turbo dnase  (Thermo Fisher)


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    TURBO DNase
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    Structured Review

    Thermo Fisher turbo dnase
    RNase H selective depletion of poly(rA) carrier from Lassa samples. (A) Native polyacrylamide gel depicting library PCR and side products of LASV preparations with poly(rA) carrier present (middle) or depleted (right panel). No free poly(rA) was present in control library (left). (B) Median base qualities per MiSeq cycle of poly(rA)-contaminated LASV libraries (solid line) and control (no carrier observed in library, dashed) from FastQC report. Both read 1 and read 2 of paired end reads are merged in the library BAM file and the quality scores are shown at each base. (C) Schematic of carrier <t>RNA</t> selective depletion and <t>DNase</t> treatment of oligo (dT).

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    Images

    1) Product Images from "Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples"

    Article Title: Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

    Journal: Genome Biology

    doi: 10.1186/s13059-014-0519-7

    RNase H selective depletion of poly(rA) carrier from Lassa samples. (A) Native polyacrylamide gel depicting library PCR and side products of LASV preparations with poly(rA) carrier present (middle) or depleted (right panel). No free poly(rA) was present in control library (left). (B) Median base qualities per MiSeq cycle of poly(rA)-contaminated LASV libraries (solid line) and control (no carrier observed in library, dashed) from FastQC report. Both read 1 and read 2 of paired end reads are merged in the library BAM file and the quality scores are shown at each base. (C) Schematic of carrier RNA selective depletion and DNase treatment of oligo (dT).
    Figure Legend Snippet: RNase H selective depletion of poly(rA) carrier from Lassa samples. (A) Native polyacrylamide gel depicting library PCR and side products of LASV preparations with poly(rA) carrier present (middle) or depleted (right panel). No free poly(rA) was present in control library (left). (B) Median base qualities per MiSeq cycle of poly(rA)-contaminated LASV libraries (solid line) and control (no carrier observed in library, dashed) from FastQC report. Both read 1 and read 2 of paired end reads are merged in the library BAM file and the quality scores are shown at each base. (C) Schematic of carrier RNA selective depletion and DNase treatment of oligo (dT).

    Techniques Used: Polymerase Chain Reaction

    2) Product Images from "Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA"

    Article Title: Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA

    Journal:

    doi: 10.1128/JVI.03288-14

    Modulation of cytokine expression by selected E3 mutants. HeLa cells were infected with selected E3 mutant recombinant viruses at an MOI of 10, and the total RNA was collected at 8 h postinfection. Trace DNA contamination was removed with DNase. The mRNA
    Figure Legend Snippet: Modulation of cytokine expression by selected E3 mutants. HeLa cells were infected with selected E3 mutant recombinant viruses at an MOI of 10, and the total RNA was collected at 8 h postinfection. Trace DNA contamination was removed with DNase. The mRNA

    Techniques Used: Expressing, Infection, Mutagenesis, Recombinant

    3) Product Images from "Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions"

    Article Title: Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions

    Journal: Biological Procedures Online

    doi: 10.1251/bpo122

    Demonstration of typical inhibitory qPCR profiles exhibited on qPCR Test Plates by the more concentrated RNA samples (on the right hand side of each graph) in a progressive dilution series. Targets here were Gallus gallus Gallinacin 1, Gallinacin 2 and Gallus gallus 18S ribosomal RNA (the single housekeeper). Stock I here was an equivolumetric mixture of the 26 total tissue RNA samples used in this study: just after their isolation by Trizol method, each RNA pellet was resolubilized in 150 ?l of 0.1 mM EDTA pH 6.75, warmed to 65°C for 5 minutes, and their 260 nm and 260 nm /280 nm measurements at 1:50 were taken. 70 ?l of each resolubilized RNA was then Turbo-DNAse treated [70 ?l RNA isolate + 10 ?l 10X Turbo DNase Buffer + 20 ?l Turbo DNase enzyme (40 Units); and finally 10 ?l Inactivation Reagent] and 80 ?l of each was then diluted 1:10 with nuclease-free water. Subsequently, 50 ?l of each of these 1:10 RNA isolates was mixed together into a single tube attaining a final volume of 1,300 ?l. This was the Stock I RNA solution from which all standards and inter-plate calibrators were prepared. It was also the mixture which served as the source of the serially-diluted template samples for the Test Plate which we ran early on to identify the best RNA dilution ranges for each of the 3 targets. All calculations for this study were quickly performed by the FF2-6-001 qPCR set-up tool.
    Figure Legend Snippet: Demonstration of typical inhibitory qPCR profiles exhibited on qPCR Test Plates by the more concentrated RNA samples (on the right hand side of each graph) in a progressive dilution series. Targets here were Gallus gallus Gallinacin 1, Gallinacin 2 and Gallus gallus 18S ribosomal RNA (the single housekeeper). Stock I here was an equivolumetric mixture of the 26 total tissue RNA samples used in this study: just after their isolation by Trizol method, each RNA pellet was resolubilized in 150 ?l of 0.1 mM EDTA pH 6.75, warmed to 65°C for 5 minutes, and their 260 nm and 260 nm /280 nm measurements at 1:50 were taken. 70 ?l of each resolubilized RNA was then Turbo-DNAse treated [70 ?l RNA isolate + 10 ?l 10X Turbo DNase Buffer + 20 ?l Turbo DNase enzyme (40 Units); and finally 10 ?l Inactivation Reagent] and 80 ?l of each was then diluted 1:10 with nuclease-free water. Subsequently, 50 ?l of each of these 1:10 RNA isolates was mixed together into a single tube attaining a final volume of 1,300 ?l. This was the Stock I RNA solution from which all standards and inter-plate calibrators were prepared. It was also the mixture which served as the source of the serially-diluted template samples for the Test Plate which we ran early on to identify the best RNA dilution ranges for each of the 3 targets. All calculations for this study were quickly performed by the FF2-6-001 qPCR set-up tool.

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation

    Different approaches to fluorogenic qPCR; we use the highlighted TaqMan® hydrolysis probe-based real-time qPCR method. All primers and probes are optimized and validated according to ABI procedural guidelines ( 31 ) using all-target-inclusive ( Stock I ) cDNA prepared from Turbo DNase-treated total RNA isolated (using Trizol®) from whole tissue homogenates as described previously ( 17 - 20 ). Our optimization approach is a very common/well-known procedure whereby one first studies different combinations of primer concentrations in the range of 50 nM-900 nM while keeping the probe at a constant 200 or 225 nM, after which the probe is studied by challenging it from 25 nM to 225 nM while primers are used at their optimal concentrations. All samples are performed in triplicate or quadruplicate during these evaluations to bolster significance of final evaluations. After optimization, a 'validation plate' or Test Plate is performed on up to eleven serial dilutions of the same cDNA or RNA (starting with full-strength cDNA or RNA which is assigned a relative dilution strength value of
    Figure Legend Snippet: Different approaches to fluorogenic qPCR; we use the highlighted TaqMan® hydrolysis probe-based real-time qPCR method. All primers and probes are optimized and validated according to ABI procedural guidelines ( 31 ) using all-target-inclusive ( Stock I ) cDNA prepared from Turbo DNase-treated total RNA isolated (using Trizol®) from whole tissue homogenates as described previously ( 17 - 20 ). Our optimization approach is a very common/well-known procedure whereby one first studies different combinations of primer concentrations in the range of 50 nM-900 nM while keeping the probe at a constant 200 or 225 nM, after which the probe is studied by challenging it from 25 nM to 225 nM while primers are used at their optimal concentrations. All samples are performed in triplicate or quadruplicate during these evaluations to bolster significance of final evaluations. After optimization, a 'validation plate' or Test Plate is performed on up to eleven serial dilutions of the same cDNA or RNA (starting with full-strength cDNA or RNA which is assigned a relative dilution strength value of "1") using the optimal primer and probe concentrations established during optimization for each target. The highest Rn (normalized reporter fluorescence) value achieved using the lowest primer concentrations is the indicator by which one selects the appropriate optimal primer concentrations in each case; the higher the Rn, the higher the magnitude of real-time fluorescent signal. Once the Rn value no longer increases with increasing primer concentrations, one has effectively attained the useful optimal primer concentrations. C T values (not Rn values) are evaluated during probe optimizations, and the lowest C T (threshold cycle) value with the lowest probe concentration is the criteria by which one chooses optimal probe concentrations. Once C T values no longer decrease with increasing probe concentration, one has effectively attained the useful optimal probe concentration. Little known is the fact that most real-time target signals can be found with greater than 75% amplification efficiency simply using 'saturating concentrations' of primers (1 ?M) and probes (150 nM) in most experimental situations if optimal RNA dilution ranges are established for each target and inhibition is entirely avoided (unpublished multiple observations from our lab, 2001-2006).

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Fluorescence, Concentration Assay, Amplification, Inhibition

    4) Product Images from "H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage"

    Article Title: H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00301-17

    Stage-specific expression of H-IPSE mRNA. Shown are RT-PCR results for H-IPSE obtained from cDNAs prepared by reverse transcription of DNase-treated RNAs isolated at various life stages of S. haematobium . Ladder, 100-bp DNA ladder; egg, S. haematobium egg cDNA; mir, miracidial cDNA; cer, cercarial cDNA; som, in vitro mechanically transformed schistosomulum cDNA; AdF, AdM, and Ad mix, mixed cDNAs from female, male, and mixed adult worms, respectively; ShTub, S. haematobium tubulin (control housekeeping gene).
    Figure Legend Snippet: Stage-specific expression of H-IPSE mRNA. Shown are RT-PCR results for H-IPSE obtained from cDNAs prepared by reverse transcription of DNase-treated RNAs isolated at various life stages of S. haematobium . Ladder, 100-bp DNA ladder; egg, S. haematobium egg cDNA; mir, miracidial cDNA; cer, cercarial cDNA; som, in vitro mechanically transformed schistosomulum cDNA; AdF, AdM, and Ad mix, mixed cDNAs from female, male, and mixed adult worms, respectively; ShTub, S. haematobium tubulin (control housekeeping gene).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, In Vitro, Transformation Assay

    5) Product Images from "Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells"

    Article Title: Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr085

    Nuclear localization of Sam68 requires the integrity of nucleic acids. Purified pachytene spermatocytes were permeabilized on microscope slides in a buffer containing 0.1% Triton X-100 and incubated for 15 min with medium alone (Control) or DNase or Rnase as indicated. At the end of the incubation, cells were washed three times with PBS and fixed for immunofluorescence analysis with the anti-Sam68 and H5 ( A ) or H14 ( B ) antibodies. DNA was stained by Hoechst dye. ( C ) The western blot analysis of Sam68 and β-tubulin in control or treated (DNase or RNase) pachytene spermatocytes after sequential extractions with the indicated buffers.
    Figure Legend Snippet: Nuclear localization of Sam68 requires the integrity of nucleic acids. Purified pachytene spermatocytes were permeabilized on microscope slides in a buffer containing 0.1% Triton X-100 and incubated for 15 min with medium alone (Control) or DNase or Rnase as indicated. At the end of the incubation, cells were washed three times with PBS and fixed for immunofluorescence analysis with the anti-Sam68 and H5 ( A ) or H14 ( B ) antibodies. DNA was stained by Hoechst dye. ( C ) The western blot analysis of Sam68 and β-tubulin in control or treated (DNase or RNase) pachytene spermatocytes after sequential extractions with the indicated buffers.

