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  • 99
    Thermo Fisher turbo dnase thermo fisher scientific cat
    OhrR binds specifically to ohr and ohrR promoter <t>DNA.</t> (A) Electropherogram traces of <t>DNase</t> I-digested ohr promoter DNA. (B) Electropherogram traces of DNase I-digested ohrR promoter DNA. Red, DNA only; blue, DNA incubated with OhrR at a stoichiometric DNA/protein ratio of 1:4. The protected regions are expanded in the lower panels, with the sequence being identified at the bottom. Numbering is relative to the translational start, defined as position +1. For ohrR promoter DNA, the ATG codon is underlined. For ohr promoter DNA, a sequence resembling the published OhrR consensus sequence is identified by a dashed underline; the asterisk marks a hypersensitive site. The results are representative of those from at least three replicates.
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    Thermo Fisher n2111 turbo dnase thermo fisher scientific
    OhrR binds specifically to ohr and ohrR promoter <t>DNA.</t> (A) Electropherogram traces of <t>DNase</t> I-digested ohr promoter DNA. (B) Electropherogram traces of DNase I-digested ohrR promoter DNA. Red, DNA only; blue, DNA incubated with OhrR at a stoichiometric DNA/protein ratio of 1:4. The protected regions are expanded in the lower panels, with the sequence being identified at the bottom. Numbering is relative to the translational start, defined as position +1. For ohrR promoter DNA, the ATG codon is underlined. For ohr promoter DNA, a sequence resembling the published OhrR consensus sequence is identified by a dashed underline; the asterisk marks a hypersensitive site. The results are representative of those from at least three replicates.
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    Thermo Fisher dnase
    Detection of MDV transcripts in BMMs and BMDCs infected with MDV in vitro . BMMs and BMDCs were infected in vitro with EGFP-expressing MDV. After 3 days, EGFP-positive cells were sorted and RT-PCR was carried out for the detection of (a) immediate early ICP4 (200 bp), (b) early pp38 (198 bp), (c) late gB (193 bp) and (d) MDV-specific l -Meq (200 bp) transcripts. L, ladder; +, positive control MDV-infected CEFs; −, negative control, nuclease-free H 2 O; M, infected BMMs (cDNA); MN, infected BMMs no-RT control <t>(DNase-treated</t> <t>RNA);</t> D, infected BMDCs (cDNA); DN, infected BMDCs no-RT control (DNase-treated RNA).
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    Thermo Fisher turbo dnase
    Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with <t>DNase</t> I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 <t>RNA</t> was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.
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    Thermo Fisher dnase treatment
    Depletion of rRNA from <t>RNA</t> samples isolated from dual species P. aeruginosa / S. aureus cultures. A total of 2 μg of <t>DNAse-treated</t> RNA, isolated form a dual species P. aeruginosa PAO1 and S. aureus ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of P. aeruginosa and/or S. aureus treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of P. aeruginosa ( E , G ) or S. aureus ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of P. aeruginosa or S. aureus 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.
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    Thermo Fisher dnase digested
    Depletion of rRNA from <t>RNA</t> samples isolated from dual species P. aeruginosa / S. aureus cultures. A total of 2 μg of <t>DNAse-treated</t> RNA, isolated form a dual species P. aeruginosa PAO1 and S. aureus ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of P. aeruginosa and/or S. aureus treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of P. aeruginosa ( E , G ) or S. aureus ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of P. aeruginosa or S. aureus 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.
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    Thermo Fisher dnase digestion step
    Depletion of rRNA from <t>RNA</t> samples isolated from dual species P. aeruginosa / S. aureus cultures. A total of 2 μg of <t>DNAse-treated</t> RNA, isolated form a dual species P. aeruginosa PAO1 and S. aureus ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of P. aeruginosa and/or S. aureus treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of P. aeruginosa ( E , G ) or S. aureus ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of P. aeruginosa or S. aureus 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.
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    Image Search Results


    OhrR binds specifically to ohr and ohrR promoter DNA. (A) Electropherogram traces of DNase I-digested ohr promoter DNA. (B) Electropherogram traces of DNase I-digested ohrR promoter DNA. Red, DNA only; blue, DNA incubated with OhrR at a stoichiometric DNA/protein ratio of 1:4. The protected regions are expanded in the lower panels, with the sequence being identified at the bottom. Numbering is relative to the translational start, defined as position +1. For ohrR promoter DNA, the ATG codon is underlined. For ohr promoter DNA, a sequence resembling the published OhrR consensus sequence is identified by a dashed underline; the asterisk marks a hypersensitive site. The results are representative of those from at least three replicates.

