early stationary phase (OD 600 of 2.0) in LB medium supplemented with 0.02% l -arabinose. Data information: Upper two panels: Total protein was analyzed by Western blot to quantify OppA expression. GroEL served as a loading control. Lower three panels: Total RNA was analyzed by Northern blot, and expression of GcvB and SroC was monitored. 5S rRNA served as a loading control. Estimated size from pUC8 marker is indicated on the right. Source data are available online for this figure.

    Article Snippet: At optical density (OD600 ) of 1.5, l -arabinose was added to cultures at a final concentration of 0.2% to induced SroC expression for 10 min, and total RNA was prepared with SV total RNA isolation system (Promega).

    Techniques: Expressing, Over Expression, Western Blot, Northern Blot, Marker

    Capillary electrophoresis analysis of pig LV biopsy RNA. ( A) Bioanalyzer nano chip traces of all tested samples. Inspection of electropherograms provides additional information to assess RNA quality and protocol performance. (B) Representative traces from each protocol with respective RIN values. Numbers 1–4 correspond to numbered boxes in A. Note that trace 3 has a high RIN despite a large smear in the gel. (C) Summary of RIN values of all the samples tested (mean±SEM). The RNAqueous protocol had undetectable RINs. The mir Vana and miRCURY tissue protocols gave consistently high RIN values, while despite high average RIN, the samples from the miRNeasy micro protocol are more variable. Numbers in brackets indicate the number of samples in each group. (D) Bioanalyzer small RNA chip traces for each kit. Although the RIN of samples marked * were similar (8.6–8.7), the spectrum of small RNAs recovered is highly heterogeneous.

    Journal: PLoS ONE

    Article Title: Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance

    doi: 10.1371/journal.pone.0213685

    Figure Lengend Snippet: Capillary electrophoresis analysis of pig LV biopsy RNA. ( A) Bioanalyzer nano chip traces of all tested samples. Inspection of electropherograms provides additional information to assess RNA quality and protocol performance. (B) Representative traces from each protocol with respective RIN values. Numbers 1–4 correspond to numbered boxes in A. Note that trace 3 has a high RIN despite a large smear in the gel. (C) Summary of RIN values of all the samples tested (mean±SEM). The RNAqueous protocol had undetectable RINs. The mir Vana and miRCURY tissue protocols gave consistently high RIN values, while despite high average RIN, the samples from the miRNeasy micro protocol are more variable. Numbers in brackets indicate the number of samples in each group. (D) Bioanalyzer small RNA chip traces for each kit. Although the RIN of samples marked * were similar (8.6–8.7), the spectrum of small RNAs recovered is highly heterogeneous.

    Article Snippet: The miRNeasy micro protocol gave variable RIN values , while the RNAqueous-micro kit had undetectable RINs in all samples, and inspection of the electropherograms reveals an unexplained shift in mobility for 4/6 samples ( ).

    Techniques: Electrophoresis, Chromatin Immunoprecipitation

    Spectrophotometry analysis of pig LV biopsy RNA. RNA extracted according to each manufacturer’s protocol, with the addition of a proteinase K digestion in half the samples. n = 3 biopsies/experimental condition, mean±SEM. N . B . Exiqon miRCURY tissue protocol requires proteinase K, therefore no–proteinase K condition was done with this kit. (A) RNA yield, assessed as ng RNA recovered per mg input tissue, was not significantly different between the protocols (Kruskal-Wallis test with Dunn correction), and additional proteinase K digestion in the RNAqueous micro, mir Vana and miRNeasy micro protocols did not significantly improve RNA yield (Kruskal-Wallis test with Dunn correction). (B) 260/280 and 260/230 ratios provide an assessment of the purity of the RNA. Ratio

    Journal: PLoS ONE

    Article Title: Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance

    doi: 10.1371/journal.pone.0213685

    Figure Lengend Snippet: Spectrophotometry analysis of pig LV biopsy RNA. RNA extracted according to each manufacturer’s protocol, with the addition of a proteinase K digestion in half the samples. n = 3 biopsies/experimental condition, mean±SEM. N . B . Exiqon miRCURY tissue protocol requires proteinase K, therefore no–proteinase K condition was done with this kit. (A) RNA yield, assessed as ng RNA recovered per mg input tissue, was not significantly different between the protocols (Kruskal-Wallis test with Dunn correction), and additional proteinase K digestion in the RNAqueous micro, mir Vana and miRNeasy micro protocols did not significantly improve RNA yield (Kruskal-Wallis test with Dunn correction). (B) 260/280 and 260/230 ratios provide an assessment of the purity of the RNA. Ratio

    Article Snippet: The miRNeasy micro protocol gave variable RIN values , while the RNAqueous-micro kit had undetectable RINs in all samples, and inspection of the electropherograms reveals an unexplained shift in mobility for 4/6 samples ( ).

    Techniques: Spectrophotometry

    Summary of experimental design for Figs 2 , 3 and 4 . (A) Left ventricular biopsies were taken from three pigs undergoing cardio-pulmonary bypass (CPB). 7 biopsies were taken per pig. RNA was isolated from the biopsies using four commercially available kits; RNAqueous micro, mir Vana, miRCURY tissue, miRNeasy micro. Three kits (RNAqueous micro, mir Vana, miRNeasy micro) were tested with and without an additional proteinase K digestion step, giving 7 protocols in total. The miRCURY tissue protocol includes a proteinase K digestion, therefore this kit was not tested without this. RNA samples were characterised by spectrophotometry and capillary electrophoresis. (B) Blood samples were taken from three pigs undergoing CPB and processed to plasma. RNA was isolated from the plasma using four commercially-available kits; miRNeasy serum/plasma, mir Vana, miRCURY biofluids, Norgen Biotek plasma/serum. Each kit was tested with and without glycogen as a co-precipitant, giving 8 protocols in total. RNA samples were evaluated by RT-qPCR for the exogenous spike-in cel-miR-39-3p, and endogenous miRNAs miR-16-5p, miR-21-5p, miR-92a-3p.

    Journal: PLoS ONE

    Article Title: Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance

    doi: 10.1371/journal.pone.0213685

    Figure Lengend Snippet: Summary of experimental design for Figs 2 , 3 and 4 . (A) Left ventricular biopsies were taken from three pigs undergoing cardio-pulmonary bypass (CPB). 7 biopsies were taken per pig. RNA was isolated from the biopsies using four commercially available kits; RNAqueous micro, mir Vana, miRCURY tissue, miRNeasy micro. Three kits (RNAqueous micro, mir Vana, miRNeasy micro) were tested with and without an additional proteinase K digestion step, giving 7 protocols in total. The miRCURY tissue protocol includes a proteinase K digestion, therefore this kit was not tested without this. RNA samples were characterised by spectrophotometry and capillary electrophoresis. (B) Blood samples were taken from three pigs undergoing CPB and processed to plasma. RNA was isolated from the plasma using four commercially-available kits; miRNeasy serum/plasma, mir Vana, miRCURY biofluids, Norgen Biotek plasma/serum. Each kit was tested with and without glycogen as a co-precipitant, giving 8 protocols in total. RNA samples were evaluated by RT-qPCR for the exogenous spike-in cel-miR-39-3p, and endogenous miRNAs miR-16-5p, miR-21-5p, miR-92a-3p.

    Article Snippet: The miRNeasy micro protocol gave variable RIN values , while the RNAqueous-micro kit had undetectable RINs in all samples, and inspection of the electropherograms reveals an unexplained shift in mobility for 4/6 samples ( ).