    Techniques Used: Purification, Microscopy, Incubation, Immunofluorescence, Staining, Western Blot

    Sam68 regulates alternative splicing of Sgce exon 8 in male germ cells. RT–PCR analysis of Sgce exon 8 inclusion in total RNA extracted from Sam68 wild-type or knockout testes at 8, 16 and 30 days post partum (dpp) ( A ) or from isolated wild-type or knockout spermatocytes and spermatids. ( B ) Bands corresponding to mRNAs containing or not, exon 8 are indicated on the right of the panels. ( C and D ) Chromatin immunoprecipitation (ChIP) analysis of Sam68 wild-type and knockout germ cells. Sonicated chromatin (100 µg of DNA/sample) was immunoprecipitated with 2µg of H5, H14 antibodies or control rabbit IgGs and co-precipitated DNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( E ) CLIP analysis of the binding of Sam68 to Sgce pre-mRNA. After UV crosslink, Sam68 wild-type and knockout germ cell extracts were sonicated and treated with DNase and RNase to yield RNA fragments of ∼200 to 250 nt. Wild-type and knockout germ cell extracts were immunoprecipitated with 2 µg of rabbit IgGs or anti-Sam68 antibodies and co-precipitated RNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( F ) Electrophoretic mobility shift assays (EMSAs) of the binding of purified GST-Sam68 1–277 to a labelled Sgce probe containing the sequences encoded at the intron 7/exon 8 boundary. The position of the free probe and the probe complexed with GST-Sam68 1–277 are shown on the left side. Competition with the wild-type (middle panel) or mutated (right panel) cold probes is shown. The scheme above the gels shows the wild-type and mutated sequence used in the EMSAs. The mutated bases that interfere with the Sam68 consensus are shown in violet. ( G ) The western blot analysis of RNA pulldown assay of U2AF65 binding to Sgce exon 8. Biotinylated RNAs encoding the Sgce wild-type and mutated exon 8 sequences (from −32 to +63) were bound to Streptavidine agarose beads and incubated with testicular nuclear extract (100 µg) in the presence of 1 µg of purified GST or GST-Sam68 1–277, as indicated.
    Figure Legend Snippet: Sam68 regulates alternative splicing of Sgce exon 8 in male germ cells. RT–PCR analysis of Sgce exon 8 inclusion in total RNA extracted from Sam68 wild-type or knockout testes at 8, 16 and 30 days post partum (dpp) ( A ) or from isolated wild-type or knockout spermatocytes and spermatids. ( B ) Bands corresponding to mRNAs containing or not, exon 8 are indicated on the right of the panels. ( C and D ) Chromatin immunoprecipitation (ChIP) analysis of Sam68 wild-type and knockout germ cells. Sonicated chromatin (100 µg of DNA/sample) was immunoprecipitated with 2µg of H5, H14 antibodies or control rabbit IgGs and co-precipitated DNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( E ) CLIP analysis of the binding of Sam68 to Sgce pre-mRNA. After UV crosslink, Sam68 wild-type and knockout germ cell extracts were sonicated and treated with DNase and RNase to yield RNA fragments of ∼200 to 250 nt. Wild-type and knockout germ cell extracts were immunoprecipitated with 2 µg of rabbit IgGs or anti-Sam68 antibodies and co-precipitated RNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( F ) Electrophoretic mobility shift assays (EMSAs) of the binding of purified GST-Sam68 1–277 to a labelled Sgce probe containing the sequences encoded at the intron 7/exon 8 boundary. The position of the free probe and the probe complexed with GST-Sam68 1–277 are shown on the left side. Competition with the wild-type (middle panel) or mutated (right panel) cold probes is shown. The scheme above the gels shows the wild-type and mutated sequence used in the EMSAs. The mutated bases that interfere with the Sam68 consensus are shown in violet. ( G ) The western blot analysis of RNA pulldown assay of U2AF65 binding to Sgce exon 8. Biotinylated RNAs encoding the Sgce wild-type and mutated exon 8 sequences (from −32 to +63) were bound to Streptavidine agarose beads and incubated with testicular nuclear extract (100 µg) in the presence of 1 µg of purified GST or GST-Sam68 1–277, as indicated.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Knock-Out, Isolation, Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, Real-time Polymerase Chain Reaction, Cross-linking Immunoprecipitation, Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Sequencing, Western Blot, Incubation

    6) Product Images from "Toll-Like Receptor 3/TRIF-Dependent IL-12p70 Secretion Mediated by Streptococcus pneumoniae RNA and Its Priming by Influenza A Virus Coinfection in Human Dendritic Cells"

    Article Title: Toll-Like Receptor 3/TRIF-Dependent IL-12p70 Secretion Mediated by Streptococcus pneumoniae RNA and Its Priming by Influenza A Virus Coinfection in Human Dendritic Cells

    Journal: mBio

    doi: 10.1128/mBio.00168-16

    RNA is required as a pneumococcal stimulus to induce IL-12p70 production. DCs were challenged with live, UV-killed, or heat-killed (HK) T4R (A), with live or UV-killed T4R at the indicated MOI (B), with UV-killed T4R (MOI, 10), with LPS pretreated with a cocktail of RNase A (200 to 20 U/ml) and RNase T1 (8,000 to 800 U/ml) (C), or with UV-killed T4R (MOI, 10) pretreated with DNase I (1,000 to 250 U/ml) (D). IL-12p70 production in the cell supernatant was measured in an ELISA (A to D), and DC viability was measured by flow cytometry (B). Values represent means ± standard errors of the means for results from 4 (A), 3 (B), 7 (C), or 4 (D) experiments. Statistical analysis was performed using a one-way analysis of variance and a Bonferroni posttest. *, P
    Figure Legend Snippet: RNA is required as a pneumococcal stimulus to induce IL-12p70 production. DCs were challenged with live, UV-killed, or heat-killed (HK) T4R (A), with live or UV-killed T4R at the indicated MOI (B), with UV-killed T4R (MOI, 10), with LPS pretreated with a cocktail of RNase A (200 to 20 U/ml) and RNase T1 (8,000 to 800 U/ml) (C), or with UV-killed T4R (MOI, 10) pretreated with DNase I (1,000 to 250 U/ml) (D). IL-12p70 production in the cell supernatant was measured in an ELISA (A to D), and DC viability was measured by flow cytometry (B). Values represent means ± standard errors of the means for results from 4 (A), 3 (B), 7 (C), or 4 (D) experiments. Statistical analysis was performed using a one-way analysis of variance and a Bonferroni posttest. *, P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    7) Product Images from "Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification"

    Article Title: Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification

    Journal: BioMed Research International

    doi: 10.1155/2019/2756516

    Amplification of cDNA by Phi29 DNA polymerase . Total RNA from N. benthamiana (a) and O. sativa (b) was treated with DNase and RNase R to enrich circRNAs. The enriched circRNAs were converted into cDNA using random hexamer and subjected to amplification by Phi29 DNA polymerase.
    Figure Legend Snippet: Amplification of cDNA by Phi29 DNA polymerase . Total RNA from N. benthamiana (a) and O. sativa (b) was treated with DNase and RNase R to enrich circRNAs. The enriched circRNAs were converted into cDNA using random hexamer and subjected to amplification by Phi29 DNA polymerase.

    Techniques Used: Amplification, Random Hexamer Labeling

    8) Product Images from "In Vivo T-Box Transcription Factor Profiling Reveals Joint Regulation of Embryonic Neuromesodermal Bipotency"

    Article Title: In Vivo T-Box Transcription Factor Profiling Reveals Joint Regulation of Embryonic Neuromesodermal Bipotency

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2013.08.012

    Combined Loss of T-box TFs Causes Embryos to Produce Excess Neural Tissue at the Expense of Axial and Paraxial Mesoderm and in the Absence of Apoptosis, Related to Figure 6 (A) WMISH on control, Xbra/Xbra3, Xbra/Xbra3/Eomes, Xbra/Xbra3/zVegT and Xbra/Xbra3/Eomes/zVegT KD embryos for neural differentiation marker N-tubulin at early tailbud stage (stage 20-21, lateral view). 1, 2 and 3 mark the position of cross-sections through control, Xbra/Xbra3 KD and Xbra/Xbra3/zVegT KD embryos. Abbreviations: no, notochord; nt, neural tube (d, dorsal; v, ventral); pm, paraxial mesoderm. (B) WMISH for actc1 , hoxd8 and tal1 at late tailbud stage illustrates the loss of mesodermal derivatives such as skeletal muscle (sm), heart (he), pronephros (pn) and ventral blood island (vbi) upon Xbra/Xbra3/Eomes/zVegT KD. Statistics in bottom right corner indicates the number of embryos observed with the depicted WMISH pattern versus the number of embryos analyzed in total. (C) TUNEL staining on control, Xbra/Xbra3 and Xbra/Xbra3/Eomes/zVegT KD embryos (cleared with Murray’s clear) at early tailbud stage (stage 20). Positive controls, embryos treated for 4 hr in 35 μM cycloheximide (chx) and fixed embryos incubated with DNase I. Arrowheads mark apoptotic cells in the brain region (where apoptosis can occasionally be observed in embryos even under normal conditions) and the posterior nervous system induced by cycloheximide. Scale bar, 0.2 mm.
    Figure Legend Snippet: Combined Loss of T-box TFs Causes Embryos to Produce Excess Neural Tissue at the Expense of Axial and Paraxial Mesoderm and in the Absence of Apoptosis, Related to Figure 6 (A) WMISH on control, Xbra/Xbra3, Xbra/Xbra3/Eomes, Xbra/Xbra3/zVegT and Xbra/Xbra3/Eomes/zVegT KD embryos for neural differentiation marker N-tubulin at early tailbud stage (stage 20-21, lateral view). 1, 2 and 3 mark the position of cross-sections through control, Xbra/Xbra3 KD and Xbra/Xbra3/zVegT KD embryos. Abbreviations: no, notochord; nt, neural tube (d, dorsal; v, ventral); pm, paraxial mesoderm. (B) WMISH for actc1 , hoxd8 and tal1 at late tailbud stage illustrates the loss of mesodermal derivatives such as skeletal muscle (sm), heart (he), pronephros (pn) and ventral blood island (vbi) upon Xbra/Xbra3/Eomes/zVegT KD. Statistics in bottom right corner indicates the number of embryos observed with the depicted WMISH pattern versus the number of embryos analyzed in total. (C) TUNEL staining on control, Xbra/Xbra3 and Xbra/Xbra3/Eomes/zVegT KD embryos (cleared with Murray’s clear) at early tailbud stage (stage 20). Positive controls, embryos treated for 4 hr in 35 μM cycloheximide (chx) and fixed embryos incubated with DNase I. Arrowheads mark apoptotic cells in the brain region (where apoptosis can occasionally be observed in embryos even under normal conditions) and the posterior nervous system induced by cycloheximide. Scale bar, 0.2 mm.

    Techniques Used: Marker, TUNEL Assay, Staining, Incubation

    9) Product Images from "A Re-Examination of Global Suppression of RNA Interference by HIV-1"

    Article Title: A Re-Examination of Global Suppression of RNA Interference by HIV-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017246

    The effect of Tat over-expression on the silencing potency of miEGFP. (A) Immunoblot analysis of protein extracts obtained from P4R5 cells 2 days post-transfection with indicated plasmids. The blot was analyzed using antibodies specific to EGFP and β-actin, which serves as a loading control. (B) Spot-densitometry analysis of two individual experiments, as described in (A). The data are shown as the ratio of EGFP to β-actin and presented as the percentage of control (no miEGFP, no Tat). Error bars represents standard deviation from 3 replicates. (C) Total RNA was extracted from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to quantitate EGFP mRNA. The data are normalized to β-actin and presented as fold change over control (no miEGFP). Error bars represent standard deviation from 3 replicates. (D) Lower panel: the RNA preparation from (C) was subjected to primer extension reaction to detect mature miEGFP. Upper panel: U6 RNA was detected by northern blotting to confirm RNA integrity and quantification.
    Figure Legend Snippet: The effect of Tat over-expression on the silencing potency of miEGFP. (A) Immunoblot analysis of protein extracts obtained from P4R5 cells 2 days post-transfection with indicated plasmids. The blot was analyzed using antibodies specific to EGFP and β-actin, which serves as a loading control. (B) Spot-densitometry analysis of two individual experiments, as described in (A). The data are shown as the ratio of EGFP to β-actin and presented as the percentage of control (no miEGFP, no Tat). Error bars represents standard deviation from 3 replicates. (C) Total RNA was extracted from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to quantitate EGFP mRNA. The data are normalized to β-actin and presented as fold change over control (no miEGFP). Error bars represent standard deviation from 3 replicates. (D) Lower panel: the RNA preparation from (C) was subjected to primer extension reaction to detect mature miEGFP. Upper panel: U6 RNA was detected by northern blotting to confirm RNA integrity and quantification.