    Journal: Infection and Immunity

    Article Title: Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans

    doi: 10.1128/IAI.00322-18

    Figure Lengend Snippet: OhrR binds specifically to ohr and ohrR promoter DNA. (A) Electropherogram traces of DNase I-digested ohr promoter DNA. (B) Electropherogram traces of DNase I-digested ohrR promoter DNA. Red, DNA only; blue, DNA incubated with OhrR at a stoichiometric DNA/protein ratio of 1:4. The protected regions are expanded in the lower panels, with the sequence being identified at the bottom. Numbering is relative to the translational start, defined as position +1. For ohrR promoter DNA, the ATG codon is underlined. For ohr promoter DNA, a sequence resembling the published OhrR consensus sequence is identified by a dashed underline; the asterisk marks a hypersensitive site. The results are representative of those from at least three replicates.

    Article Snippet: DNA contamination was removed using a Turbo DNase kit (Ambion), and the absence of DNA was verified by PCR.

    Techniques: Incubation, Sequencing

    Effect of OhrR oxidation on binding to ohr promoter DNA. (A) Electropherogram traces of DNase I-digested ohr promoter DNA and DNA incubated with reduced OhrR. (B) DNA incubated with reduced OhrR or with OhrR oxidized with 7 μM CHP. (C) DNA incubated with reduced OhrR or with OhrR oxidized with 70 μM CHP. (D) DNA incubated with OhrR oxidized with 70 μM CHP or with 70 μM CHP-oxidized OhrR subsequently rereduced with DTT. (E) DNA incubated with reduced OhrR or with OhrR oxidized with 0.7 mM H 2 O 2 . (F) Free ohr promoter DNA and DNA incubated with OhrR oxidized with 7 μM NaOCl. Asterisks mark hypersensitive sites (positions −56 and −49). The results are representative of those from at least three replicates.

    Journal: Infection and Immunity

    Article Title: Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans

    doi: 10.1128/IAI.00322-18

    Figure Lengend Snippet: Effect of OhrR oxidation on binding to ohr promoter DNA. (A) Electropherogram traces of DNase I-digested ohr promoter DNA and DNA incubated with reduced OhrR. (B) DNA incubated with reduced OhrR or with OhrR oxidized with 7 μM CHP. (C) DNA incubated with reduced OhrR or with OhrR oxidized with 70 μM CHP. (D) DNA incubated with OhrR oxidized with 70 μM CHP or with 70 μM CHP-oxidized OhrR subsequently rereduced with DTT. (E) DNA incubated with reduced OhrR or with OhrR oxidized with 0.7 mM H 2 O 2 . (F) Free ohr promoter DNA and DNA incubated with OhrR oxidized with 7 μM NaOCl. Asterisks mark hypersensitive sites (positions −56 and −49). The results are representative of those from at least three replicates.

    Article Snippet: DNA contamination was removed using a Turbo DNase kit (Ambion), and the absence of DNA was verified by PCR.

