rnasin  (Promega)

 
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    RNasin
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    Catalog Number:
    R2394
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    Score:
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    Structured Review

    Promega rnasin
    Anti-prion activity of r(GGAGGAGGAGGA) (R12) and d(GGAGGAGGAGGA) (D12). Western blotting of PrP Sc in GT + FK cells after treatment with either 10 μM R12 or D12. Two independent experiments, #1 and #2, are shown. The control was treated with just the buffer solution. The treatment with R12 was also performed in the presence of an <t>RNase</t> inhibitor, <t>RNasin.</t> Molecular mass markers are shown at the left.

    https://www.bioz.com/result/rnasin/product/Promega
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    Images

    1) Product Images from "Anti-prion activity of an RNA aptamer and its structural basis"

    Article Title: Anti-prion activity of an RNA aptamer and its structural basis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1132

    Anti-prion activity of r(GGAGGAGGAGGA) (R12) and d(GGAGGAGGAGGA) (D12). Western blotting of PrP Sc in GT + FK cells after treatment with either 10 μM R12 or D12. Two independent experiments, #1 and #2, are shown. The control was treated with just the buffer solution. The treatment with R12 was also performed in the presence of an RNase inhibitor, RNasin. Molecular mass markers are shown at the left.
    Figure Legend Snippet: Anti-prion activity of r(GGAGGAGGAGGA) (R12) and d(GGAGGAGGAGGA) (D12). Western blotting of PrP Sc in GT + FK cells after treatment with either 10 μM R12 or D12. Two independent experiments, #1 and #2, are shown. The control was treated with just the buffer solution. The treatment with R12 was also performed in the presence of an RNase inhibitor, RNasin. Molecular mass markers are shown at the left.

    Techniques Used: Activity Assay, Western Blot

    2) Product Images from "Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing"

    Article Title: Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing

    Journal:

    doi: 10.1074/jbc.M113.530717

    ARVCF is part of an RNP containing classical mRNAs and other RNA-binding proteins. Sucrose gradient centrifugation with lysates from HEK 293 cells was done with the addition of RNasin ( A ) or RNase A ( B ). Western blot was performed with antibodies against
    Figure Legend Snippet: ARVCF is part of an RNP containing classical mRNAs and other RNA-binding proteins. Sucrose gradient centrifugation with lysates from HEK 293 cells was done with the addition of RNasin ( A ) or RNase A ( B ). Western blot was performed with antibodies against

    Techniques Used: RNA Binding Assay, Gradient Centrifugation, Western Blot

    3) Product Images from "Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus"

    Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus

    Journal: The ISME Journal

    doi: 10.1038/ismej.2015.65

    RT–PCR amplification of nifH mRNA from wood-fed P. nigrolineatus GI tract regions. After dissection, RNA was extracted from fore (lane 2), hind (lane 3) and midgut (lane 4) regions, as described in the materials and methods section. RNA (3 μg) was first treated with DNaseI in the presence of RNasin and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (see Methods). Complementary DNA synthesis was followed by amplification with nifH32 and nifH623 primers and a second round amplification using nifH1 and nifH2 primers as described in the materials and methods section. Reaction products were separated on a 1.5% agarose gel. RNA preparations treated with DNase in the absence of RT and amplifications without DNase and RT treatments are shown. M is a 100 bp ladder; lane 1 is a negative control that did not contain RNA.
    Figure Legend Snippet: RT–PCR amplification of nifH mRNA from wood-fed P. nigrolineatus GI tract regions. After dissection, RNA was extracted from fore (lane 2), hind (lane 3) and midgut (lane 4) regions, as described in the materials and methods section. RNA (3 μg) was first treated with DNaseI in the presence of RNasin and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (see Methods). Complementary DNA synthesis was followed by amplification with nifH32 and nifH623 primers and a second round amplification using nifH1 and nifH2 primers as described in the materials and methods section. Reaction products were separated on a 1.5% agarose gel. RNA preparations treated with DNase in the absence of RT and amplifications without DNase and RT treatments are shown. M is a 100 bp ladder; lane 1 is a negative control that did not contain RNA.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Dissection, DNA Synthesis, Agarose Gel Electrophoresis, Negative Control