    Techniques Used: Over Expression, Transfection, Standard Deviation, Quantitative RT-PCR, Northern Blot

    Efficacy of EGFP silencing by miEGFP in the presence of HIV-1 replication. (A) Protein extracts were prepared 2d post-transfection from 293T cells transfected with pCMV-dsEGFP and the indicated plasmids, and then analyzed by immunoblotting with antibodies specific to EGFP and β-actin. Results show two replicates from the same experiment. (B) Total RNA was isolated from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to determine the level of EGFP mRNA. The data are normalized to β-actin mRNA and presented as fold change over control. Two individual replicates from the same experiment are shown. Error bars represent standard deviation from three qPCR replicates of the same sample.
    Figure Legend Snippet: Efficacy of EGFP silencing by miEGFP in the presence of HIV-1 replication. (A) Protein extracts were prepared 2d post-transfection from 293T cells transfected with pCMV-dsEGFP and the indicated plasmids, and then analyzed by immunoblotting with antibodies specific to EGFP and β-actin. Results show two replicates from the same experiment. (B) Total RNA was isolated from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to determine the level of EGFP mRNA. The data are normalized to β-actin mRNA and presented as fold change over control. Two individual replicates from the same experiment are shown. Error bars represent standard deviation from three qPCR replicates of the same sample.

    Techniques Used: Transfection, Isolation, Quantitative RT-PCR, Standard Deviation, Real-time Polymerase Chain Reaction

    The impact of Dicer depletion on HIV-1 replication in 293T cells. (A) Dicer knockdown was confirmed by immunoblotting total protein extracts prepared 2d post-transfection from 293T cells transfected with indicated amounts of pU6-miDicer. The immunoblot was incubated with antibody specific to Dicer and β-actin that serves as a loading control. (B) 293T cells were transfected with pLAI, together with pmiEGFP or pU6-miDicer, as indicated. 2d post transfection infectious virus released into the supernatant was assayed using P4R5 indicator cells (see Materials and Methods ). Error bars represent standard deviation from 6 replicates. (C) Total RNA isolated from cells in (B) was subjected to semi-quantitative RT-PCR to determine the mRNA levels of Dicer, HIV-1 Tat, and β-actin. Following PCR, products were analyzed by electrophoresis in 2% agarose and ethidium bromide staining. PCR products are resolved on the same gel and irrelevant samples are cropped out. D) Total RNA isolated in (C) was treated with DNase, and following cDNA synthesis with a Gag mRNA specific primer, semi-quantitative PCR was performed and products were analyzed as in (C). PCR for β-actin following oligo(dT)-primed cDNA synthesis serves as a loading control.
    Figure Legend Snippet: The impact of Dicer depletion on HIV-1 replication in 293T cells. (A) Dicer knockdown was confirmed by immunoblotting total protein extracts prepared 2d post-transfection from 293T cells transfected with indicated amounts of pU6-miDicer. The immunoblot was incubated with antibody specific to Dicer and β-actin that serves as a loading control. (B) 293T cells were transfected with pLAI, together with pmiEGFP or pU6-miDicer, as indicated. 2d post transfection infectious virus released into the supernatant was assayed using P4R5 indicator cells (see Materials and Methods ). Error bars represent standard deviation from 6 replicates. (C) Total RNA isolated from cells in (B) was subjected to semi-quantitative RT-PCR to determine the mRNA levels of Dicer, HIV-1 Tat, and β-actin. Following PCR, products were analyzed by electrophoresis in 2% agarose and ethidium bromide staining. PCR products are resolved on the same gel and irrelevant samples are cropped out. D) Total RNA isolated in (C) was treated with DNase, and following cDNA synthesis with a Gag mRNA specific primer, semi-quantitative PCR was performed and products were analyzed as in (C). PCR for β-actin following oligo(dT)-primed cDNA synthesis serves as a loading control.

    Techniques Used: Transfection, Incubation, Standard Deviation, Isolation, Quantitative RT-PCR, Polymerase Chain Reaction, Electrophoresis, Staining, Real-time Polymerase Chain Reaction

    10) Product Images from "C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci"

    Article Title: C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci

    Journal: Acta Neuropathologica

    doi: 10.1007/s00401-013-1200-z

    Antisense RNA foci are a consistent and specific feature of C9FTLD. RNA FISH for antisense foci ( green ) was combined with immunostaining for neurons with NeuN ( red ) and nuclear DNA staining with DAPI ( blue ). a Representative images from the frontal cortex, hippocampus and cerebellum from a neurologically normal control and heterozygous (C9 Het) and homozygous (C9 Hom) C9FTLD cases. Blind quantification of antisense RNA foci in frontal cortical neurons in C9FTLD cases and controls ( b ) and granule cell neurons of the hippocampus and cerebellum in C9FTLD cases only ( c ). d Representative images from a heterozygous C9FTLD case show antisense RNA foci are present in astrocytes, microglia, and oligodendrocytes. e RNA FISH for antisense foci was combined with RNase or DNase treatment, confirming the antisense probe detects RNA and not DNA. Scale bar represents 2 μm in all panels. In b , c , each dot represents an individual case with the homozygous C9FTLD case shown in red , and the average and SEM of heterozygous cases shown as long and short horizontal bars, respectively
    Figure Legend Snippet: Antisense RNA foci are a consistent and specific feature of C9FTLD. RNA FISH for antisense foci ( green ) was combined with immunostaining for neurons with NeuN ( red ) and nuclear DNA staining with DAPI ( blue ). a Representative images from the frontal cortex, hippocampus and cerebellum from a neurologically normal control and heterozygous (C9 Het) and homozygous (C9 Hom) C9FTLD cases. Blind quantification of antisense RNA foci in frontal cortical neurons in C9FTLD cases and controls ( b ) and granule cell neurons of the hippocampus and cerebellum in C9FTLD cases only ( c ). d Representative images from a heterozygous C9FTLD case show antisense RNA foci are present in astrocytes, microglia, and oligodendrocytes. e RNA FISH for antisense foci was combined with RNase or DNase treatment, confirming the antisense probe detects RNA and not DNA. Scale bar represents 2 μm in all panels. In b , c , each dot represents an individual case with the homozygous C9FTLD case shown in red , and the average and SEM of heterozygous cases shown as long and short horizontal bars, respectively

    Techniques Used: Fluorescence In Situ Hybridization, Immunostaining, Staining

    DNase and RNase treatments confirm specificity of RNA foci. a RNA FISH for sense foci ( red ) or CTG repeats [(CAG) 7 probe, bottom right panel ] was combined with nuclear DNA staining with DAPI ( blue ) in the hippocampus of the homozygous C9FTLD case. RNA foci were not detected after RNase treatment but were still present after DNase treatment, which was confirmed by blind quantification. b Loss of DAPI staining ( bottom left panel ) confirmed the DNase was effective. No signal was observed for the (CAG) 7 probe, suggesting the sense RNA foci are not due to non-specific binding of the RNA probe. Scale bar represents 5 μm
    Figure Legend Snippet: DNase and RNase treatments confirm specificity of RNA foci. a RNA FISH for sense foci ( red ) or CTG repeats [(CAG) 7 probe, bottom right panel ] was combined with nuclear DNA staining with DAPI ( blue ) in the hippocampus of the homozygous C9FTLD case. RNA foci were not detected after RNase treatment but were still present after DNase treatment, which was confirmed by blind quantification. b Loss of DAPI staining ( bottom left panel ) confirmed the DNase was effective. No signal was observed for the (CAG) 7 probe, suggesting the sense RNA foci are not due to non-specific binding of the RNA probe. Scale bar represents 5 μm

    Techniques Used: Fluorescence In Situ Hybridization, CTG Assay, Staining, Binding Assay

    11) Product Images from "One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles"

    Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17951-5

    His-tagged MS2 phage-like (His-tagged MS2 PLP) particles stability testing. Agarose gel electrophoresis of His-tagged MS2 PLP, DNA and RNA treated (+) and not treated (−) with DNase and/or RNase; M, marker, 2-log DNA ladder (New England Biolabs, UK); the marker values are in kb. The figure was cropped, full-length gel is presented in Supplementary Figure 5 .
    Figure Legend Snippet: His-tagged MS2 phage-like (His-tagged MS2 PLP) particles stability testing. Agarose gel electrophoresis of His-tagged MS2 PLP, DNA and RNA treated (+) and not treated (−) with DNase and/or RNase; M, marker, 2-log DNA ladder (New England Biolabs, UK); the marker values are in kb. The figure was cropped, full-length gel is presented in Supplementary Figure 5 .

    Techniques Used: Plasmid Purification, Agarose Gel Electrophoresis, Marker

    12) Product Images from "Strand-specific deep sequencing of the transcriptome"

    Article Title: Strand-specific deep sequencing of the transcriptome

    Journal: Genome Research

    doi: 10.1101/gr.094318.109

    Comparative evaluation of deep sequencing. ( A ) Cumulative coverage of read data set, expressed as the ratio of sequenced bases located in annotated genes and the total number of bases in annotated genes. Exhaustive coverage is reached after 14 lanes (Supplemental Fig. 5). ( B ) Comparison of dynamic range of expression measurements (protein coding genes only) on tiling array and DSSS expressed on a log2 scale. ( C ) Validation of DSSS results using qPCR. DNase-treated RNA was reverse transcribed and subjected to SYBR green real-time PCR. Non-reverse-transcribed controls were included for each gene, as well as a genomic DNA dilution series. DSSS signal corresponds to the log2 transformed mean number of counts along the gene.
    Figure Legend Snippet: Comparative evaluation of deep sequencing. ( A ) Cumulative coverage of read data set, expressed as the ratio of sequenced bases located in annotated genes and the total number of bases in annotated genes. Exhaustive coverage is reached after 14 lanes (Supplemental Fig. 5). ( B ) Comparison of dynamic range of expression measurements (protein coding genes only) on tiling array and DSSS expressed on a log2 scale. ( C ) Validation of DSSS results using qPCR. DNase-treated RNA was reverse transcribed and subjected to SYBR green real-time PCR. Non-reverse-transcribed controls were included for each gene, as well as a genomic DNA dilution series. DSSS signal corresponds to the log2 transformed mean number of counts along the gene.