    Techniques: Binding Assay, Incubation

    Dissection of the consensus binding motif of PhoP Xcc . (A) Venn diagram showing the number of PhoP Xcc -binding promoter regions identified by the ChIP-seq analysis. (B) Functional categories of all genes with promoters bound by PhoP Xcc under PhoQ Xcc induction and repression conditions. (C) Consensus PhoP Xcc -binding DNA motifs predicted by ChIP-seq and MEME. The height of each nucleotide is proportional to the level of conservation at that site. Upper and lower panels: motifs predicted under PhoQ Xcc induction and repression conditions, respectively. (D–F) Binding sites of PhoP Xcc on the promoter regions of (D) oar , (E), phoP , and (F) ftsA . DNase I footprinting assays were conducted. The amounts of PhoP Xcc protein used in the reactions were 1: zero; 2: 40 μM, 3: 80 μM, 4: 120 μM, and 5: 160 μM. The DNA regions protected by PhoP Xcc are shown on the right of the footprinting results, with the PhoP Xcc -binding sites shown in red. (G–I) PhoP Xcc –DNA-binding affinities were quantified by MST. (G) PhoP Xcc –P ftsA Xcc . (H) PhoP Xcc –P phoP Xcc . (I) PhoP Xcc –P oar Xcc . DNA probes were labeled with FAM and used for MST. The FAM-labeled DNA (20 nM) was incubated with the PhoP Xcc protein in an NT standard capillary in the MST assay. Titrations of the PhoP Xcc protein ranged from 1.5 to 50 μM. The solid curve is the fit of the data points to the standard KD-Fit function. The black bars represent SD ( n = 3). K d = dissociation constant. ChIP-seq, chromatin immunoprecipitation sequencing; FAM, fluorescein; MST, microscale thermophoresis; NT, NanoTemper.

    Journal: Genetics

    Article Title: An Essential Regulatory System Originating from Polygenic Transcriptional Rewiring of PhoP-PhoQ of Xanthomonas campestris

    doi: 10.1534/genetics.117.200204

    Figure Lengend Snippet: Dissection of the consensus binding motif of PhoP Xcc . (A) Venn diagram showing the number of PhoP Xcc -binding promoter regions identified by the ChIP-seq analysis. (B) Functional categories of all genes with promoters bound by PhoP Xcc under PhoQ Xcc induction and repression conditions. (C) Consensus PhoP Xcc -binding DNA motifs predicted by ChIP-seq and MEME. The height of each nucleotide is proportional to the level of conservation at that site. Upper and lower panels: motifs predicted under PhoQ Xcc induction and repression conditions, respectively. (D–F) Binding sites of PhoP Xcc on the promoter regions of (D) oar , (E), phoP , and (F) ftsA . DNase I footprinting assays were conducted. The amounts of PhoP Xcc protein used in the reactions were 1: zero; 2: 40 μM, 3: 80 μM, 4: 120 μM, and 5: 160 μM. The DNA regions protected by PhoP Xcc are shown on the right of the footprinting results, with the PhoP Xcc -binding sites shown in red. (G–I) PhoP Xcc –DNA-binding affinities were quantified by MST. (G) PhoP Xcc –P ftsA Xcc . (H) PhoP Xcc –P phoP Xcc . (I) PhoP Xcc –P oar Xcc . DNA probes were labeled with FAM and used for MST. The FAM-labeled DNA (20 nM) was incubated with the PhoP Xcc protein in an NT standard capillary in the MST assay. Titrations of the PhoP Xcc protein ranged from 1.5 to 50 μM. The solid curve is the fit of the data points to the standard KD-Fit function. The black bars represent SD ( n = 3). K d = dissociation constant. ChIP-seq, chromatin immunoprecipitation sequencing; FAM, fluorescein; MST, microscale thermophoresis; NT, NanoTemper.

    Article Snippet: One microgram of RNA from each sample was treated with Turbo DNA-free DNase (Ambion) before RT-PCR, to exclude DNA contamination.

    Techniques: Dissection, Binding Assay, Chromatin Immunoprecipitation, Functional Assay, Footprinting, Microscale Thermophoresis, Labeling, Incubation, ChIP-sequencing

    Detection of MDV transcripts in BMMs and BMDCs infected with MDV in vitro . BMMs and BMDCs were infected in vitro with EGFP-expressing MDV. After 3 days, EGFP-positive cells were sorted and RT-PCR was carried out for the detection of (a) immediate early ICP4 (200 bp), (b) early pp38 (198 bp), (c) late gB (193 bp) and (d) MDV-specific l -Meq (200 bp) transcripts. L, ladder; +, positive control MDV-infected CEFs; −, negative control, nuclease-free H 2 O; M, infected BMMs (cDNA); MN, infected BMMs no-RT control (DNase-treated RNA); D, infected BMDCs (cDNA); DN, infected BMDCs no-RT control (DNase-treated RNA).