    Techniques: Isolation, Spectrophotometry, Electrophoresis, Quantitative RT-PCR

    The UME6 5′ UTR functions to inhibit translational efficiency. The indicated strains were grown overnight in YEPD medium at 30°C and diluted into prewarmed YEPD at 30°C (non-filament-inducing conditions) or YEPD + 10% serum at 37°C (filament-inducing conditions). At 30 minutes following serum and temperature induction, cells were treated with 0.1 mg mL −1 cycloheximide, lysed (in the presence or absence of 25 mM EDTA for cells grown in YEPD + 10% serum at 37°C), and subjected to sucrose gradient centrifugation for polysome isolation. (A) Polysome profiles across sucrose gradients for the indicated strains grown in YEPD at 30°C or YEPD + 10% serum at 37°C (+/− EDTA treatment) are shown; please note that profiles for each strain are offset. (B) RNA was extracted from each fraction of the indicated sucrose gradients to determine the abundance of UME6 transcript by qRT-PCR analysis. Data shown represents normalized mean UME6 transcript levels based on two independent experiments (± SEM). Please note that in the presence of serum at 37°C there is a statistically significant increase in UME6 transcript abundance in UME6 5′ utrΔ/Δ vs. wild-type (WT) strains for the polysome fractions 7,10 and 11 (p ≤ 0.01 as determined by Student’s t test).

    Journal: Molecular microbiology

    Article Title: A 5′ UTR-mediated Translational Efficiency Mechanism Inhibits the Candida albicans Morphological Transition

    doi: 10.1111/mmi.12576

    Figure Lengend Snippet: The UME6 5′ UTR functions to inhibit translational efficiency. The indicated strains were grown overnight in YEPD medium at 30°C and diluted into prewarmed YEPD at 30°C (non-filament-inducing conditions) or YEPD + 10% serum at 37°C (filament-inducing conditions). At 30 minutes following serum and temperature induction, cells were treated with 0.1 mg mL −1 cycloheximide, lysed (in the presence or absence of 25 mM EDTA for cells grown in YEPD + 10% serum at 37°C), and subjected to sucrose gradient centrifugation for polysome isolation. (A) Polysome profiles across sucrose gradients for the indicated strains grown in YEPD at 30°C or YEPD + 10% serum at 37°C (+/− EDTA treatment) are shown; please note that profiles for each strain are offset. (B) RNA was extracted from each fraction of the indicated sucrose gradients to determine the abundance of UME6 transcript by qRT-PCR analysis. Data shown represents normalized mean UME6 transcript levels based on two independent experiments (± SEM). Please note that in the presence of serum at 37°C there is a statistically significant increase in UME6 transcript abundance in UME6 5′ utrΔ/Δ vs. wild-type (WT) strains for the polysome fractions 7,10 and 11 (p ≤ 0.01 as determined by Student’s t test).

    Article Snippet: RNA for qRT-PCR analysis and for 5′ RACE analysis was prepared using the SV Total RNA Isolation Kit (Promega) according to the manufacturer’s directions with the following modification: cells were resuspended in 225 μL buffer RLT and placed in a bead beater for 2.5 minutes (yeast) or 5 minutes (hyphae); cells were rested on ice for 1 minute per every 2.5 minutes in the bead beater.

    Techniques: Gradient Centrifugation, Isolation, Quantitative RT-PCR

    The UME6 5′ UTR is sufficient to inhibit translation of a heterologous GFP reporter. (A) Cells of the indicated strains were grown in YNB minimal medium at 30°C. Fluorescence units were determined by dividing the fluorescence value (485 nm excitation, 535 nm emission) by the optical density (600 nm) of each sample. Data shown represents the average of three biological replicates (mean ± SEM). (B) Cells from part (A) were harvested for RNA isolation and GFP transcript levels were measured by qRT-PCR. Fluorescence units determined in (A) were normalized to the relative GFP expression of each biological replicate. Data shown is for three biological replicates of each strain. Statistical significance was determined by one-way ANOVA analysis. * = p

    Journal: Molecular microbiology

    Article Title: A 5′ UTR-mediated Translational Efficiency Mechanism Inhibits the Candida albicans Morphological Transition

    doi: 10.1111/mmi.12576

    Figure Lengend Snippet: The UME6 5′ UTR is sufficient to inhibit translation of a heterologous GFP reporter. (A) Cells of the indicated strains were grown in YNB minimal medium at 30°C. Fluorescence units were determined by dividing the fluorescence value (485 nm excitation, 535 nm emission) by the optical density (600 nm) of each sample. Data shown represents the average of three biological replicates (mean ± SEM). (B) Cells from part (A) were harvested for RNA isolation and GFP transcript levels were measured by qRT-PCR. Fluorescence units determined in (A) were normalized to the relative GFP expression of each biological replicate. Data shown is for three biological replicates of each strain. Statistical significance was determined by one-way ANOVA analysis. * = p

    Article Snippet: RNA for qRT-PCR analysis and for 5′ RACE analysis was prepared using the SV Total RNA Isolation Kit (Promega) according to the manufacturer’s directions with the following modification: cells were resuspended in 225 μL buffer RLT and placed in a bead beater for 2.5 minutes (yeast) or 5 minutes (hyphae); cells were rested on ice for 1 minute per every 2.5 minutes in the bead beater.

    Techniques: Fluorescence, Isolation, Quantitative RT-PCR, Expressing

    The 5′ UTR inhibits UME6 -driven hyphal growth in the absence of filament-inducing conditions but does not cause a reduction in UME6 transcript levels. (A) The indicated strains were grown overnight in YEPD medium at 30°C in the presence of 1 μg mL −1 Dox, washed twice with ddH 2 O and inoculated into fresh prewarmed YEPD medium at 30°C in the absence of Dox. Cell aliquots were harvested at the indicated time points, fixed with 4.5% formaldehyde, washed twice with 1X PBS and visualized by DIC microscopy. Bar = 10 μm. (B) Total RNA was isolated from cells grown as described in (A) as well as cells inoculated into fresh prewarmed YEPD medium at 30°C in the presence of 1 μg mL −1 Dox as a control. UME6 transcript levels were determined by qRT-PCR analysis and normalized to those of an ACT1 internal control. Fold induction was determined by dividing normalized UME6 expression values for each time point by the normalized UME6 expression value for the zero time point. Data shown represents the average of three biological replicates run in technical duplicate (mean ± SEM).

    Journal: Molecular microbiology

    Article Title: A 5′ UTR-mediated Translational Efficiency Mechanism Inhibits the Candida albicans Morphological Transition

    doi: 10.1111/mmi.12576

    Figure Lengend Snippet: The 5′ UTR inhibits UME6 -driven hyphal growth in the absence of filament-inducing conditions but does not cause a reduction in UME6 transcript levels. (A) The indicated strains were grown overnight in YEPD medium at 30°C in the presence of 1 μg mL −1 Dox, washed twice with ddH 2 O and inoculated into fresh prewarmed YEPD medium at 30°C in the absence of Dox. Cell aliquots were harvested at the indicated time points, fixed with 4.5% formaldehyde, washed twice with 1X PBS and visualized by DIC microscopy. Bar = 10 μm. (B) Total RNA was isolated from cells grown as described in (A) as well as cells inoculated into fresh prewarmed YEPD medium at 30°C in the presence of 1 μg mL −1 Dox as a control. UME6 transcript levels were determined by qRT-PCR analysis and normalized to those of an ACT1 internal control. Fold induction was determined by dividing normalized UME6 expression values for each time point by the normalized UME6 expression value for the zero time point. Data shown represents the average of three biological replicates run in technical duplicate (mean ± SEM).