    Techniques Used: Sequencing, Expressing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Transformation Assay

    13) Product Images from "Thiol reductive stress induces cellulose-anchored biofilm formation in Mycobacterium tuberculosis"

    Article Title: Thiol reductive stress induces cellulose-anchored biofilm formation in Mycobacterium tuberculosis

    Journal: Nature Communications

    doi: 10.1038/ncomms11392

    Mtb biofilms are disrupted by cellulase and protease treatments. Mature Mtb biofilms were treated with cellulase (5 mg ml −1 ), alpha-amylase, proteinase K (100 μg ml −1 ), lipase (1 mg ml −1 ) and DNase (2 U). ( a ) CLSM of mature Mtb biofilms overexpressing GFP following treatment with cellulase, lipase, proteinase K, DNase or their respective controls. EPS of Mtb biofilm was destroyed after treatment with cellulase and proteinase K, as depicted by significantly small cross-section compared with the control. ( b ) CV assays of Mtb biofilms after treatment with cellulase, lipase, proteinase K or DNase. Cellulase and proteinase K disrupted biofilm, whereas lipase, DNase and α-amylase had little effect on the biofilms. ( c ) Concentration of glucose released on treating Mtb biofilms with cellulase as measured by a glucose assay kit (Sigma). ( d ) Cellulase treatment of biofilms resulted in significantly higher reduction of 3,5-dinitrosalicylic acid (DNS) by the reducing sugars released on the cellulase treatment. ( e ) The biomass in the untreated biofilms and the biofilms independently treated with the cellulase, lipase, proteinase K or DNase was estimated using COMSTAT. The data presented in b – e are expressed as the mean (±s.e.m.). Statistical significance was determined using Student's t -test. * P
    Figure Legend Snippet: Mtb biofilms are disrupted by cellulase and protease treatments. Mature Mtb biofilms were treated with cellulase (5 mg ml −1 ), alpha-amylase, proteinase K (100 μg ml −1 ), lipase (1 mg ml −1 ) and DNase (2 U). ( a ) CLSM of mature Mtb biofilms overexpressing GFP following treatment with cellulase, lipase, proteinase K, DNase or their respective controls. EPS of Mtb biofilm was destroyed after treatment with cellulase and proteinase K, as depicted by significantly small cross-section compared with the control. ( b ) CV assays of Mtb biofilms after treatment with cellulase, lipase, proteinase K or DNase. Cellulase and proteinase K disrupted biofilm, whereas lipase, DNase and α-amylase had little effect on the biofilms. ( c ) Concentration of glucose released on treating Mtb biofilms with cellulase as measured by a glucose assay kit (Sigma). ( d ) Cellulase treatment of biofilms resulted in significantly higher reduction of 3,5-dinitrosalicylic acid (DNS) by the reducing sugars released on the cellulase treatment. ( e ) The biomass in the untreated biofilms and the biofilms independently treated with the cellulase, lipase, proteinase K or DNase was estimated using COMSTAT. The data presented in b – e are expressed as the mean (±s.e.m.). Statistical significance was determined using Student's t -test. * P

    Techniques Used: Confocal Laser Scanning Microscopy, Concentration Assay, Glucose Assay

    Effect of ECM-degrading enzymes on the development of Mtb biofilms. ( a ) Mtb cells overexpressing GFP were treated with 6 mM DTT to induce TRS. After 3 h of DTT exposure, the cultures were treated with proteinase K, cellulase, α-amylase, lipase or DNase. After 29 h, the effects of various enzymes on the formation of Mtb biofilms were observed through CLSM. Cellulase and proteinase K inhibited the Mtb biofilm formation, suggesting that cellulose fibres and unidentified structural proteins play a critical role in the early stages of biofilm attachment. In contrast, amylase, lipase and DNase had no effect on the structural integrity of biofilm initiation and maturation. ( b ) The biomass of biofilms developed in the presence of enzymes capable of degrading the ECM was estimated using COMSTAT. ( c ) CV assays of Mtb biofilms developed despite the presence of cellulase, lipase, proteinase K and DNase. Cellulase and proteinase K inhibited biofilm development, whereas lipase, DNase and α-amylase had no effect on the biofilm formation and hence on the CV staining. The data presented in b , c are expressed as the mean (±s.e.m.). Statistical significance was determined using Student's t -test. * P
    Figure Legend Snippet: Effect of ECM-degrading enzymes on the development of Mtb biofilms. ( a ) Mtb cells overexpressing GFP were treated with 6 mM DTT to induce TRS. After 3 h of DTT exposure, the cultures were treated with proteinase K, cellulase, α-amylase, lipase or DNase. After 29 h, the effects of various enzymes on the formation of Mtb biofilms were observed through CLSM. Cellulase and proteinase K inhibited the Mtb biofilm formation, suggesting that cellulose fibres and unidentified structural proteins play a critical role in the early stages of biofilm attachment. In contrast, amylase, lipase and DNase had no effect on the structural integrity of biofilm initiation and maturation. ( b ) The biomass of biofilms developed in the presence of enzymes capable of degrading the ECM was estimated using COMSTAT. ( c ) CV assays of Mtb biofilms developed despite the presence of cellulase, lipase, proteinase K and DNase. Cellulase and proteinase K inhibited biofilm development, whereas lipase, DNase and α-amylase had no effect on the biofilm formation and hence on the CV staining. The data presented in b , c are expressed as the mean (±s.e.m.). Statistical significance was determined using Student's t -test. * P

    Techniques Used: Confocal Laser Scanning Microscopy, Staining

    14) Product Images from "Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula"

    Article Title: Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula

    Journal: Scientific Reports

    doi: 10.1038/srep27587

    DNase and RNase activities and degradation of DNA by Nezara viridula nucleases. ( a ) Total and specific nuclease activities in gut, salivary gland and saliva (letters indicate significantly different groups; P ≤ 0.05; Student’s t -test). ( b ) Degradation of DNA by DNases from gut, salivary gland and saliva. Samples (gut, salivary gland or saliva) were incubated with DNA for 5, 10 and 30 min. Samples were then run on 2% agarose gels and DNA visualized by ethidium bromide staining. TURBO™ DNase (DNase) was used as a positive control.
    Figure Legend Snippet: DNase and RNase activities and degradation of DNA by Nezara viridula nucleases. ( a ) Total and specific nuclease activities in gut, salivary gland and saliva (letters indicate significantly different groups; P ≤ 0.05; Student’s t -test). ( b ) Degradation of DNA by DNases from gut, salivary gland and saliva. Samples (gut, salivary gland or saliva) were incubated with DNA for 5, 10 and 30 min. Samples were then run on 2% agarose gels and DNA visualized by ethidium bromide staining. TURBO™ DNase (DNase) was used as a positive control.

    Techniques Used: Incubation, Staining, Positive Control

    15) Product Images from "Programmed synthesis of 3D tissues"

    Article Title: Programmed synthesis of 3D tissues

    Journal: Nature methods

    doi: 10.1038/nmeth.3553

    Programming the reconstitution of fully ECM-embedded 3D microtissues by DNA-programmed assembly (DPAC) (a) Scheme showing the relationship between DNA spots (colored squares), DNA-programmed connectivity (colored lines), and multistep assembly. (b) Incubation of cells with lipid-modified oligonucleotides results in chemical remodeling of cell surfaces. Combining cells bearing complementary cell-surface oligonucleotides forms a temporary chemical adhesion. (c) 7 μm amino-modified DNA spots are patterned onto aldehyde-coated glass slides and covalently linked to the surface by reductive amination. Cells bearing complementary cell-surface oligonucleotides are introduced above the patterned substrate at high concentration and at controlled flow rate using a flow cell. Cells adhere to the appropriate DNA spot, and excess cells are removed by gentle washing. Iteration of this process assembles the microtissue into the third dimension. Addition of liquid ECM incorporating DNase releases the assembled microtissues from the template where they are trapped in the embedding ECM as it gels. The gel is peeled off the glass, releasing the tissues. Underlay of the gel with additional ECM results in a fully embedded 3D culture. Cells interact with each other and their microenvironment as they condense into 3D microtissues. (d) Implementation of the scheme described in Figure 1a–c using MCF10A mammary epithelial cells showing (i) DNA spots, (ii) cells in flow cell, and (iii) single cell array followed by additional rounds of programmed assembly. X,Z reconstructions show an unstained MCF10A cell aggregate embedded between Alexa Fluor-488 and Alexa Fluor 555-stained layers of Matrigel at (iv) 0 and (v) 24 hr. All scale bars are 100 μm.
    Figure Legend Snippet: Programming the reconstitution of fully ECM-embedded 3D microtissues by DNA-programmed assembly (DPAC) (a) Scheme showing the relationship between DNA spots (colored squares), DNA-programmed connectivity (colored lines), and multistep assembly. (b) Incubation of cells with lipid-modified oligonucleotides results in chemical remodeling of cell surfaces. Combining cells bearing complementary cell-surface oligonucleotides forms a temporary chemical adhesion. (c) 7 μm amino-modified DNA spots are patterned onto aldehyde-coated glass slides and covalently linked to the surface by reductive amination. Cells bearing complementary cell-surface oligonucleotides are introduced above the patterned substrate at high concentration and at controlled flow rate using a flow cell. Cells adhere to the appropriate DNA spot, and excess cells are removed by gentle washing. Iteration of this process assembles the microtissue into the third dimension. Addition of liquid ECM incorporating DNase releases the assembled microtissues from the template where they are trapped in the embedding ECM as it gels. The gel is peeled off the glass, releasing the tissues. Underlay of the gel with additional ECM results in a fully embedded 3D culture. Cells interact with each other and their microenvironment as they condense into 3D microtissues. (d) Implementation of the scheme described in Figure 1a–c using MCF10A mammary epithelial cells showing (i) DNA spots, (ii) cells in flow cell, and (iii) single cell array followed by additional rounds of programmed assembly. X,Z reconstructions show an unstained MCF10A cell aggregate embedded between Alexa Fluor-488 and Alexa Fluor 555-stained layers of Matrigel at (iv) 0 and (v) 24 hr. All scale bars are 100 μm.

    Techniques Used: Incubation, Modification, Concentration Assay, Flow Cytometry, Staining

    16) Product Images from "Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes"

    Article Title: Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt356

    Reverse transcriptase–PCR of tRNA Arg I CG from M. capricolum and B. subtilis . ( A ) Comparison of the nucleotide sequences of M. capricolum (Mca) and B. subtilis (Bsu) tRNA Arg I CG , obtained from ( 15 ). The cloverleaf structures are shown. I, 4, D, K, P, 7 and T represent inosine, 4-thio-uridine, dihydrouridine, 1-methylguanosine, pseudouridine, 7-methylguanosine and 5-methyluridine (ribosylthymine), respectively. Regions of primers for reverse transcription of the first strand (and first primers for PCR) are shown with black arrows. Regions of the second primers for PCR are shown with gray arrows. ( B ) Summary of sequences of cDNA clones for M. capricolum and B. subtilis tRNA Arg I CG . The DNA sequences of the cDNA clones, except for the PCR primer regions, are shown in brackets. The RNA sequences corresponding to the obtained DNA sequences are shown in parentheses. I (inosine) in the RNA sequence corresponds to G in the DNA sequence obtained by reverse transcription. ( C ) Agarose gel electrophoresis of reverse transcriptase–PCR products. Lane M: size marker (100-bp ladder, the position of 100 bp is shown with an arrow). Lanes 1–10: PCR products of various templates. Lane 1: reverse-transcribed McatRNA Arg I CG solution treated with DNase before reverse transcription. Lane 2: total McatRNA solution with DNase treatment. Lane 3: Reverse-transcribed McatRNA Arg I CG solution without DNase treatment before reverse transcription. Lane 4: total McatRNA solution without DNase treatment. Lane 6: reverse-transcribed BsutRNA Arg I CG solution with DNase treatment before reverse transcription. Lane 7: total BsutRNA solution with DNase treatment. Lane 8: reverse-transcribed BsutRNA Arg I CG solution without DNase treatment before reverse transcription. Lane 9: total BsutRNA solution without DNase treatment. Lanes 5 and 10: control (no RNA/DNA).
    Figure Legend Snippet: Reverse transcriptase–PCR of tRNA Arg I CG from M. capricolum and B. subtilis . ( A ) Comparison of the nucleotide sequences of M. capricolum (Mca) and B. subtilis (Bsu) tRNA Arg I CG , obtained from ( 15 ). The cloverleaf structures are shown. I, 4, D, K, P, 7 and T represent inosine, 4-thio-uridine, dihydrouridine, 1-methylguanosine, pseudouridine, 7-methylguanosine and 5-methyluridine (ribosylthymine), respectively. Regions of primers for reverse transcription of the first strand (and first primers for PCR) are shown with black arrows. Regions of the second primers for PCR are shown with gray arrows. ( B ) Summary of sequences of cDNA clones for M. capricolum and B. subtilis tRNA Arg I CG . The DNA sequences of the cDNA clones, except for the PCR primer regions, are shown in brackets. The RNA sequences corresponding to the obtained DNA sequences are shown in parentheses. I (inosine) in the RNA sequence corresponds to G in the DNA sequence obtained by reverse transcription. ( C ) Agarose gel electrophoresis of reverse transcriptase–PCR products. Lane M: size marker (100-bp ladder, the position of 100 bp is shown with an arrow). Lanes 1–10: PCR products of various templates. Lane 1: reverse-transcribed McatRNA Arg I CG solution treated with DNase before reverse transcription. Lane 2: total McatRNA solution with DNase treatment. Lane 3: Reverse-transcribed McatRNA Arg I CG solution without DNase treatment before reverse transcription. Lane 4: total McatRNA solution without DNase treatment. Lane 6: reverse-transcribed BsutRNA Arg I CG solution with DNase treatment before reverse transcription. Lane 7: total BsutRNA solution with DNase treatment. Lane 8: reverse-transcribed BsutRNA Arg I CG solution without DNase treatment before reverse transcription. Lane 9: total BsutRNA solution without DNase treatment. Lanes 5 and 10: control (no RNA/DNA).