    Journal: The Journal of General Virology

    Article Title: Marek's disease virus infection of phagocytes: a de novo in vitro infection model

    doi: 10.1099/jgv.0.000763

    Figure Lengend Snippet: Detection of MDV transcripts in BMMs and BMDCs infected with MDV in vitro . BMMs and BMDCs were infected in vitro with EGFP-expressing MDV. After 3 days, EGFP-positive cells were sorted and RT-PCR was carried out for the detection of (a) immediate early ICP4 (200 bp), (b) early pp38 (198 bp), (c) late gB (193 bp) and (d) MDV-specific l -Meq (200 bp) transcripts. L, ladder; +, positive control MDV-infected CEFs; −, negative control, nuclease-free H 2 O; M, infected BMMs (cDNA); MN, infected BMMs no-RT control (DNase-treated RNA); D, infected BMDCs (cDNA); DN, infected BMDCs no-RT control (DNase-treated RNA).

    Article Snippet: RT-PCR RNA samples were extracted using RNeasy Mini Kits (Qiagen) and treated with DNase (Ambion Turbo DNA-free Kits, Life Technologies).

    Techniques: Infection, In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control

    Depletion of linear RNA by RNase R digestion. RT-qPCR analysis of subsets of linear rRNA, mRNAs and circRNAs (A), as well as snRNAs and 5S rRNA (B) in HeLa cell RNA samples left untreated or treated with RNase R. In (A) since the circRNA levels were not affected by RNase R treatment, mRNAs left after RNase R treatment were normalized to the levels of the corresponding circRNAs. Data represent the means ± SEM from 4 or more experiments.

    Journal: Methods (San Diego, Calif.)

    Article Title: RPAD (RNase R Treatment, Polyadenylation, and Poly(A)+ RNA Depletion) Method to Isolate Highly Pure Circular RNA

    doi: 10.1016/j.ymeth.2018.10.022

    Figure Lengend Snippet: Depletion of linear RNA by RNase R digestion. RT-qPCR analysis of subsets of linear rRNA, mRNAs and circRNAs (A), as well as snRNAs and 5S rRNA (B) in HeLa cell RNA samples left untreated or treated with RNase R. In (A) since the circRNA levels were not affected by RNase R treatment, mRNAs left after RNase R treatment were normalized to the levels of the corresponding circRNAs. Data represent the means ± SEM from 4 or more experiments.

    Article Snippet: Total RNA isolated from HeLa cells Nuclease-free microcentrifuge tubes RNase R (Lucigen, Epicentre) TURBO DNA-free™ Kit (Thermo Fisher Scientific) RiboLock RNase inhibitor (Thermo Fisher Scientific) Thermomixer (Eppendorf) miRNeasy Kit (QIAGEN) Vortexer Microcentrifuge (Eppendorf) NanoDrop spectrophotometer (Thermo Fisher Scientific)

    Techniques: Quantitative RT-PCR

    Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with DNase I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 RNA was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.

    Journal: International Journal of Molecular Sciences

    Article Title: Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs

    doi: 10.3390/ijms21082812

    Figure Lengend Snippet: Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with DNase I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 RNA was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.

    Article Snippet: Subsequently, the RNA was treated with Turbo DNase (Thermo Fisher Scientific) for 30 min at 37 °C, to remove any contaminating DNA.

    Techniques: Northern Blot, Hybridization, Quantitation Assay, Standard Deviation, Marker, Reverse Transcription Polymerase Chain Reaction, Fractionation, Quantitative RT-PCR

    Depletion of rRNA from RNA samples isolated from dual species P. aeruginosa / S. aureus cultures. A total of 2 μg of DNAse-treated RNA, isolated form a dual species P. aeruginosa PAO1 and S. aureus ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of P. aeruginosa and/or S. aureus treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of P. aeruginosa ( E , G ) or S. aureus ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of P. aeruginosa or S. aureus 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Journal: Scientific Reports

    Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    doi: 10.1038/srep41114

    Figure Lengend Snippet: Depletion of rRNA from RNA samples isolated from dual species P. aeruginosa / S. aureus cultures. A total of 2 μg of DNAse-treated RNA, isolated form a dual species P. aeruginosa PAO1 and S. aureus ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of P. aeruginosa and/or S. aureus treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of P. aeruginosa ( E , G ) or S. aureus ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of P. aeruginosa or S. aureus 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Article Snippet: The RNA was subsequently subjected to DNase treatment (TURBO DNA-free DNase Treatment, Ambion) according to the manufacturer’s protocol.