    Article Snippet: RNA for qRT-PCR analysis and for 5′ RACE analysis was prepared using the SV Total RNA Isolation Kit (Promega) according to the manufacturer’s directions with the following modification: cells were resuspended in 225 μL buffer RLT and placed in a bead beater for 2.5 minutes (yeast) or 5 minutes (hyphae); cells were rested on ice for 1 minute per every 2.5 minutes in the bead beater.

    Techniques: Microscopy, Isolation, Quantitative RT-PCR, Expressing

    Characterization of the UME6 upstream region identifies an exceptionally long 5′ UTR. (A) Schematic representation of the UME6 locus, and surrounding region, based on Candida ). (B) 3 × 10 3 cells of the indicated strains were spotted on solid YEPD + 10% serum medium, grown at 37°C for 3 days and visualized by light microscopy. Images on the right are enlarged to show spot edges. Please note that only a single UME6 allele is shown in the schematic representations; the second allele, along with 19.3 kb of the UME6 upstream intergenic region, has been deleted in all strains except ume6Δ/Δ . (C) C. albicans strains bearing the indicated reporter constructs were grown overnight in YEPD at 30°C and diluted into pre-warmed YEPD + 10% serum at 37°C (filament-inducing conditions) or YEPD only at 30°C (non-filament-inducing conditions). At 30 minutes post-induction, cells were harvested for total RNA preparation and cDNA synthesis. lacZ expression levels were determined by qRT-PCR and normalized to levels of an ACT1 internal control. Fold induction was determined by dividing normalized lacZ values in induced cells by those observed in cells grown under non-filament-inducing conditions. Data shown represents the average of three biological replicates run in technical duplicate (mean ± SEM). (D) Schematic representation of the immediate UME6 upstream intergenic region (note: not drawn exactly to scale). Transcript start sites were determined by 5′ RACE analysis using cDNA prepared from wild-type (SC5314) cells harvested following induction in YEPD + 10% serum at 37°C.

    Journal: Molecular microbiology

    Article Title: A 5′ UTR-mediated Translational Efficiency Mechanism Inhibits the Candida albicans Morphological Transition

    doi: 10.1111/mmi.12576

    Figure Lengend Snippet: Characterization of the UME6 upstream region identifies an exceptionally long 5′ UTR. (A) Schematic representation of the UME6 locus, and surrounding region, based on Candida ). (B) 3 × 10 3 cells of the indicated strains were spotted on solid YEPD + 10% serum medium, grown at 37°C for 3 days and visualized by light microscopy. Images on the right are enlarged to show spot edges. Please note that only a single UME6 allele is shown in the schematic representations; the second allele, along with 19.3 kb of the UME6 upstream intergenic region, has been deleted in all strains except ume6Δ/Δ . (C) C. albicans strains bearing the indicated reporter constructs were grown overnight in YEPD at 30°C and diluted into pre-warmed YEPD + 10% serum at 37°C (filament-inducing conditions) or YEPD only at 30°C (non-filament-inducing conditions). At 30 minutes post-induction, cells were harvested for total RNA preparation and cDNA synthesis. lacZ expression levels were determined by qRT-PCR and normalized to levels of an ACT1 internal control. Fold induction was determined by dividing normalized lacZ values in induced cells by those observed in cells grown under non-filament-inducing conditions. Data shown represents the average of three biological replicates run in technical duplicate (mean ± SEM). (D) Schematic representation of the immediate UME6 upstream intergenic region (note: not drawn exactly to scale). Transcript start sites were determined by 5′ RACE analysis using cDNA prepared from wild-type (SC5314) cells harvested following induction in YEPD + 10% serum at 37°C.

    Article Snippet: RNA for qRT-PCR analysis and for 5′ RACE analysis was prepared using the SV Total RNA Isolation Kit (Promega) according to the manufacturer’s directions with the following modification: cells were resuspended in 225 μL buffer RLT and placed in a bead beater for 2.5 minutes (yeast) or 5 minutes (hyphae); cells were rested on ice for 1 minute per every 2.5 minutes in the bead beater.

    Techniques: Light Microscopy, Construct, Expressing, Quantitative RT-PCR

    Transcriptional analysis of the bleomycin transcript in WE8. Total RNA was isolated from the deletion strain WE8 (Fig ), and cDNA was produced using a gene-specific primer (primer 15). After cDNA synthesis, PCR was performed using the

    Journal: Journal of Bacteriology

    Article Title: A Novel Heme a Insertion Factor Gene Cotranscribes with the Thermus thermophilus Cytochrome ba3 Oxidase Locus ▿

    doi: 10.1128/JB.00548-10

    Figure Lengend Snippet: Transcriptional analysis of the bleomycin transcript in WE8. Total RNA was isolated from the deletion strain WE8 (Fig ), and cDNA was produced using a gene-specific primer (primer 15). After cDNA synthesis, PCR was performed using the

    Article Snippet: Total RNAs from different Thermus strains were isolated using the SV total RNA isolation system kit (Promega, Germany), and cDNA synthesis was performed with a RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas) using primer 15, each according to the manuals.

    Techniques: Isolation, Produced, Polymerase Chain Reaction

    Transcriptional analysis of cbaX . Total RNA was isolated from T. thermophilus HB27, and cDNA was produced using the gene-specific primer 15. After cDNA synthesis, PCR was performed using the primer pair 14 and 15 on different templates. Lane 1, cDNA as

    Journal: Journal of Bacteriology

    Article Title: A Novel Heme a Insertion Factor Gene Cotranscribes with the Thermus thermophilus Cytochrome ba3 Oxidase Locus ▿

    doi: 10.1128/JB.00548-10

    Figure Lengend Snippet: Transcriptional analysis of cbaX . Total RNA was isolated from T. thermophilus HB27, and cDNA was produced using the gene-specific primer 15. After cDNA synthesis, PCR was performed using the primer pair 14 and 15 on different templates. Lane 1, cDNA as

    Article Snippet: Total RNAs from different Thermus strains were isolated using the SV total RNA isolation system kit (Promega, Germany), and cDNA synthesis was performed with a RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas) using primer 15, each according to the manuals.

    Techniques: Isolation, Produced, Polymerase Chain Reaction

    Dual exon skipping of myostatin and dystrophin in DL589.2 DMD patient cells . DL589.2 myotubes were transfected with 200 nM of myostatin AON and h50AON1 DMD AON. RNA was isolated two days post-transfection and analyzed for myostatin and dystrophin skips by RT-PCR.

    Journal: BMC Medical Genomics

    Article Title: Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy

    doi: 10.1186/1755-8794-4-36

    Figure Lengend Snippet: Dual exon skipping of myostatin and dystrophin in DL589.2 DMD patient cells . DL589.2 myotubes were transfected with 200 nM of myostatin AON and h50AON1 DMD AON. RNA was isolated two days post-transfection and analyzed for myostatin and dystrophin skips by RT-PCR.

    Article Snippet: RNA Isolation, RT-PCR and Quantitative PCR analysis Cell lysates were prepared using lysis buffer provided in the NucleoSpin RNA II kit (Macherey-Nagel, Germany ) and RNA was isolated using the same kit according to manufacturer's instructions.