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Sequencing, Agarose Gel Electrophoresis, Electrophoresis, Marker

    17) Product Images from "Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants"

    Article Title: Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00680-17

    Effect of nuclease treatment on extracted HBV DNA and RNA from the supernatant of infected HepaRG cells treated with HBV inhibitors. Cells were incubated with DMSO or 30 μM NVR 3-1983 or LMV for 6 days. HBV DNA and RNA from culture supernatants were extracted and treated with DNase or RNase, prior to quantitative assays. Extracellular HBV DNA (A) and HBV RNA (B) levels were normalized to those for DMSO-treated samples without nuclease treatment. Results and error bars represented means and standard deviations from at least three independent experiments, respectively.
    Figure Legend Snippet: Effect of nuclease treatment on extracted HBV DNA and RNA from the supernatant of infected HepaRG cells treated with HBV inhibitors. Cells were incubated with DMSO or 30 μM NVR 3-1983 or LMV for 6 days. HBV DNA and RNA from culture supernatants were extracted and treated with DNase or RNase, prior to quantitative assays. Extracellular HBV DNA (A) and HBV RNA (B) levels were normalized to those for DMSO-treated samples without nuclease treatment. Results and error bars represented means and standard deviations from at least three independent experiments, respectively.

    Techniques Used: Infection, Incubation

    18) Product Images from "N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection"

    Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006995

    KSHV mRNA contains m 6 A modifications. (A) Two independent replicates of iSLK.219 cells containing latent KSHV were treated with dox for 5 days to induce the viral lytic cycle (induced) or left untreated to preserve viral latency (uninduced). DNase-treated RNA was isolated and subjected to m 6 Aseq. Displayed are peaks with a fold change of four or higher, comparing reads in the m 6 A-IP to the corresponding input. Numbers above peaks correspond to the base position within the KSHV genome. (B) Overview of sequencing reads from induced and uninduced m 6 A IP samples, aligned to the ORF50 transcript and the annotated GG(m 6 A)C consensus motifs found in exon 2 of ORF50. Numbers to the left of sequencing reads indicate the scale of the read count. (C) Cells were induced as in (A), and total RNA was subjected to m 6 A RIP, followed by RT-qPCR using primers for the indicated viral and cellular genes. Values are displayed as fold change over input, normalized to GAPDH. (D) Quantification of cellular m 6 A peaks from m 6 Aseq analysis.
    Figure Legend Snippet: KSHV mRNA contains m 6 A modifications. (A) Two independent replicates of iSLK.219 cells containing latent KSHV were treated with dox for 5 days to induce the viral lytic cycle (induced) or left untreated to preserve viral latency (uninduced). DNase-treated RNA was isolated and subjected to m 6 Aseq. Displayed are peaks with a fold change of four or higher, comparing reads in the m 6 A-IP to the corresponding input. Numbers above peaks correspond to the base position within the KSHV genome. (B) Overview of sequencing reads from induced and uninduced m 6 A IP samples, aligned to the ORF50 transcript and the annotated GG(m 6 A)C consensus motifs found in exon 2 of ORF50. Numbers to the left of sequencing reads indicate the scale of the read count. (C) Cells were induced as in (A), and total RNA was subjected to m 6 A RIP, followed by RT-qPCR using primers for the indicated viral and cellular genes. Values are displayed as fold change over input, normalized to GAPDH. (D) Quantification of cellular m 6 A peaks from m 6 Aseq analysis.

    Techniques Used: Isolation, Sequencing, Quantitative RT-PCR

    19) Product Images from "Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development"

    Article Title: Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development

    Journal:

    doi: 10.1128/IAI.05861-11

    Py-MIF transcript is expressed in liver-stage (LS) and blood-stage (BS) parasites. The expression of Py- mif was determined by RT-PCR on RNA derived from P. yoelii 17XNL. RNA was isolated from salivary gland sporozoites, mixed blood-stage parasites, or mouse livers infected with 1 × 106 sporozoites (isolated at 24 or 44 hpi). Genomic DNA was removed by DNase treatment, and cDNA was generated by reverse transcriptase single-strand DNA synthesis. Either Py- mif or 18S rRNA was amplified from cDNAs via 35 cycles of PCR. Non-RT controls (−RT) were included to confirm that mif amplification was due to cDNA and not to residual genomic DNA.
    Figure Legend Snippet: Py-MIF transcript is expressed in liver-stage (LS) and blood-stage (BS) parasites. The expression of Py- mif was determined by RT-PCR on RNA derived from P. yoelii 17XNL. RNA was isolated from salivary gland sporozoites, mixed blood-stage parasites, or mouse livers infected with 1 × 106 sporozoites (isolated at 24 or 44 hpi). Genomic DNA was removed by DNase treatment, and cDNA was generated by reverse transcriptase single-strand DNA synthesis. Either Py- mif or 18S rRNA was amplified from cDNAs via 35 cycles of PCR. Non-RT controls (−RT) were included to confirm that mif amplification was due to cDNA and not to residual genomic DNA.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Isolation, Infection, Generated, DNA Synthesis, Amplification, Polymerase Chain Reaction

    20) Product Images from "Integrated Analysis of Dysregulated lncRNA Expression in Fetal Cardiac Tissues with Ventricular Septal Defect"

    Article Title: Integrated Analysis of Dysregulated lncRNA Expression in Fetal Cardiac Tissues with Ventricular Septal Defect

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077492

    Bioinformatics analysis of ENST00000513542. ( A ) ENST00000513542 is a natural antisense lncRNA, transcribed from 2,105 bp downstream of the second exon of the Smad1 gene. ( B ) Several tracks of interest, including conservation, histone markings, DNase hypersensitivity, and TFBS are displayed. Integrated Regulation track data for a region spanning the ENST0000051354 are shown. The red box shows a region of overlap between the various tracks. ( C ) Transcription factor binding sites (TFBS) prediction indicated that ENST0000051354 loci combine with AP-1 as a cis-acting element. ( D ) Further catRAPID analysis indicated a strong RNA-protein interaction between ENST0000051354 and TCF-4, which is the TF of SMAD1.
    Figure Legend Snippet: Bioinformatics analysis of ENST00000513542. ( A ) ENST00000513542 is a natural antisense lncRNA, transcribed from 2,105 bp downstream of the second exon of the Smad1 gene. ( B ) Several tracks of interest, including conservation, histone markings, DNase hypersensitivity, and TFBS are displayed. Integrated Regulation track data for a region spanning the ENST0000051354 are shown. The red box shows a region of overlap between the various tracks. ( C ) Transcription factor binding sites (TFBS) prediction indicated that ENST0000051354 loci combine with AP-1 as a cis-acting element. ( D ) Further catRAPID analysis indicated a strong RNA-protein interaction between ENST0000051354 and TCF-4, which is the TF of SMAD1.

    Techniques Used: Binding Assay

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    Real-time Polymerase Chain Reaction:

    Article Title: Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions
    Article Snippet: Above 12,500 pg total RNA per 25 ?l reaction, we begin to observe problematic qPCR inhibitory phenomena (with Trizol® -isolated tissue total RNA) of Type 1, Type 2, Type 3 (and presumably Type 4) varieties. .. Interestingly, at first, the qPCR inhibition we observed seemed to be either a byproduct of Turbo-DNase (Ambion) treatment (Type 5 inhibition), or rRNA and tRNA inhibition of the RT enzyme during reverse transcription (Type 1 inhibition). .. But, then it became apparent that this inhibition was more likely due to the method of total RNA isolation (our final Turbo-DNase treated RNA samples never comprised more than 26% of each final one-step real-time qPCR reaction volume; an amount that is safely within Ambion product literature guidelines regarding the proper use of Turbo DNase-treated RNA in qPCR reactions).

    Article Title: Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development
    Article Snippet: Total RNA was extracted using TRIzol reagent (Invitrogen) from P. yoelii salivary gland sporozoites (2 × 106 ), mixed blood-stage parasites (1 × 107 infected red blood cells were isolated from an infected mouse by heart puncture bleeding and serial limiting dilution of infected blood), or infected livers and then treated with Turbo DNase (Ambion). cDNA synthesis was performed using a SuperScript III Platinum two-step quantitative reverse transcription-PCR (qRT-PCR) kit according to the manufacturer's instructions (Invitrogen). .. Thirty-five PCR amplification cycles were performed (95°C for 30 s for DNA denaturation and 60°C for 4 min for primer annealing and DNA strand extension).

    Article Title: Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA
    Article Snippet: Paragraph title: Quantitative real-time PCR. ... Residual DNA contamination in the RNA sample was removed using Turbo DNase (Ambion Biosystems).

    Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection
    Article Snippet: Total cellular RNA (containing KSHV RNA) was extracted and purified by TRIzol and then DNAse treated with Turbo DNase (Ambion). .. Total cellular RNA (containing KSHV RNA) was extracted and purified by TRIzol and then DNAse treated with Turbo DNase (Ambion).

    Plasmid Purification:

    Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles
    Article Snippet: Ferulic acid (Sigma-Aldrich) (12.5 mg/ml in water:acetonitrile:formic acid, 50:33:17, v/v mixture) was used as the MALDI matrix in combination with a stainless steel sample plate. .. Stability of His-tagged MS2 PLP against nucleases was verified by their incubation with the combination of 10 U of Turbo DNase (Ambion) and/or 10 U of RNase A (Qiagen) at 37 °C for 1 hour. .. As controls for the reaction, DNA (500 ng of purified PCR fragment encoding the maturase and single-chain version of the coat protein dimer with His-tag modification) and RNA (500 ng of IAC in vitro transcript ), were added to the tested His-tagged MS2 PLP.

    Incubation:

    Article Title: A Re-Examination of Global Suppression of RNA Interference by HIV-1
    Article Snippet: For downstream applications that required DNase treatment, RNA samples were treated with Turbo DNase (Ambion). .. For downstream applications that required DNase treatment, RNA samples were treated with Turbo DNase (Ambion).