    4) Product Images from "Inhibition of HIV-1 Gag–membrane interactions by specific RNAs"

    Article Title: Inhibition of HIV-1 Gag–membrane interactions by specific RNAs

    Journal:

    doi: 10.1261/rna.058453.116

    Yeast total tRNA inhibits Gag binding to PC + PS liposomes. [35 S]-labeled Gag synthesized using rabbit reticulocyte lysates was treated (or left untreated) with RNase A at 37°C for 20 min. After inactivation of RNase A with RNasin, the mixtures were further incubated with varying concentrations of yeast tRNA at 37°C for 30 min. Subsequently, PC + PS liposomes were added and incubated further for 15 min before performing equilibrium flotation centrifugation. Fractions were analyzed using SDS-PAGE followed by phosphorimager analysis. (M) Membrane-bound, (NM) non-membrane-bound. The liposome binding efficiency was calculated as the amount of membrane-bound Gag as a fraction of total Gag. Data from three different experiments are shown as mean ± standard deviation. P- values were determined in comparison to the no RNA add-back condition using Student's t -test. (**) P < 0.01; (***) P < 0.001. Reduction of liposome binding efficiency to 50% was observed when RNase-treated Gag was incubated with 91 ng/µL yeast tRNA prior to mixing with liposomes.
    Figure Legend Snippet: Yeast total tRNA inhibits Gag binding to PC + PS liposomes. [35 S]-labeled Gag synthesized using rabbit reticulocyte lysates was treated (or left untreated) with RNase A at 37°C for 20 min. After inactivation of RNase A with RNasin, the mixtures were further incubated with varying concentrations of yeast tRNA at 37°C for 30 min. Subsequently, PC + PS liposomes were added and incubated further for 15 min before performing equilibrium flotation centrifugation. Fractions were analyzed using SDS-PAGE followed by phosphorimager analysis. (M) Membrane-bound, (NM) non-membrane-bound. The liposome binding efficiency was calculated as the amount of membrane-bound Gag as a fraction of total Gag. Data from three different experiments are shown as mean ± standard deviation. P- values were determined in comparison to the no RNA add-back condition using Student's t -test. (**) P < 0.01; (***) P < 0.001. Reduction of liposome binding efficiency to 50% was observed when RNase-treated Gag was incubated with 91 ng/µL yeast tRNA prior to mixing with liposomes.

    Techniques Used: Binding Assay, Labeling, Synthesized, Incubation, Centrifugation, SDS Page, Standard Deviation

    Related Articles

    Amplification:

    Article Title: Chicken Cells Sense Influenza A Virus Infection through MDA5 and CARDIF Signaling Involving LGP2
    Article Snippet: For silencing, DF-1 or HD-11 cells were transfected with siRNAs at a 100 nM final concentration using TransIT-TKO (Mirus) according to the manufacturer's protocol. .. RNA was extracted using TRIzol (Invitrogen), DNase I (Ambion), and RNase inhibitor (RNasin Plus; Promega) or with the Nucleospin RNA II kit (Macherey-Nagel) and amplified with the SuperScript III platinum one-step quantitative RT-PCR system (Invitrogen) using oligonucleotide primers and probes ( ) purchased from Microsynth. .. Primers and probes for chIFN-β were published earlier ( ).

    Synthesized:

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: WT and mutant RNAs started at the transcription initiation site from the iraD P2 promoter and were synthesized using an RNAMaxx kit (Agilent Technologies) and PCR-generated DNA templates. .. Binding reaction mixtures (10 µl) contained 0.1 nM labeled RNA, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 100 mM KCl, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithiothreitol, 4 U of RNase inhibitor (Promega), 0.1 mg/ml xylene cyanol, and various concentrations of purified CsrA-H6.

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: Biotin-labeled RNA was synthesized using the mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Ambion), with template plasmid (1 μg) and biotin-CTP (Invitrogen). .. After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega).