    Techniques: Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction

    Myostatin exon 2 skip in several myotubes cultures . Human primary control (KM109) and DMD patient derived- (DL589.2) myoblasts were differentiated for 7 days before transfection with MSTN AON. Immortalized control (7304.1) myoblasts were differentiated for 2-3 days. A non-targeting, fluorescently-labeled AONs were transfected as control. Fluorescent nuclei were observed three hours post-transfection (A). RNA was isolated 2 days post-transfection. cDNA was synthesized using random hexamer (N6) primers and subjected for PCR using primers in exon 1 and 3 (B). Note the inverse dose-dependent skips in KM109 samples. Skip fragment was confirmed by sequencing analysis (C). Quantitative real-time PCR was performed using primers in MSTN exon 1 and 2, thereby depicting the expression of remaining full length or non-skipped transcript (D). Data are means ± SD from 3 to 4 independent experiments. Expression was normalized with GAPDH. Statistical analysis was performed using Student's t-test, using the 500 nM control AON-transfected samples as reference. *P

    Journal: BMC Medical Genomics

    Article Title: Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy

    doi: 10.1186/1755-8794-4-36

    Figure Lengend Snippet: Myostatin exon 2 skip in several myotubes cultures . Human primary control (KM109) and DMD patient derived- (DL589.2) myoblasts were differentiated for 7 days before transfection with MSTN AON. Immortalized control (7304.1) myoblasts were differentiated for 2-3 days. A non-targeting, fluorescently-labeled AONs were transfected as control. Fluorescent nuclei were observed three hours post-transfection (A). RNA was isolated 2 days post-transfection. cDNA was synthesized using random hexamer (N6) primers and subjected for PCR using primers in exon 1 and 3 (B). Note the inverse dose-dependent skips in KM109 samples. Skip fragment was confirmed by sequencing analysis (C). Quantitative real-time PCR was performed using primers in MSTN exon 1 and 2, thereby depicting the expression of remaining full length or non-skipped transcript (D). Data are means ± SD from 3 to 4 independent experiments. Expression was normalized with GAPDH. Statistical analysis was performed using Student's t-test, using the 500 nM control AON-transfected samples as reference. *P

    Article Snippet: RNA Isolation, RT-PCR and Quantitative PCR analysis Cell lysates were prepared using lysis buffer provided in the NucleoSpin RNA II kit (Macherey-Nagel, Germany ) and RNA was isolated using the same kit according to manufacturer's instructions.

    Techniques: Derivative Assay, Transfection, Labeling, Isolation, Synthesized, Random Hexamer Labeling, Polymerase Chain Reaction, Sequencing, Real-time Polymerase Chain Reaction, Expressing

    The expression myogenic regulatory factors MYOG and MYF5 upon exon 2 skipping . Cells were fused and transfected with different concentrations of AON as described in Figure 2. Total RNA was isolated and N6-primed cDNA was subjected to quantitative real-time PCR for MYOG (A) and MYF5 (B). Data are means ± SD from 3 to 4 independent experiments. Expression was normalized with GAPDH. Statistical analysis was performed using Student's t-test, using the 500 nM control AON-transfected samples as reference. *P

    Journal: BMC Medical Genomics

    Article Title: Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy

    doi: 10.1186/1755-8794-4-36

    Figure Lengend Snippet: The expression myogenic regulatory factors MYOG and MYF5 upon exon 2 skipping . Cells were fused and transfected with different concentrations of AON as described in Figure 2. Total RNA was isolated and N6-primed cDNA was subjected to quantitative real-time PCR for MYOG (A) and MYF5 (B). Data are means ± SD from 3 to 4 independent experiments. Expression was normalized with GAPDH. Statistical analysis was performed using Student's t-test, using the 500 nM control AON-transfected samples as reference. *P

    Article Snippet: RNA Isolation, RT-PCR and Quantitative PCR analysis Cell lysates were prepared using lysis buffer provided in the NucleoSpin RNA II kit (Macherey-Nagel, Germany ) and RNA was isolated using the same kit according to manufacturer's instructions.

    Techniques: Expressing, Transfection, Isolation, Real-time Polymerase Chain Reaction

    Dual exon skipping of myostatin and dystrophin in control cells . KM109 (A) and 7304-1 (B) myotubes were transfected with 200 nM of myostatin AON and AON targeting different DMD exons, namely exon 8, 44 and 54. The AONs were premixed (boxed) before complexing with the transfection reagent, or directly complexed (not boxed). RNA was isolated two days post-transfection and analyzed for myostatin or dystrophin skips by RT-PCR.

    Journal: BMC Medical Genomics

    Article Title: Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy

    doi: 10.1186/1755-8794-4-36

    Figure Lengend Snippet: Dual exon skipping of myostatin and dystrophin in control cells . KM109 (A) and 7304-1 (B) myotubes were transfected with 200 nM of myostatin AON and AON targeting different DMD exons, namely exon 8, 44 and 54. The AONs were premixed (boxed) before complexing with the transfection reagent, or directly complexed (not boxed). RNA was isolated two days post-transfection and analyzed for myostatin or dystrophin skips by RT-PCR.

    Article Snippet: RNA Isolation, RT-PCR and Quantitative PCR analysis Cell lysates were prepared using lysis buffer provided in the NucleoSpin RNA II kit (Macherey-Nagel, Germany ) and RNA was isolated using the same kit according to manufacturer's instructions.

    Techniques: Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction

    Administration of myostatin AON in mdx mice . Single dose of 40 μg MSTN AON was injected into the gastrocnemius muscles of mdx mice. Control M23 DMD AON were injected in the contralateral muscles. The animals were sacrificed 4 days after injection. RNA was isolated from three different parts of the muscles relative to the tendon: proximal (P), medial (M) and distal (E). RT-PCR analysis was performed to detect dystrophin (upper) or myostatin (lower) skips (A). Four times consecutive injections were performed with cocktails of AON containing 40 μg of m23 DMD AON and 40 μg of either myostatin or control AON into the gastrocnemius muscles of mdx mice. Injections were varied between contralateral muscles. The mice were sacrificed at 6 hours, 1 day and 2 days after the last injection. RNA was isolated from multiple areas within the muscles and analyzed for dystrophin (upper) or myostatin (lower) skips by RT-PCR (B).

    Journal: BMC Medical Genomics

    Article Title: Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy

    doi: 10.1186/1755-8794-4-36

    Figure Lengend Snippet: Administration of myostatin AON in mdx mice . Single dose of 40 μg MSTN AON was injected into the gastrocnemius muscles of mdx mice. Control M23 DMD AON were injected in the contralateral muscles. The animals were sacrificed 4 days after injection. RNA was isolated from three different parts of the muscles relative to the tendon: proximal (P), medial (M) and distal (E). RT-PCR analysis was performed to detect dystrophin (upper) or myostatin (lower) skips (A). Four times consecutive injections were performed with cocktails of AON containing 40 μg of m23 DMD AON and 40 μg of either myostatin or control AON into the gastrocnemius muscles of mdx mice. Injections were varied between contralateral muscles. The mice were sacrificed at 6 hours, 1 day and 2 days after the last injection. RNA was isolated from multiple areas within the muscles and analyzed for dystrophin (upper) or myostatin (lower) skips by RT-PCR (B).

    Article Snippet: RNA Isolation, RT-PCR and Quantitative PCR analysis Cell lysates were prepared using lysis buffer provided in the NucleoSpin RNA II kit (Macherey-Nagel, Germany ) and RNA was isolated using the same kit according to manufacturer's instructions.