    Article Title: C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci
    Article Snippet: For FISH with both probes, the sense probe was applied first as above, and after the three 30 min 80 °C washes in 50 % formamide/0.5×SSC, the antisense probe was added for 2 h, followed by the protocol above. .. For DNase and RNase treatments the standard protocol above was followed with additional steps: after rehydration in PBS, sections were incubated in 0.5 % Triton/PBS for 10 min, washed once in PBS, and treated with either RNase A (1 mg/ml in PBS/0.05 % Tween-20) or Turbo DNase (Ambion, 200 U/ml in supplied buffer) for 2 h at 37 °C. .. After the treatment sections were washed three times in PBS, once in 2×SSC and then placed in pre-hybridisation solution and the standard FISH protocol resumed.

    Article Title: Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells
    Article Snippet: Sam68-cross-linked in vivo immunoprecipitation (CLIP) was performed as previously described , with minor modifications. .. Cross-linked germ cells were re-suspended in PXL buffer (1 × PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40), sonicated (3 × 30″ with BioRuptor) and incubated with Turbo DNase and Cocktail RNase (Ambion) for 15 min at 37°C. .. Length of digested RNA was ∼200–250 nucleotides.

    Article Title: In Vivo T-Box Transcription Factor Profiling Reveals Joint Regulation of Embryonic Neuromesodermal Bipotency
    Article Snippet: To induce apoptosis in vivo, embryos were treated for 4 hr in 35 μM cycloheximide at 23°C. .. As a positive control for the TUNEL assay fixed embryos were incubated in 0.1 U/μl Turbo DNase (Ambion) for 20 min at room temperature. .. G.E.G. conceived the study and carried out most experiments and postsequencing analysis.

    Article Title: Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA
    Article Snippet: Following 1 h of incubation at 37°C, 2 ml of DMEM containing 2% FBS was added to each well. .. Residual DNA contamination in the RNA sample was removed using Turbo DNase (Ambion Biosystems).

    Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles
    Article Snippet: Ferulic acid (Sigma-Aldrich) (12.5 mg/ml in water:acetonitrile:formic acid, 50:33:17, v/v mixture) was used as the MALDI matrix in combination with a stainless steel sample plate. .. Stability of His-tagged MS2 PLP against nucleases was verified by their incubation with the combination of 10 U of Turbo DNase (Ambion) and/or 10 U of RNase A (Qiagen) at 37 °C for 1 hour. .. As controls for the reaction, DNA (500 ng of purified PCR fragment encoding the maturase and single-chain version of the coat protein dimer with His-tag modification) and RNA (500 ng of IAC in vitro transcript ), were added to the tested His-tagged MS2 PLP.

    Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection
    Article Snippet: Total cellular RNA (containing KSHV RNA) was extracted and purified by TRIzol and then DNAse treated with Turbo DNase (Ambion). .. 30 μl protein G magnetic beads (Invitrogen) were blocked in 1% BSA solution for 1 hour, followed by incubation with 12.5 μg affinity-purified anti-m6 A polyclonal antibody (Millipore) at 4°C for 2 hr with head-over-tail rotation.

    Article Title: Microbial-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management
    Article Snippet: The pellet was resuspended in 180 µL of pre-warmed nuclease-free water plus 20 µL 10× DNase buffer, and incubated at room temperature for 30 minutes. .. Then, after adding 2 µL Turbo DNase and 2 µL RNase (Thermo Scientific) to the tube, the mixture was incubated at 37°C for 1 hour to destroy remaining DNA and single-stranded RNA and then stored at −20°C until use. .. DsRNA was isolated using 200 µL of phenol/chloroform/isoamyl alcohol by vigorously vortexing, followed by centrifugation at 12,000 g for 15 minutes at room temperature.

    Article Title: Toll-Like Receptor 3/TRIF-Dependent IL-12p70 Secretion Mediated by Streptococcus pneumoniae RNA and Its Priming by Influenza A Virus Coinfection in Human Dendritic Cells
    Article Snippet: Total RNA was isolated with TRIzol (Life Technologies) followed by DNA digestion with Turbo DNase (Life Technologies). .. RNA concentration and quality were determined spectrophotometrically with the NanoDrop ND 1000 apparatus, and the absence of DNA contamination was confirmed by PCR.

    Article Title: Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes
    Article Snippet: Twenty micrograms of total tRNA from either M. capricolum or B. subtilis was treated with 4 U of Turbo DNase (Ambion), in the presence of 80 U of RNaseOUT (Invitrogen) for 30 min at 37°C. .. In addition to the first strand cDNA synthesis primers, the following primers 5′-GCCCG-TAGAT-CAATT-GGATA-GATCG-CTTGA-3′ (Mca-2nd) and 5′-GCCCG-TAGCT-CAATG-GATAG-AGCGT-TTGA-3′ (Bsu-2nd) were used for further polymerase chain reaction (PCR) amplification of the cDNAs (gray arrows in A).

    Article Title: Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants
    Article Snippet: To evaluate the specificity of RNA and DNA detection, a viral nucleic acid extraction step was performed on the supernatants of infected HepaRG cells using a ZR viral DNA/RNA kit (Zymo Research). .. Extracted HBV nucleic acids were then incubated with 1 unit of Turbo DNase (Life Technologies) and 0.5 unit of DNase I (Thermo Fisher) or 25 units of RNase If (New England BioLabs) for 1 h at 37°C. .. Both DNase and RNase If were inactivated by incubation at 70°C for 20 min, prior to performing quantitation assays as described above.

    Amplification:

    Article Title: Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development
    Article Snippet: Total RNA was extracted using TRIzol reagent (Invitrogen) from P. yoelii salivary gland sporozoites (2 × 106 ), mixed blood-stage parasites (1 × 107 infected red blood cells were isolated from an infected mouse by heart puncture bleeding and serial limiting dilution of infected blood), or infected livers and then treated with Turbo DNase (Ambion). cDNA synthesis was performed using a SuperScript III Platinum two-step quantitative reverse transcription-PCR (qRT-PCR) kit according to the manufacturer's instructions (Invitrogen). .. The sequences of specific primers used for amplification from cDNA are listed in Table S2 in the supplemental material.

    Article Title: Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification
    Article Snippet: Extracted RNA was treated with 4U of Turbo DNase (2U/μ L, Ambion, Austin, TX, USA) at 37°C for 30 minutes and then inactivated at 72°C for 10 minutes, followed by phenol/chloroform purification. .. Extracted RNA was treated with 4U of Turbo DNase (2U/μ L, Ambion, Austin, TX, USA) at 37°C for 30 minutes and then inactivated at 72°C for 10 minutes, followed by phenol/chloroform purification.

    Article Title: Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes
    Article Snippet: Twenty micrograms of total tRNA from either M. capricolum or B. subtilis was treated with 4 U of Turbo DNase (Ambion), in the presence of 80 U of RNaseOUT (Invitrogen) for 30 min at 37°C. .. Twenty micrograms of total tRNA from either M. capricolum or B. subtilis was treated with 4 U of Turbo DNase (Ambion), in the presence of 80 U of RNaseOUT (Invitrogen) for 30 min at 37°C.

    Activity Assay:

    Article Title: Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula
    Article Snippet: Paragraph title: Nuclease activity assays ... Gut, salivary gland extracts and saliva (5 μg protein per sample in a volume of 10 μL) were added to 200 μL DNA or RNA (100 μg/mL) and the reaction was carried out at 37 °C for 30 min. TURBO™ DNase (5 units) (Life Technologies, Carlsbad, CA, USA) and RNase A (20 μg) (Thermo Scientific, Waltham, MA, USA) were used as positive controls.

    Cell Culture:

    Article Title: Programmed synthesis of 3D tissues
    Article Snippet: Given the capacity of DPAC to directly link complex tissue structural features with specific single and collective cell behaviors, we anticipate that this method will find utility in a variety of contexts, both basic and applied. .. Aldehyde-silanized glass slides (Nexterion® Aldehyde AL, Schott), Sigmacote® (Sigma-Aldrich), Slygard® 184 (Fisher Scientific), sodium borohydride (NaBH4 , ACROS, 98%), Pluronic® F108 NF (BASF), ethanol (Fisher Scientific), trypsin inhibitor from Glycine max (Sigma-Aldrich), Matrigel® (BD Biosciences), rat-tail collagen 1 (BD Biosciences), Turbo DNase (Life Technologies), amine-modified ssDNA (5′-amine-X20 , Operon), PBS (UCSF Cell-Culture Facility), PBS-CMF (UCSF Cell-Culture Facility), trypsin (UCSF Cell-Culture Facility), 100x penicillin/streptomycin, heat-inactivated fetal bovine serum (UCSF Cell-Culture Facility), RPMI media (UCSF Cell-Culture Facility) were used as received without further purification. .. Lipid-modified ssDNA (5′-lipid-T80 -X20 ) was synthesized as previously described .

    Expressing:

    Article Title: H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage
    Article Snippet: Paragraph title: RT-PCR and stage-specific expression of H-IPSE. ... Contaminating genomic DNA was removed by DNase treatment using Turbo DNase (Invitrogen Ambion, USA) and chemical DNase inactivation, according to the manufacturer's instructions.

    Article Title: Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA
    Article Snippet: Residual DNA contamination in the RNA sample was removed using Turbo DNase (Ambion Biosystems). .. Residual DNA contamination in the RNA sample was removed using Turbo DNase (Ambion Biosystems).

    Hybridization:

    Article Title: C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci
    Article Snippet: Paragraph title: RNA fluorescence in situ hybridisation (FISH) with protein immunostaining ... For DNase and RNase treatments the standard protocol above was followed with additional steps: after rehydration in PBS, sections were incubated in 0.5 % Triton/PBS for 10 min, washed once in PBS, and treated with either RNase A (1 mg/ml in PBS/0.05 % Tween-20) or Turbo DNase (Ambion, 200 U/ml in supplied buffer) for 2 h at 37 °C.

    TUNEL Assay:

    Article Title: In Vivo T-Box Transcription Factor Profiling Reveals Joint Regulation of Embryonic Neuromesodermal Bipotency
    Article Snippet: To induce apoptosis in vivo, embryos were treated for 4 hr in 35 μM cycloheximide at 23°C. .. As a positive control for the TUNEL assay fixed embryos were incubated in 0.1 U/μl Turbo DNase (Ambion) for 20 min at room temperature. .. G.E.G. conceived the study and carried out most experiments and postsequencing analysis.

    Transfection:

    Article Title: Toll-Like Receptor 3/TRIF-Dependent IL-12p70 Secretion Mediated by Streptococcus pneumoniae RNA and Its Priming by Influenza A Virus Coinfection in Human Dendritic Cells
    Article Snippet: Paragraph title: RNA transfection. ... Total RNA was isolated with TRIzol (Life Technologies) followed by DNA digestion with Turbo DNase (Life Technologies).

    Concentration Assay:

    Article Title: Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions
    Article Snippet: Final, in-well RNA concentrations were never greater than 0.5 ng/?l in any of these qPCR studies (~0.3 ng/?l seemed to work the best), so inhibition of RT enzyme and/or Taq DNA polymerase by excess RNA in the reaction wells (Type 1 inhibition) was reasonably eliminated as a source of any of the inhibition phenomena witnessed (since by the time most samples reached this final in-well concentration, they had already incurred dilutions of 1:3,000 or greater – certainly outside the range where most forms of inhibition would be reasonably expected, with the possible exception of inhibition Type 4) (See Appendix 2). .. Interestingly, at first, the qPCR inhibition we observed seemed to be either a byproduct of Turbo-DNase (Ambion) treatment (Type 5 inhibition), or rRNA and tRNA inhibition of the RT enzyme during reverse transcription (Type 1 inhibition).