    Techniques: Isolation, Software, Real-time Polymerase Chain Reaction, Standard Deviation

    Quantitative and qualitative comparison of RNA recovered following rRNA depletion. A total of 4 μg of DNAse-treated RNA, isolated form 3-day-old P. aeruginosa PAO1 biofilms, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using Qubit fluorimetric quantitation with the Qubit RNA HS Assay Kit. Yields are reported as total RNA recovered ( A ) and as percentage of the input RNA ( B ). The RNA samples were also assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( C ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( D ) MICROBExpress, ( E ) RiboMinus, or ( F ) Ribo-Zero kits are shown. Dashed lines indicate peaks corresponding to 16S and 23S RNA traces, which were detected in total RNA, MICROBExpress, and RiboMins samples. ( G ) The area of 16S and 23S rRNA peaks as percent of the total detected RNA was estimated, as determined using the 2100 Expert Software. RFU, relative fluorescence units. Experiments were repeated using three biological replicates.

    Journal: Scientific Reports

    Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    doi: 10.1038/srep41114

    Figure Lengend Snippet: Quantitative and qualitative comparison of RNA recovered following rRNA depletion. A total of 4 μg of DNAse-treated RNA, isolated form 3-day-old P. aeruginosa PAO1 biofilms, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using Qubit fluorimetric quantitation with the Qubit RNA HS Assay Kit. Yields are reported as total RNA recovered ( A ) and as percentage of the input RNA ( B ). The RNA samples were also assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( C ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( D ) MICROBExpress, ( E ) RiboMinus, or ( F ) Ribo-Zero kits are shown. Dashed lines indicate peaks corresponding to 16S and 23S RNA traces, which were detected in total RNA, MICROBExpress, and RiboMins samples. ( G ) The area of 16S and 23S rRNA peaks as percent of the total detected RNA was estimated, as determined using the 2100 Expert Software. RFU, relative fluorescence units. Experiments were repeated using three biological replicates.

    Article Snippet: The RNA was subsequently subjected to DNase treatment (TURBO DNA-free DNase Treatment, Ambion) according to the manufacturer’s protocol.

    Techniques: Isolation, Quantitation Assay, RNA HS Assay, Software, Fluorescence

    rRNA depletion treatments of RNA derived from planktonic P. aeruginosa PAO1 and Staphylococcus aureus ATCC6538 cells. A total of 2 μg of DNAse-treated RNA, isolated from exponential phase P. aeruginosa PAO1 or Staphylococcus aureus ATCC6538 cells, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of starting total RNA material and aliquots of the RNA samples that have been processed using the MICROBExpress or Ribo-Zero kits are shown for the P. aeruginosa ( A ) and S. aureus ( B ) samples. The samples were subsequently subjected to qPCR analysis of rRNA transcript abundance, with qPCR threshold cycle (Cq) for the indicated rRNA transcripts shown for P. aeruginosa ( C ) and S. aureus ( E ). Copy numbers of P. aeruginosa ( D ) and S. aureus ( F ) 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Journal: Scientific Reports

    Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    doi: 10.1038/srep41114

    Figure Lengend Snippet: rRNA depletion treatments of RNA derived from planktonic P. aeruginosa PAO1 and Staphylococcus aureus ATCC6538 cells. A total of 2 μg of DNAse-treated RNA, isolated from exponential phase P. aeruginosa PAO1 or Staphylococcus aureus ATCC6538 cells, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of starting total RNA material and aliquots of the RNA samples that have been processed using the MICROBExpress or Ribo-Zero kits are shown for the P. aeruginosa ( A ) and S. aureus ( B ) samples. The samples were subsequently subjected to qPCR analysis of rRNA transcript abundance, with qPCR threshold cycle (Cq) for the indicated rRNA transcripts shown for P. aeruginosa ( C ) and S. aureus ( E ). Copy numbers of P. aeruginosa ( D ) and S. aureus ( F ) 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Article Snippet: The RNA was subsequently subjected to DNase treatment (TURBO DNA-free DNase Treatment, Ambion) according to the manufacturer’s protocol.