    Techniques: Mouse Assay, Injection, Isolation, Reverse Transcription Polymerase Chain Reaction

    Global cDNA read count distribution and Hfq levels in Y. pseudotuberculosis during growth under environmental and infection-relevant conditions. (A) Percentage of uniquely mapped reads of the virulence plasmid pYV obtained by RNA-seq of Y . pseudotuberculosis YPIII and YPIIIΔ crp cDNA libraries. Libraries were generated from TAP-treated (+TAP) rRNA depleted RNA obtained from cultures grown to exponential (E) or stationary (S) growth phase at 25°C (25) and 37°C (37) (for detailed statistics see S1 Dataset ). The data represent the mean ± SEM from three independent biological replicates and were analyzed with Student’s t-test. **: P

    Journal: PLoS Genetics

    Article Title: Transcriptomic Profiling of Yersinia pseudotuberculosis Reveals Reprogramming of the Crp Regulon by Temperature and Uncovers Crp as a Master Regulator of Small RNAs

    doi: 10.1371/journal.pgen.1005087

    Figure Lengend Snippet: Global cDNA read count distribution and Hfq levels in Y. pseudotuberculosis during growth under environmental and infection-relevant conditions. (A) Percentage of uniquely mapped reads of the virulence plasmid pYV obtained by RNA-seq of Y . pseudotuberculosis YPIII and YPIIIΔ crp cDNA libraries. Libraries were generated from TAP-treated (+TAP) rRNA depleted RNA obtained from cultures grown to exponential (E) or stationary (S) growth phase at 25°C (25) and 37°C (37) (for detailed statistics see S1 Dataset ). The data represent the mean ± SEM from three independent biological replicates and were analyzed with Student’s t-test. **: P

    Article Snippet: RNA isolation, rRNA depletion, fragmentation, RNA adapter ligation and strand-specific library preparation for Illumina sequencing is illustrated.

    Techniques: Infection, Plasmid Preparation, RNA Sequencing Assay, Generated

    CRP-dependent non-coding RNAs of Y. pseudotuberculosis . (A) Northern blot analyses of selected CRP-regulated non-coding RNAs. RNA samples were prepared from YPIII and YP89 (YPIII Δ crp ) bacteria grown to stationary growth phase (stat) at 25°C and 37°C. 5S rRNA served as loading control. The size marker (nt) is indicated. (B) Interaction of CRP with the regulatory regions of selected CRP-regulated sRNA genes. Individual DNA fragments with the predicted CRP-binding site(s) (yellow boxes; S3 Dataset ) used for electrophoretic mobility shift assays are illustrated. An individual sRNA promoter fragment ( ysr204 ) for which no CRP-binding site was predicted was included as negative control. Respective DNA fragments were incubated with increasing concentrations of CRP and 0.2 mM cAMP. As a negative control, cAMP was omitted in samples with the highest CRP concentration (right lane). The CRP-DNA complexes were separated on 4% polyacrylamide gels. The position of specific higher molecular weight complexes is marked with an asterisk. A molecular weight standard (M) was loaded, and the corresponding molecular weights are indicated. A csiD PCR fragment amplified from E . coli served as an internal negative control.

    Journal: PLoS Genetics

    Article Title: Transcriptomic Profiling of Yersinia pseudotuberculosis Reveals Reprogramming of the Crp Regulon by Temperature and Uncovers Crp as a Master Regulator of Small RNAs

    doi: 10.1371/journal.pgen.1005087

    Figure Lengend Snippet: CRP-dependent non-coding RNAs of Y. pseudotuberculosis . (A) Northern blot analyses of selected CRP-regulated non-coding RNAs. RNA samples were prepared from YPIII and YP89 (YPIII Δ crp ) bacteria grown to stationary growth phase (stat) at 25°C and 37°C. 5S rRNA served as loading control. The size marker (nt) is indicated. (B) Interaction of CRP with the regulatory regions of selected CRP-regulated sRNA genes. Individual DNA fragments with the predicted CRP-binding site(s) (yellow boxes; S3 Dataset ) used for electrophoretic mobility shift assays are illustrated. An individual sRNA promoter fragment ( ysr204 ) for which no CRP-binding site was predicted was included as negative control. Respective DNA fragments were incubated with increasing concentrations of CRP and 0.2 mM cAMP. As a negative control, cAMP was omitted in samples with the highest CRP concentration (right lane). The CRP-DNA complexes were separated on 4% polyacrylamide gels. The position of specific higher molecular weight complexes is marked with an asterisk. A molecular weight standard (M) was loaded, and the corresponding molecular weights are indicated. A csiD PCR fragment amplified from E . coli served as an internal negative control.

    Article Snippet: RNA isolation, rRNA depletion, fragmentation, RNA adapter ligation and strand-specific library preparation for Illumina sequencing is illustrated.

    Techniques: Northern Blot, Marker, Binding Assay, Electrophoretic Mobility Shift Assay, Negative Control, Incubation, Concentration Assay, Molecular Weight, Polymerase Chain Reaction, Amplification

    Temperature- and growth phase-responsive protein-encoding genes and trans -encoded RNAs. (A) Differential expression of trans -encoded sRNAs of YPIII shown by Northern blot analyses. RNA samples were prepared from bacteria grown to exponential (exp) or stationary phase (stat) at 25°C and 37°C. sRNAs were detected by specific radioactive labeled probes. 5S rRNA served as loading control. The size marker (nt) is indicated. Venn diagrams illustrating temperature and growth phase-regulated (B) protein-encoding genes and (C) trans -encoded sRNAs in Y . pseudotuberculosis YPIII. The regulons were obtained by comparative RNA-seq using DESeq from triplicate experiments ( S3 and S4 Datasets). Protein- and trans -encoded RNAs which are differentially regulated by at least 4-fold (p-value ≤0.05) are included.

    Journal: PLoS Genetics

    Article Title: Transcriptomic Profiling of Yersinia pseudotuberculosis Reveals Reprogramming of the Crp Regulon by Temperature and Uncovers Crp as a Master Regulator of Small RNAs

    doi: 10.1371/journal.pgen.1005087

    Figure Lengend Snippet: Temperature- and growth phase-responsive protein-encoding genes and trans -encoded RNAs. (A) Differential expression of trans -encoded sRNAs of YPIII shown by Northern blot analyses. RNA samples were prepared from bacteria grown to exponential (exp) or stationary phase (stat) at 25°C and 37°C. sRNAs were detected by specific radioactive labeled probes. 5S rRNA served as loading control. The size marker (nt) is indicated. Venn diagrams illustrating temperature and growth phase-regulated (B) protein-encoding genes and (C) trans -encoded sRNAs in Y . pseudotuberculosis YPIII. The regulons were obtained by comparative RNA-seq using DESeq from triplicate experiments ( S3 and S4 Datasets). Protein- and trans -encoded RNAs which are differentially regulated by at least 4-fold (p-value ≤0.05) are included.

    Article Snippet: RNA isolation, rRNA depletion, fragmentation, RNA adapter ligation and strand-specific library preparation for Illumina sequencing is illustrated.