    Article Title: Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples
    Article Snippet: RNA (from AVL) was isolated using the QIAamp Viral RNA Minikit (Qiagen) according to the manufacturer’s protocol, except that 0.1 M final concentration of β-mercaptoethanol was added to each sample. .. All extracted RNA was resuspended in water and treated with Turbo DNase (Ambion) to digest contaminating DNA.

    Article Title: Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification
    Article Snippet: Extracted RNA was treated with 4U of Turbo DNase (2U/μ L, Ambion, Austin, TX, USA) at 37°C for 30 minutes and then inactivated at 72°C for 10 minutes, followed by phenol/chloroform purification. .. Extracted RNA was treated with 4U of Turbo DNase (2U/μ L, Ambion, Austin, TX, USA) at 37°C for 30 minutes and then inactivated at 72°C for 10 minutes, followed by phenol/chloroform purification.

    Protease Inhibitor:

    Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection
    Article Snippet: Total cellular RNA (containing KSHV RNA) was extracted and purified by TRIzol and then DNAse treated with Turbo DNase (Ambion). .. 30 μl protein G magnetic beads (Invitrogen) were blocked in 1% BSA solution for 1 hour, followed by incubation with 12.5 μg affinity-purified anti-m6 A polyclonal antibody (Millipore) at 4°C for 2 hr with head-over-tail rotation.

    Northern Blot:

    Article Title: A Re-Examination of Global Suppression of RNA Interference by HIV-1
    Article Snippet: For downstream applications that required DNase treatment, RNA samples were treated with Turbo DNase (Ambion). .. For downstream applications that required DNase treatment, RNA samples were treated with Turbo DNase (Ambion).

    Infection:

    Article Title: H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage
    Article Snippet: Total RNA was isolated from the following S. haematobium life cycle stages (obtained from the NIAID Schistosomiasis Resource Center for distribution through BEI Resources, NIAID, NIH), using the RNAzol kit (Molecular Research Center) according to the manufacturer's instructions: purified eggs, retrieved from the liver of S. haematobium -infected hamsters; miracidia; cercariae; schistosomula; adult females; adult males; and mixed-sex adult worms. .. Contaminating genomic DNA was removed by DNase treatment using Turbo DNase (Invitrogen Ambion, USA) and chemical DNase inactivation, according to the manufacturer's instructions.

    Article Title: Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development
    Article Snippet: The MIF open reading frame (ORF) was amplified using the primers Sp/PyMIF and Asp/PyMIF (primer sequences are provided in Table S1). .. Total RNA was extracted using TRIzol reagent (Invitrogen) from P. yoelii salivary gland sporozoites (2 × 106 ), mixed blood-stage parasites (1 × 107 infected red blood cells were isolated from an infected mouse by heart puncture bleeding and serial limiting dilution of infected blood), or infected livers and then treated with Turbo DNase (Ambion). cDNA synthesis was performed using a SuperScript III Platinum two-step quantitative reverse transcription-PCR (qRT-PCR) kit according to the manufacturer's instructions (Invitrogen). .. The sequences of specific primers used for amplification from cDNA are listed in Table S2 in the supplemental material.

    Article Title: Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA
    Article Snippet: Confluent HeLa monolayers in 6-well plates were infected with virus at an MOI of 10 in a 350-μl inoculum. .. Residual DNA contamination in the RNA sample was removed using Turbo DNase (Ambion Biosystems).

    Article Title: Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants
    Article Snippet: To evaluate the specificity of RNA and DNA detection, a viral nucleic acid extraction step was performed on the supernatants of infected HepaRG cells using a ZR viral DNA/RNA kit (Zymo Research). .. Extracted HBV nucleic acids were then incubated with 1 unit of Turbo DNase (Life Technologies) and 0.5 unit of DNase I (Thermo Fisher) or 25 units of RNase If (New England BioLabs) for 1 h at 37°C.

    Generated:

    Article Title: Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development
    Article Snippet: Total RNA was extracted using TRIzol reagent (Invitrogen) from P. yoelii salivary gland sporozoites (2 × 106 ), mixed blood-stage parasites (1 × 107 infected red blood cells were isolated from an infected mouse by heart puncture bleeding and serial limiting dilution of infected blood), or infected livers and then treated with Turbo DNase (Ambion). cDNA synthesis was performed using a SuperScript III Platinum two-step quantitative reverse transcription-PCR (qRT-PCR) kit according to the manufacturer's instructions (Invitrogen). .. Thirty-five PCR amplification cycles were performed (95°C for 30 s for DNA denaturation and 60°C for 4 min for primer annealing and DNA strand extension).

    Inhibition:

    Article Title: Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions
    Article Snippet: Above 12,500 pg total RNA per 25 ?l reaction, we begin to observe problematic qPCR inhibitory phenomena (with Trizol® -isolated tissue total RNA) of Type 1, Type 2, Type 3 (and presumably Type 4) varieties. .. Interestingly, at first, the qPCR inhibition we observed seemed to be either a byproduct of Turbo-DNase (Ambion) treatment (Type 5 inhibition), or rRNA and tRNA inhibition of the RT enzyme during reverse transcription (Type 1 inhibition). .. But, then it became apparent that this inhibition was more likely due to the method of total RNA isolation (our final Turbo-DNase treated RNA samples never comprised more than 26% of each final one-step real-time qPCR reaction volume; an amount that is safely within Ambion product literature guidelines regarding the proper use of Turbo DNase-treated RNA in qPCR reactions).

    Enzyme Inhibition Assay:

    Article Title: Thiol reductive stress induces cellulose-anchored biofilm formation in Mycobacterium tuberculosis
    Article Snippet: Paragraph title: Enzyme inhibition assay ... Mtb biofilms were treated with cellulase (T. viride , Calbiochem) at 5 mg ml−1 in citrate buffer, 0.05 M, pH 4 α-amylase (672 U ml−1 ) from Bacillus licheniformis (Sigma), Turbo DNase (0.8 U ml−1 ) (Invitrogen), proteinase K (0.1 mg ml−1 , from Tritirachium album , Sigma) or lipase (0.5 mg ml−1 , from Chromobacterium viscosum , Calbiochem).

    Sequencing:

    Article Title: Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula
    Article Snippet: Gut, salivary gland extracts and saliva (5 μg protein per sample in a volume of 10 μL) were added to 200 μL DNA or RNA (100 μg/mL) and the reaction was carried out at 37 °C for 30 min. TURBO™ DNase (5 units) (Life Technologies, Carlsbad, CA, USA) and RNase A (20 μg) (Thermo Scientific, Waltham, MA, USA) were used as positive controls. .. For dsRNase activity assays, dsRNAs were prepared using the Ambion® MEGAscript® RNAi Kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol.

    Sonication:

    Article Title: Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells
    Article Snippet: Sam68-cross-linked in vivo immunoprecipitation (CLIP) was performed as previously described , with minor modifications. .. Cross-linked germ cells were re-suspended in PXL buffer (1 × PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40), sonicated (3 × 30″ with BioRuptor) and incubated with Turbo DNase and Cocktail RNase (Ambion) for 15 min at 37°C. .. Length of digested RNA was ∼200–250 nucleotides.

    In Vivo:

    Article Title: Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells
    Article Snippet: Paragraph title: Cross-linked in vivo immunoprecipitation ... Cross-linked germ cells were re-suspended in PXL buffer (1 × PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40), sonicated (3 × 30″ with BioRuptor) and incubated with Turbo DNase and Cocktail RNase (Ambion) for 15 min at 37°C.

    Article Title: In Vivo T-Box Transcription Factor Profiling Reveals Joint Regulation of Embryonic Neuromesodermal Bipotency
    Article Snippet: To induce apoptosis in vivo, embryos were treated for 4 hr in 35 μM cycloheximide at 23°C. .. As a positive control for the TUNEL assay fixed embryos were incubated in 0.1 U/μl Turbo DNase (Ambion) for 20 min at room temperature.

    Fluorescence:

    Article Title: C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci
    Article Snippet: Paragraph title: RNA fluorescence in situ hybridisation (FISH) with protein immunostaining ... For DNase and RNase treatments the standard protocol above was followed with additional steps: after rehydration in PBS, sections were incubated in 0.5 % Triton/PBS for 10 min, washed once in PBS, and treated with either RNase A (1 mg/ml in PBS/0.05 % Tween-20) or Turbo DNase (Ambion, 200 U/ml in supplied buffer) for 2 h at 37 °C.

    Multiple Displacement Amplification:

    Article Title: Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification
    Article Snippet: Paragraph title: 2.2. RNA Extraction, Reverse Transcription (RT), and Template-Dependent Multiple Displacement Amplification (tdMDA) ... Extracted RNA was treated with 4U of Turbo DNase (2U/μ L, Ambion, Austin, TX, USA) at 37°C for 30 minutes and then inactivated at 72°C for 10 minutes, followed by phenol/chloroform purification.

    Isolation:

    Article Title: A Re-Examination of Global Suppression of RNA Interference by HIV-1
    Article Snippet: Paragraph title: RNA isolation, primer extension, and RT-PCR ... For downstream applications that required DNase treatment, RNA samples were treated with Turbo DNase (Ambion).

    Article Title: Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions
    Article Snippet: Above 12,500 pg total RNA per 25 ?l reaction, we begin to observe problematic qPCR inhibitory phenomena (with Trizol® -isolated tissue total RNA) of Type 1, Type 2, Type 3 (and presumably Type 4) varieties. .. Interestingly, at first, the qPCR inhibition we observed seemed to be either a byproduct of Turbo-DNase (Ambion) treatment (Type 5 inhibition), or rRNA and tRNA inhibition of the RT enzyme during reverse transcription (Type 1 inhibition).

    Article Title: H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage
    Article Snippet: Total RNA was isolated from the following S. haematobium life cycle stages (obtained from the NIAID Schistosomiasis Resource Center for distribution through BEI Resources, NIAID, NIH), using the RNAzol kit (Molecular Research Center) according to the manufacturer's instructions: purified eggs, retrieved from the liver of S. haematobium -infected hamsters; miracidia; cercariae; schistosomula; adult females; adult males; and mixed-sex adult worms. .. Contaminating genomic DNA was removed by DNase treatment using Turbo DNase (Invitrogen Ambion, USA) and chemical DNase inactivation, according to the manufacturer's instructions.

    Article Title: Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples
    Article Snippet: Paragraph title: Viral RNA isolation ... All extracted RNA was resuspended in water and treated with Turbo DNase (Ambion) to digest contaminating DNA.

    Article Title: Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development
    Article Snippet: The MIF open reading frame (ORF) was amplified using the primers Sp/PyMIF and Asp/PyMIF (primer sequences are provided in Table S1). .. Total RNA was extracted using TRIzol reagent (Invitrogen) from P. yoelii salivary gland sporozoites (2 × 106 ), mixed blood-stage parasites (1 × 107 infected red blood cells were isolated from an infected mouse by heart puncture bleeding and serial limiting dilution of infected blood), or infected livers and then treated with Turbo DNase (Ambion). cDNA synthesis was performed using a SuperScript III Platinum two-step quantitative reverse transcription-PCR (qRT-PCR) kit according to the manufacturer's instructions (Invitrogen). .. The sequences of specific primers used for amplification from cDNA are listed in Table S2 in the supplemental material.

    Article Title: Microbial-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management
    Article Snippet: Paragraph title: Isolation of dsRNA using conventional method ... Then, after adding 2 µL Turbo DNase and 2 µL RNase (Thermo Scientific) to the tube, the mixture was incubated at 37°C for 1 hour to destroy remaining DNA and single-stranded RNA and then stored at −20°C until use.