    Techniques: Derivative Assay, Isolation, Real-time Polymerase Chain Reaction, Standard Deviation

    Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*,  p

    Journal: PLoS Pathogens

    Article Title: Mono-ubiquitylated ORF45 Mediates Association of KSHV Particles with Internal Lipid Rafts for Viral Assembly and Egress

    doi: 10.1371/journal.ppat.1005332

    Figure Lengend Snippet: Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*, p

    Article Snippet: For the preparation of DNA from intact virions, 200 μl of virus stocks was pretreated with 2 μl of Turbo DNase I (Ambion) for 1 h at 37°C.

    Techniques: Mutagenesis, BAC Assay, Real-time Polymerase Chain Reaction

    Bacterial messenger RNA constitutes an active vita -PAMP a–c and e–g . LDH, IL-1β and IL-6 at 24h. a. Total EC RNA treated with RNAse I and RNAse III, RNAse III alone, or DNAse prior to stimulation of BMDC. b. BMM treated with viable or HKEC, or HKEC with 0.1µg/ml of different bacterial RNA; ribosomal (rRNA), messenger (mRNA), small (sRNA) or eukaryotic RNA (eukRNA). c. BMDC responses. Gro-RNA; in vitro transcribed EC Gro-operon RNA. d. Predicted secondary structure of Gro-RNA. Colour code indicates base pairing probability. e. BMM treated with in vitro transcribed Gro-RNA or Gro dsRNA alone or with HKEC. f. BMDC responses. Era-RNA and DNApol-RNA; in vitro transcribed EC Era-GTPase and DNA-polymerase-III RNA, respectively. g . BMM treated with different doses of unmodified (control), or modified Gro-RNA with HKEC (5’cap, 5’ m 7 G capping; CIP, calf intestinal phosphatase; 3’poly(A), 3’-polyadenylation). a–g. #, ‘not detected’, all RNA at 10µg/ml except as noted, data represent ≥5 experiments. h. Mice vaccinated and boosted twice with viable EC, HKEC or HKEC with 30µg total purified bacterial RNA (HKEC+RNA) (vaccination regimen in suppl. Fig. 22 ). Class-specific anti- E. coli antibody serum titers at 25 days. *; p≤0.05, **; p≤0.01, ***; p≤0.001. All bars represent mean ± s.e.m.

    Journal: Nature

    Article Title: Sensing prokaryotic mRNA signifies microbial viability and promotes immunity

    doi: 10.1038/nature10072

    Figure Lengend Snippet: Bacterial messenger RNA constitutes an active vita -PAMP a–c and e–g . LDH, IL-1β and IL-6 at 24h. a. Total EC RNA treated with RNAse I and RNAse III, RNAse III alone, or DNAse prior to stimulation of BMDC. b. BMM treated with viable or HKEC, or HKEC with 0.1µg/ml of different bacterial RNA; ribosomal (rRNA), messenger (mRNA), small (sRNA) or eukaryotic RNA (eukRNA). c. BMDC responses. Gro-RNA; in vitro transcribed EC Gro-operon RNA. d. Predicted secondary structure of Gro-RNA. Colour code indicates base pairing probability. e. BMM treated with in vitro transcribed Gro-RNA or Gro dsRNA alone or with HKEC. f. BMDC responses. Era-RNA and DNApol-RNA; in vitro transcribed EC Era-GTPase and DNA-polymerase-III RNA, respectively. g . BMM treated with different doses of unmodified (control), or modified Gro-RNA with HKEC (5’cap, 5’ m 7 G capping; CIP, calf intestinal phosphatase; 3’poly(A), 3’-polyadenylation). a–g. #, ‘not detected’, all RNA at 10µg/ml except as noted, data represent ≥5 experiments. h. Mice vaccinated and boosted twice with viable EC, HKEC or HKEC with 30µg total purified bacterial RNA (HKEC+RNA) (vaccination regimen in suppl. Fig. 22 ). Class-specific anti- E. coli antibody serum titers at 25 days. *; p≤0.05, **; p≤0.01, ***; p≤0.001. All bars represent mean ± s.e.m.

    Article Snippet: Contaminating DNA was removed by DNase digestion (TURBO DNase, Ambion/Applied Biosystems).

    Techniques: In Vitro, Modification, Mouse Assay, Purification