    Techniques: Expressing, Northern Blot, Labeling, Marker, RNA Sequencing Assay, Significance Assay

    Silencing of NbCUL1 enhances CLCuMuV DNA accumulation and results in typical disease symptoms. (A1 and A2) Six- to seven-week-old N . benthamiana plants were agroinoculated with CLCuMuV (CA) and βM2- CUL1 F1 (A1), βM2- CUL1 F2 (A2) or βM2- βC1 F (as the control). (B1 and B2) Silencing of NbCUL1 enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1 and C2) Real-time RT-PCR confirmed silencing of NbCUL1 . Total RNA was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify NbCUL1 mRNA level. Actin was used as the internal reference. The raw data of (B1 and B2) and (C1 and C2) were analysed by two-sample t -test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1 and D2) 100% plants infected with CA+βM2- CUL1 F1 (D1) or CA+βM2- CUL1 F2 (D2) show severe symptoms at 21 dpi.

    Journal: PLoS Pathogens

    Article Title: CLCuMuB βC1 Subverts Ubiquitination by Interacting with NbSKP1s to Enhance Geminivirus Infection in Nicotiana benthamiana

    doi: 10.1371/journal.ppat.1005668

    Figure Lengend Snippet: Silencing of NbCUL1 enhances CLCuMuV DNA accumulation and results in typical disease symptoms. (A1 and A2) Six- to seven-week-old N . benthamiana plants were agroinoculated with CLCuMuV (CA) and βM2- CUL1 F1 (A1), βM2- CUL1 F2 (A2) or βM2- βC1 F (as the control). (B1 and B2) Silencing of NbCUL1 enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1 and C2) Real-time RT-PCR confirmed silencing of NbCUL1 . Total RNA was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify NbCUL1 mRNA level. Actin was used as the internal reference. The raw data of (B1 and B2) and (C1 and C2) were analysed by two-sample t -test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1 and D2) 100% plants infected with CA+βM2- CUL1 F1 (D1) or CA+βM2- CUL1 F2 (D2) show severe symptoms at 21 dpi.

    Article Snippet: DNA and RNA Isolation and Real-Time PCR or RT-PCR Analysis Total DNA was extracted from apical developing leaves using the DNAsecure Plant Kit (TIANGEN, China).

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

    CLCuMuB βC1 hinders the degradation of YFP-GAI in vivo . (A) CLCuMuB βC1 attenued degradation of YFP-GAI in vivo . YFP-GAI expression construct was coinfiltrated with constructs expressing HA-nLUC or HA-βC1 into seven to eight-week-old N . benthamiana plant leaves. Around 48 hpi, agroinfiltrated leaves were sprayed with 100 μM GA 3 or mock solution (ethonal) and visualized via a Zeiss LSM 710 laser scanning microscope. Bar scales represents 200 μm. DMSO and MG132 (50 μM) were applied into plant leaves 12 h before observation. Protein level was analyzed via SDS-PAGE and western blot analysis with the anti-GFP antibody, which also recognizes YFP. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as a loading control. (B) Real-time RT-PCR detected the mRNA level of YFP-GAI. Total RNA was extracted from each N . benthamiana leaves and then subjected to quantitative RT-PCR (means±SEM, n = 3) to quantify YFP-GAI mRNA level. eIF4a was used as the internal reference. (C) CLCuMuB βC1 didn’t affect stability of GFP in vivo . Detection of GFP (as an internal control) in N . benthamiana leaves coinfiltrated with the construct expressing GFP together with constructs expressing HA-nLUC or HA-βC1 and treated with 100 μM GA 3 or mock (ethanol) solution and visualized via a Zeiss LSM 710 laser scanning microscope. Bar scale represents 200 μm. Protein level was analyzed via SDS-PAGE and immunoblot analysis with anti-GFP. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as a loading control.

    Journal: PLoS Pathogens

    Article Title: CLCuMuB βC1 Subverts Ubiquitination by Interacting with NbSKP1s to Enhance Geminivirus Infection in Nicotiana benthamiana

    doi: 10.1371/journal.ppat.1005668

    Figure Lengend Snippet: CLCuMuB βC1 hinders the degradation of YFP-GAI in vivo . (A) CLCuMuB βC1 attenued degradation of YFP-GAI in vivo . YFP-GAI expression construct was coinfiltrated with constructs expressing HA-nLUC or HA-βC1 into seven to eight-week-old N . benthamiana plant leaves. Around 48 hpi, agroinfiltrated leaves were sprayed with 100 μM GA 3 or mock solution (ethonal) and visualized via a Zeiss LSM 710 laser scanning microscope. Bar scales represents 200 μm. DMSO and MG132 (50 μM) were applied into plant leaves 12 h before observation. Protein level was analyzed via SDS-PAGE and western blot analysis with the anti-GFP antibody, which also recognizes YFP. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as a loading control. (B) Real-time RT-PCR detected the mRNA level of YFP-GAI. Total RNA was extracted from each N . benthamiana leaves and then subjected to quantitative RT-PCR (means±SEM, n = 3) to quantify YFP-GAI mRNA level. eIF4a was used as the internal reference. (C) CLCuMuB βC1 didn’t affect stability of GFP in vivo . Detection of GFP (as an internal control) in N . benthamiana leaves coinfiltrated with the construct expressing GFP together with constructs expressing HA-nLUC or HA-βC1 and treated with 100 μM GA 3 or mock (ethanol) solution and visualized via a Zeiss LSM 710 laser scanning microscope. Bar scale represents 200 μm. Protein level was analyzed via SDS-PAGE and immunoblot analysis with anti-GFP. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as a loading control.

    Article Snippet: DNA and RNA Isolation and Real-Time PCR or RT-PCR Analysis Total DNA was extracted from apical developing leaves using the DNAsecure Plant Kit (TIANGEN, China).

    Techniques: In Vivo, Expressing, Construct, Laser-Scanning Microscopy, SDS Page, Western Blot, Staining, Quantitative RT-PCR

    Silencing of NbSKP1s enhances CLCuMuV DNA accumulation and results in typical disease symptoms. (A1, A2 and A3) Six- to seven-week-old N . benthamiana plants were agroinoculated with CLCuMuV (CA) and βM2- SKP1 F1 (A1), βM2- SKP1 F2 (A2), βM2- SKP1 F3 (A3) or βM2- βC1 F (as the control). (B1, B2 and B3) Silencing of NbSKP1s enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1, C2 and C3) Real-time RT-PCR confirmed silencing of NbSKP1s . Total RNA was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify NbSKP1s mRNA level. Actin was used as the internal reference. The raw data of (B1–B3) and (C1–C3) were analysed by two-sample t -test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1, D2 and D3) 50% plants infected with CA+βM2- SKP1 F1 (D1), 50% plants infected with CA+βM2- SKP1 F2 (D2) and 100% plants infected with CA+βM2- SKP1 F3 (D3) show severe symptoms at 21 dpi. (E1, E2, E3 and E4) Apical leaves of plants infected with CA+βM2- βC1 F (E1), CA+βM2- SKP1 F1(E2), CA+βM2- SKP1 F2 (E3) and CA+βM2- SKP1 F3 (E4) at 21 dpi.