    Article Title: Toll-Like Receptor 3/TRIF-Dependent IL-12p70 Secretion Mediated by Streptococcus pneumoniae RNA and Its Priming by Influenza A Virus Coinfection in Human Dendritic Cells
    Article Snippet: T4R was grown to mid-log phase and treated with phenol. .. Total RNA was isolated with TRIzol (Life Technologies) followed by DNA digestion with Turbo DNase (Life Technologies). .. RNA concentration and quality were determined spectrophotometrically with the NanoDrop ND 1000 apparatus, and the absence of DNA contamination was confirmed by PCR.

    Microscopy:

    Article Title: C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci
    Article Snippet: Images were obtained using a LSM710 META confocal microscope (Zeiss). .. For DNase and RNase treatments the standard protocol above was followed with additional steps: after rehydration in PBS, sections were incubated in 0.5 % Triton/PBS for 10 min, washed once in PBS, and treated with either RNase A (1 mg/ml in PBS/0.05 % Tween-20) or Turbo DNase (Ambion, 200 U/ml in supplied buffer) for 2 h at 37 °C.

    Purification:

    Article Title: H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage
    Article Snippet: Total RNA was isolated from the following S. haematobium life cycle stages (obtained from the NIAID Schistosomiasis Resource Center for distribution through BEI Resources, NIAID, NIH), using the RNAzol kit (Molecular Research Center) according to the manufacturer's instructions: purified eggs, retrieved from the liver of S. haematobium -infected hamsters; miracidia; cercariae; schistosomula; adult females; adult males; and mixed-sex adult worms. .. Contaminating genomic DNA was removed by DNase treatment using Turbo DNase (Invitrogen Ambion, USA) and chemical DNase inactivation, according to the manufacturer's instructions.

    Article Title: Programmed synthesis of 3D tissues
    Article Snippet: Given the capacity of DPAC to directly link complex tissue structural features with specific single and collective cell behaviors, we anticipate that this method will find utility in a variety of contexts, both basic and applied. .. Aldehyde-silanized glass slides (Nexterion® Aldehyde AL, Schott), Sigmacote® (Sigma-Aldrich), Slygard® 184 (Fisher Scientific), sodium borohydride (NaBH4 , ACROS, 98%), Pluronic® F108 NF (BASF), ethanol (Fisher Scientific), trypsin inhibitor from Glycine max (Sigma-Aldrich), Matrigel® (BD Biosciences), rat-tail collagen 1 (BD Biosciences), Turbo DNase (Life Technologies), amine-modified ssDNA (5′-amine-X20 , Operon), PBS (UCSF Cell-Culture Facility), PBS-CMF (UCSF Cell-Culture Facility), trypsin (UCSF Cell-Culture Facility), 100x penicillin/streptomycin, heat-inactivated fetal bovine serum (UCSF Cell-Culture Facility), RPMI media (UCSF Cell-Culture Facility) were used as received without further purification. .. Lipid-modified ssDNA (5′-lipid-T80 -X20 ) was synthesized as previously described .

    Article Title: Integrated Analysis of Dysregulated lncRNA Expression in Fetal Cardiac Tissues with Ventricular Septal Defect
    Article Snippet: RNA purification was performed on the RNA-containing aqueous phase with the RNeasy minikit (Qiagen, Valencia, CA, USA). .. RNA was then eluted with RNase-free water and treated with turbo DNase (Ambion) to remove contaminating DNA.

    Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection
    Article Snippet: The sample was then filtered (Amicon 3K cutoff spin column), and 5 μL of the flow through was analyzed by liquid chromatography (LC) coupled to an Orbitrap-XL mass spectrometer (MS) equipped with an electrospray ionization source (QB3 Chemistry facility). .. Total cellular RNA (containing KSHV RNA) was extracted and purified by TRIzol and then DNAse treated with Turbo DNase (Ambion). .. 30 μl protein G magnetic beads (Invitrogen) were blocked in 1% BSA solution for 1 hour, followed by incubation with 12.5 μg affinity-purified anti-m6 A polyclonal antibody (Millipore) at 4°C for 2 hr with head-over-tail rotation.

    Article Title: Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification
    Article Snippet: Total RNA was extracted from approximately 100 mg of leaves of N. benthamiana and O. sativa using Tri Reagent (Sigma, St. Louis, MO, USA) according to the manufacturer's instruction (TRI Reagent (T9424)-Technical bulletin). .. Extracted RNA was treated with 4U of Turbo DNase (2U/μ L, Ambion, Austin, TX, USA) at 37°C for 30 minutes and then inactivated at 72°C for 10 minutes, followed by phenol/chloroform purification. .. Ten microgram of DNase-treated RNA was subjected to 10U of RNase R (20U/μ L, Epicentre, Madison, WI, USA) digestion at 37°C for 15 minutes.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: A Re-Examination of Global Suppression of RNA Interference by HIV-1
    Article Snippet: Paragraph title: RNA isolation, primer extension, and RT-PCR ... For downstream applications that required DNase treatment, RNA samples were treated with Turbo DNase (Ambion).

    Article Title: H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage
    Article Snippet: Paragraph title: RT-PCR and stage-specific expression of H-IPSE. ... Contaminating genomic DNA was removed by DNase treatment using Turbo DNase (Invitrogen Ambion, USA) and chemical DNase inactivation, according to the manufacturer's instructions.

    Article Title: Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development
    Article Snippet: Paragraph title: RT-PCR. ... Total RNA was extracted using TRIzol reagent (Invitrogen) from P. yoelii salivary gland sporozoites (2 × 106 ), mixed blood-stage parasites (1 × 107 infected red blood cells were isolated from an infected mouse by heart puncture bleeding and serial limiting dilution of infected blood), or infected livers and then treated with Turbo DNase (Ambion). cDNA synthesis was performed using a SuperScript III Platinum two-step quantitative reverse transcription-PCR (qRT-PCR) kit according to the manufacturer's instructions (Invitrogen).

    Quantitative RT-PCR:

    Article Title: Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development
    Article Snippet: The MIF open reading frame (ORF) was amplified using the primers Sp/PyMIF and Asp/PyMIF (primer sequences are provided in Table S1). .. Total RNA was extracted using TRIzol reagent (Invitrogen) from P. yoelii salivary gland sporozoites (2 × 106 ), mixed blood-stage parasites (1 × 107 infected red blood cells were isolated from an infected mouse by heart puncture bleeding and serial limiting dilution of infected blood), or infected livers and then treated with Turbo DNase (Ambion). cDNA synthesis was performed using a SuperScript III Platinum two-step quantitative reverse transcription-PCR (qRT-PCR) kit according to the manufacturer's instructions (Invitrogen). .. The sequences of specific primers used for amplification from cDNA are listed in Table S2 in the supplemental material.

    Software:

    Article Title: Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA
    Article Snippet: Residual DNA contamination in the RNA sample was removed using Turbo DNase (Ambion Biosystems). .. Residual DNA contamination in the RNA sample was removed using Turbo DNase (Ambion Biosystems).

    RNA Extraction:

    Article Title: Integrated Analysis of Dysregulated lncRNA Expression in Fetal Cardiac Tissues with Ventricular Septal Defect
    Article Snippet: Paragraph title: RNA extraction ... RNA was then eluted with RNase-free water and treated with turbo DNase (Ambion) to remove contaminating DNA.

    Article Title: Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification
    Article Snippet: Paragraph title: 2.2. RNA Extraction, Reverse Transcription (RT), and Template-Dependent Multiple Displacement Amplification (tdMDA) ... Extracted RNA was treated with 4U of Turbo DNase (2U/μ L, Ambion, Austin, TX, USA) at 37°C for 30 minutes and then inactivated at 72°C for 10 minutes, followed by phenol/chloroform purification.

    Agarose Gel Electrophoresis:

    Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles
    Article Snippet: Stability of His-tagged MS2 PLP against nucleases was verified by their incubation with the combination of 10 U of Turbo DNase (Ambion) and/or 10 U of RNase A (Qiagen) at 37 °C for 1 hour. .. As controls for the reaction, DNA (500 ng of purified PCR fragment encoding the maturase and single-chain version of the coat protein dimer with His-tag modification) and RNA (500 ng of IAC in vitro transcript ), were added to the tested His-tagged MS2 PLP.

    Article Title: Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification
    Article Snippet: Extracted RNA was treated with 4U of Turbo DNase (2U/μ L, Ambion, Austin, TX, USA) at 37°C for 30 minutes and then inactivated at 72°C for 10 minutes, followed by phenol/chloroform purification. .. Extracted RNA was treated with 4U of Turbo DNase (2U/μ L, Ambion, Austin, TX, USA) at 37°C for 30 minutes and then inactivated at 72°C for 10 minutes, followed by phenol/chloroform purification.

    In Situ:

    Article Title: C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci
    Article Snippet: Paragraph title: RNA fluorescence in situ hybridisation (FISH) with protein immunostaining ... For DNase and RNase treatments the standard protocol above was followed with additional steps: after rehydration in PBS, sections were incubated in 0.5 % Triton/PBS for 10 min, washed once in PBS, and treated with either RNase A (1 mg/ml in PBS/0.05 % Tween-20) or Turbo DNase (Ambion, 200 U/ml in supplied buffer) for 2 h at 37 °C.

    Cross-linking Immunoprecipitation:

    Article Title: Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells
    Article Snippet: Sam68-cross-linked in vivo immunoprecipitation (CLIP) was performed as previously described , with minor modifications. .. Cross-linked germ cells were re-suspended in PXL buffer (1 × PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40), sonicated (3 × 30″ with BioRuptor) and incubated with Turbo DNase and Cocktail RNase (Ambion) for 15 min at 37°C.

    Spectrophotometry:

    Article Title: H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage
    Article Snippet: Contaminating genomic DNA was removed by DNase treatment using Turbo DNase (Invitrogen Ambion, USA) and chemical DNase inactivation, according to the manufacturer's instructions. .. Contaminating genomic DNA was removed by DNase treatment using Turbo DNase (Invitrogen Ambion, USA) and chemical DNase inactivation, according to the manufacturer's instructions.

    Immunoprecipitation:

    Article Title: Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells
    Article Snippet: Paragraph title: Cross-linked in vivo immunoprecipitation ... Cross-linked germ cells were re-suspended in PXL buffer (1 × PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40), sonicated (3 × 30″ with BioRuptor) and incubated with Turbo DNase and Cocktail RNase (Ambion) for 15 min at 37°C.

    Lysis:

    Article Title: Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants
    Article Snippet: To quantify intracellular encapsidated pgRNA, cell lysates were first treated with S7 nuclease (6 units) in lysis buffer containing 10 mM CaCl2 to remove cytoplasmic RNA, after which S7 nuclease was inactivated by the addition of EDTA (0.5 M). .. Extracted HBV nucleic acids were then incubated with 1 unit of Turbo DNase (Life Technologies) and 0.5 unit of DNase I (Thermo Fisher) or 25 units of RNase If (New England BioLabs) for 1 h at 37°C.

    Fluorescence In Situ Hybridization:

    Article Title: C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci
    Article Snippet: Paragraph title: RNA fluorescence in situ hybridisation (FISH) with protein immunostaining ... For DNase and RNase treatments the standard protocol above was followed with additional steps: after rehydration in PBS, sections were incubated in 0.5 % Triton/PBS for 10 min, washed once in PBS, and treated with either RNase A (1 mg/ml in PBS/0.05 % Tween-20) or Turbo DNase (Ambion, 200 U/ml in supplied buffer) for 2 h at 37 °C.

    Hood:

    Article Title: Microbial-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management
    Article Snippet: After removing residual ethanol, the pellet was air-dried in a sterile hood for 5 to 10 minutes (nucleic acid pellet should turn clear when dried). .. Then, after adding 2 µL Turbo DNase and 2 µL RNase (Thermo Scientific) to the tube, the mixture was incubated at 37°C for 1 hour to destroy remaining DNA and single-stranded RNA and then stored at −20°C until use.

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