    Journal: PLoS Pathogens

    Article Title: CLCuMuB βC1 Subverts Ubiquitination by Interacting with NbSKP1s to Enhance Geminivirus Infection in Nicotiana benthamiana

    doi: 10.1371/journal.ppat.1005668

    Figure Lengend Snippet: Silencing of NbSKP1s enhances CLCuMuV DNA accumulation and results in typical disease symptoms. (A1, A2 and A3) Six- to seven-week-old N . benthamiana plants were agroinoculated with CLCuMuV (CA) and βM2- SKP1 F1 (A1), βM2- SKP1 F2 (A2), βM2- SKP1 F3 (A3) or βM2- βC1 F (as the control). (B1, B2 and B3) Silencing of NbSKP1s enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1, C2 and C3) Real-time RT-PCR confirmed silencing of NbSKP1s . Total RNA was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify NbSKP1s mRNA level. Actin was used as the internal reference. The raw data of (B1–B3) and (C1–C3) were analysed by two-sample t -test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1, D2 and D3) 50% plants infected with CA+βM2- SKP1 F1 (D1), 50% plants infected with CA+βM2- SKP1 F2 (D2) and 100% plants infected with CA+βM2- SKP1 F3 (D3) show severe symptoms at 21 dpi. (E1, E2, E3 and E4) Apical leaves of plants infected with CA+βM2- βC1 F (E1), CA+βM2- SKP1 F1(E2), CA+βM2- SKP1 F2 (E3) and CA+βM2- SKP1 F3 (E4) at 21 dpi.

    Article Snippet: DNA and RNA Isolation and Real-Time PCR or RT-PCR Analysis Total DNA was extracted from apical developing leaves using the DNAsecure Plant Kit (TIANGEN, China).

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

    Association of nELAVs with the APP pre-mRNA. ( A ) Localization of primers used for the detection of APP/App pre-mRNAs ( arrows ; APPI/AppI : Forward I and Reverse I; APPII/AppII : Forward II and Reverse II). Boxes indicate exons and bold lines introns. ( B – D ) Nuclear extracts prepared from human SK-N-SH ( B ), human SH-SY5Y ( C ) and mouse Neuro-2a ( D ) cells were immunoprecipitated with a mouse anti-ELAVL antibody or mouse IgGs as a control. RNA was isolated from immunoprecipitates, as well as their supernatants and analyzed by semi-quantitative and quantitative RT-PCR, respectively, using specific primers against human or mouse APP pre-mRNA ( arrows ) and intronic GAPDH. Minus RT lanes are included as controls. Note that APP pre-mRNA was detectable in the immunoprecipitate only when lysates from cells expressing nELAVLs were used. Bars in graphs depict mean ± standard deviation of three independent experiments (*P

    Journal: Scientific Reports

    Article Title: Neuronal ELAVL proteins utilize AUF-1 as a co-partner to induce neuron-specific alternative splicing of APP

    doi: 10.1038/srep44507

    Figure Lengend Snippet: Association of nELAVs with the APP pre-mRNA. ( A ) Localization of primers used for the detection of APP/App pre-mRNAs ( arrows ; APPI/AppI : Forward I and Reverse I; APPII/AppII : Forward II and Reverse II). Boxes indicate exons and bold lines introns. ( B – D ) Nuclear extracts prepared from human SK-N-SH ( B ), human SH-SY5Y ( C ) and mouse Neuro-2a ( D ) cells were immunoprecipitated with a mouse anti-ELAVL antibody or mouse IgGs as a control. RNA was isolated from immunoprecipitates, as well as their supernatants and analyzed by semi-quantitative and quantitative RT-PCR, respectively, using specific primers against human or mouse APP pre-mRNA ( arrows ) and intronic GAPDH. Minus RT lanes are included as controls. Note that APP pre-mRNA was detectable in the immunoprecipitate only when lysates from cells expressing nELAVLs were used. Bars in graphs depict mean ± standard deviation of three independent experiments (*P

    Article Snippet: RNA isolation and RT-PCR Total RNA was extracted from cells using the RNAzol Reagent (Molecular Research Center, Inc) according to the manufacturer’s protocol, or from RIP material (see below) by acidic phenol/chlorophorm extraction.

    Techniques: Immunoprecipitation, Isolation, Quantitative RT-PCR, Expressing, Standard Deviation

    AUF-1 facilitates ELAVL4-mediated APP695-specific AS. Biotinylated RNA probes transcribed from human ( A ) and mouse ( B ) APP minigenes were tested for AUF-1 binding. Note, that AUF-1 isoforms p42 and/or p45 bind to all transcripts shown to interact with nELAVLs. ( C ) SK-N-SH cells were transfected with the DNA3.1 expression vector bearing either no insert (empty) or p42 AUF-1, or with the pENTR/U6 vector carrying a shRNA specific for LacZ or all AUF - 1 mRNAs. Inclusion of APP exons 7 and 8 was assayed by RT-qPCR with primers specific for APP770, APP695 and APP751 . Total APP cDNA was used for normalization. Bars in graphs correspond to mean ± standard deviation of three independent experiments. ( D , E ) SK-N-SH cells were co-transfected with the human ( D ) or the mouse ( E ) APPE78 minigene along with two expression vectors, one bearing ELAVL4 and the other one carrying no insert (empty) or p42 AUF-1, in ratios depicted. Splicing pathways were determined by RT-PCR, as described in Fig. 5 . Quantification of the results was performed by scanning densitometry. Note, that AUF-1 enhances ELAVL4-mediated exclusion of APP exons 7 and 8. ( F ) Neuro2a cells were transfected with the pSilencer vector bearing either no insert (empty) or a shRNA targeting all Auf - 1 transcripts. Inclusion of APP exons 7 and 8 was assayed by RT-qPCR, as described above (**P

    Journal: Scientific Reports

    Article Title: Neuronal ELAVL proteins utilize AUF-1 as a co-partner to induce neuron-specific alternative splicing of APP

    doi: 10.1038/srep44507

    Figure Lengend Snippet: AUF-1 facilitates ELAVL4-mediated APP695-specific AS. Biotinylated RNA probes transcribed from human ( A ) and mouse ( B ) APP minigenes were tested for AUF-1 binding. Note, that AUF-1 isoforms p42 and/or p45 bind to all transcripts shown to interact with nELAVLs. ( C ) SK-N-SH cells were transfected with the DNA3.1 expression vector bearing either no insert (empty) or p42 AUF-1, or with the pENTR/U6 vector carrying a shRNA specific for LacZ or all AUF - 1 mRNAs. Inclusion of APP exons 7 and 8 was assayed by RT-qPCR with primers specific for APP770, APP695 and APP751 . Total APP cDNA was used for normalization. Bars in graphs correspond to mean ± standard deviation of three independent experiments. ( D , E ) SK-N-SH cells were co-transfected with the human ( D ) or the mouse ( E ) APPE78 minigene along with two expression vectors, one bearing ELAVL4 and the other one carrying no insert (empty) or p42 AUF-1, in ratios depicted. Splicing pathways were determined by RT-PCR, as described in Fig. 5 . Quantification of the results was performed by scanning densitometry. Note, that AUF-1 enhances ELAVL4-mediated exclusion of APP exons 7 and 8. ( F ) Neuro2a cells were transfected with the pSilencer vector bearing either no insert (empty) or a shRNA targeting all Auf - 1 transcripts. Inclusion of APP exons 7 and 8 was assayed by RT-qPCR, as described above (**P

    Article Snippet: RNA isolation and RT-PCR Total RNA was extracted from cells using the RNAzol Reagent (Molecular Research Center, Inc) according to the manufacturer’s protocol, or from RIP material (see below) by acidic phenol/chlorophorm extraction.

    Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, shRNA, Quantitative RT-PCR, Standard Deviation, Reverse Transcription Polymerase Chain Reaction

    nELAVL-binding to sequences located both upstream and downstream of exon 7 is required for nELAVL-mediated APP exon 7 exclusion. ( A , B ) Schematic representation of human ( A ) and mouse ( B ) APP genomic regions used for the generation of APPE7 and AppE7 minigenes, respectively. The gray box corresponds to exon 7, bold lines to its flanking intronic sequences, whose length is indicated and triangles to segments encoding for U-rich sequences in the pre-mRNA. ( C , D ) Human SK-N-SH cells were co-transfected with either APPE7 ( C ) or AppE7 ( D ) minigenes and the pCAGGS expression vector bearing ELAVL4. Splicing pathways were determined by RT-PCR using primers specific for the artificial exons of the minigene vector. Amplification bands of transcripts lacking exon 7 are shown. Quantification of the results was performed by scanning densitometry. Bars in graphs depict the percentage of exon 7 skipping (mean ± standard deviation). Note, that ELAVL4 promoted exon 7 exclusion only from transcripts containing U-rich sequences both upstream and downstream of this exon ( E , F ) Biotinylated RNA probes transcribed from the indicated human ( E ) and mouse ( F ) APPE7 minigenes were tested for nELAVL-binding after incubation with lysates of Neuro-2a cells and pull-down using streptavidin beads. The presence of nELAVLs was assayed by immunoblotting. Note, that nELAVLs strongly associated with both flanking intronic regions of APP exon 7.

    Journal: Scientific Reports

    Article Title: Neuronal ELAVL proteins utilize AUF-1 as a co-partner to induce neuron-specific alternative splicing of APP

    doi: 10.1038/srep44507

    Figure Lengend Snippet: nELAVL-binding to sequences located both upstream and downstream of exon 7 is required for nELAVL-mediated APP exon 7 exclusion. ( A , B ) Schematic representation of human ( A ) and mouse ( B ) APP genomic regions used for the generation of APPE7 and AppE7 minigenes, respectively. The gray box corresponds to exon 7, bold lines to its flanking intronic sequences, whose length is indicated and triangles to segments encoding for U-rich sequences in the pre-mRNA. ( C , D ) Human SK-N-SH cells were co-transfected with either APPE7 ( C ) or AppE7 ( D ) minigenes and the pCAGGS expression vector bearing ELAVL4. Splicing pathways were determined by RT-PCR using primers specific for the artificial exons of the minigene vector. Amplification bands of transcripts lacking exon 7 are shown. Quantification of the results was performed by scanning densitometry. Bars in graphs depict the percentage of exon 7 skipping (mean ± standard deviation). Note, that ELAVL4 promoted exon 7 exclusion only from transcripts containing U-rich sequences both upstream and downstream of this exon ( E , F ) Biotinylated RNA probes transcribed from the indicated human ( E ) and mouse ( F ) APPE7 minigenes were tested for nELAVL-binding after incubation with lysates of Neuro-2a cells and pull-down using streptavidin beads. The presence of nELAVLs was assayed by immunoblotting. Note, that nELAVLs strongly associated with both flanking intronic regions of APP exon 7.

    Article Snippet: RNA isolation and RT-PCR Total RNA was extracted from cells using the RNAzol Reagent (Molecular Research Center, Inc) according to the manufacturer’s protocol, or from RIP material (see below) by acidic phenol/chlorophorm extraction.

    Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, Standard Deviation, Incubation

    Expression of nELAVLs correlates with the APP695-specific pre-mRNA processing. ( A ) High-throughput sequencing data from 17 human samples were analyzed for the association between APP isoforms and Hu, AUF - 1 and TIA - 1 mRNA expression. Color intensity and circle size indicate the strength of the correlation. Note that only APP695 is correlated with nELAVL expression. ( B ) Schematic representation of APP AS in neuronal and non-neuronal cells and localization of primers used in this study ( arrows , F: forward, R: reverse) Boxes indicate exons and bold lines introns. RT-PCR was carried out using total RNA isolated from five cell lines and primary cortical neurons with primers specific for human or mouse ELAVL1, ELAVL2, ELAVL3, ELAVL4, and primers for human or mouse APP that allow the simultaneous detection of all AS events of exons 7 and 8. The indicated amplification bands resulting from AS of APP exons 7 and/or 8 were identified by their respective length. Quantification of the results was performed by scanning densitometry and the percentage of exclusion of both exons 7 and 8 is indicated below each lane. ( C ) Equal amounts of total protein from lysates of five cell lines and cortical neurons were analyzed on SDS-PAGE and immunoblotted with antibodies specific for ELAVLs, APP and GAPDH as a loading control. Note that similar to cortical neurons, significant levels of APP695 were observed only in the cells lines expressing nELAVLs. ( D ) Human SH-SY5Y and mouse CAD neuroblastoma cells were differentiated into a neuronal-like phenotype. Equal amounts of total protein from lysates of the above untreated and differentiated cells were analyzed on SDS-PAGE and immunoblotted with antibodies specific for the neuronal markers β-ΙΙΙ tubulin and SAP97 as well as for APP, ELAVLs and finally GAPDH as a loading control. Note that differentiated SH-SY5Y and CAD cells displayed a concurrent upregulation of nELAVLs and APP695.

    Journal: Scientific Reports

    Article Title: Neuronal ELAVL proteins utilize AUF-1 as a co-partner to induce neuron-specific alternative splicing of APP

    doi: 10.1038/srep44507

    Figure Lengend Snippet: Expression of nELAVLs correlates with the APP695-specific pre-mRNA processing. ( A ) High-throughput sequencing data from 17 human samples were analyzed for the association between APP isoforms and Hu, AUF - 1 and TIA - 1 mRNA expression. Color intensity and circle size indicate the strength of the correlation. Note that only APP695 is correlated with nELAVL expression. ( B ) Schematic representation of APP AS in neuronal and non-neuronal cells and localization of primers used in this study ( arrows , F: forward, R: reverse) Boxes indicate exons and bold lines introns. RT-PCR was carried out using total RNA isolated from five cell lines and primary cortical neurons with primers specific for human or mouse ELAVL1, ELAVL2, ELAVL3, ELAVL4, and primers for human or mouse APP that allow the simultaneous detection of all AS events of exons 7 and 8. The indicated amplification bands resulting from AS of APP exons 7 and/or 8 were identified by their respective length. Quantification of the results was performed by scanning densitometry and the percentage of exclusion of both exons 7 and 8 is indicated below each lane. ( C ) Equal amounts of total protein from lysates of five cell lines and cortical neurons were analyzed on SDS-PAGE and immunoblotted with antibodies specific for ELAVLs, APP and GAPDH as a loading control. Note that similar to cortical neurons, significant levels of APP695 were observed only in the cells lines expressing nELAVLs. ( D ) Human SH-SY5Y and mouse CAD neuroblastoma cells were differentiated into a neuronal-like phenotype. Equal amounts of total protein from lysates of the above untreated and differentiated cells were analyzed on SDS-PAGE and immunoblotted with antibodies specific for the neuronal markers β-ΙΙΙ tubulin and SAP97 as well as for APP, ELAVLs and finally GAPDH as a loading control. Note that differentiated SH-SY5Y and CAD cells displayed a concurrent upregulation of nELAVLs and APP695.

    Article Snippet: RNA isolation and RT-PCR Total RNA was extracted from cells using the RNAzol Reagent (Molecular Research Center, Inc) according to the manufacturer’s protocol, or from RIP material (see below) by acidic phenol/chlorophorm extraction.

    Techniques: Expressing, Next-Generation Sequencing, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, SDS Page