    Mobility Shift:

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: Paragraph title: Gel mobility shift assay. ... Binding reaction mixtures (10 µl) contained 0.1 nM labeled RNA, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 100 mM KCl, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithiothreitol, 4 U of RNase inhibitor (Promega), 0.1 mg/ml xylene cyanol, and various concentrations of purified CsrA-H6.

    Construct:

    Article Title: SOX9 has distinct regulatory roles in alternative splicing and transcription
    Article Snippet: HEK293T cells were transfected with wt SOX9 or its mutants together with the ZDHHC16 minigene reporter constructs. .. Nuclear extracts were then prepared using, successively, a lysis buffer (Tris 50 mM pH8, NaCl 100 mM, MgCl2 5 mM, 0.5% NP-40, Protease inhibitor (Roche) and RNAsin 50U/ml (Promega)) and FA lysis buffer (Tris 50 mM pH8, NaCl 140 mM, ethylenediaminetetraacetic acid (EDTA) 1 mM, 1% Triton X-100, 0.1% Na-deoxycholate, Protease inhibitor (Roche) and RNAsin 50U/ml (Promega)).

    Ubiquitin Assay:

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: Paragraph title: In Vivo Ubiquitination Assay. ... The beads were washed thrice with Ubi Wash buffer (1% Tritone X-100, 50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.05% SDS) containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega) and then twice with PBS containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega).

    Incubation:

    Article Title: SOX9 has distinct regulatory roles in alternative splicing and transcription
    Article Snippet: Nuclear extracts were then prepared using, successively, a lysis buffer (Tris 50 mM pH8, NaCl 100 mM, MgCl2 5 mM, 0.5% NP-40, Protease inhibitor (Roche) and RNAsin 50U/ml (Promega)) and FA lysis buffer (Tris 50 mM pH8, NaCl 140 mM, ethylenediaminetetraacetic acid (EDTA) 1 mM, 1% Triton X-100, 0.1% Na-deoxycholate, Protease inhibitor (Roche) and RNAsin 50U/ml (Promega)). .. Extracts were then sonicated for 8 min (30s on/30s off) in a Bioruptor® twin sonicator (Diagenode).

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: Binding reaction mixtures (10 µl) contained 0.1 nM labeled RNA, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 100 mM KCl, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithiothreitol, 4 U of RNase inhibitor (Promega), 0.1 mg/ml xylene cyanol, and various concentrations of purified CsrA-H6. .. Previously frozen CsrA was thawed and activated by incubation for 15 min at 37°C.

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: Thirty microliters of Protein G Dynabeads (Invitrogen) were added and incubated for 3 h at 4 °C with gentle rotation. .. The beads were washed three times with RIP wash buffer [50 mM Hepes (pH 7.5), 150 mM KCl, 0.05% Nonidet P-40] containing RNase inhibitor (Promega) and then were washed twice with PBS containing RNase inhibitor (Promega).

    Article Title: Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells
    Article Snippet: A typical 20 µl reaction mixture contained 25 µg total WCE protein, 20 units RNase inhibitor (Promega), 1.25 mM each adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), 0.2 mM uridine triphosphate (UTP), [except B, which contained 1.25 mM each nucleotide triphosphate (NTP)], ∼2 μCi [α-32 P]-UTP, 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 10 mM dithiothreitol (DTT), 2 mM spermidine, 100 µM NaCl, 100 nM coligo template unless otherwise indicated. .. IVT with yeast RNAP II was performed with 2.5 µg purified enzyme in 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 1.25 mM NTP with 1 µM coligo template.

    Article Title: Inhibition of HIV-1 Gag–membrane interactions by specific RNAs
    Article Snippet: HB (1.5 μL) was added to the negative control while RNase A (Thermo Scientific, diluted to 1 μg/μL in HB) was added to the pool, 1.5 μL per reaction. .. Following incubation at 37°C for 20 min, RNase A was inactivated by addition of 8 μL of RNasin (Promega) per reaction and incubated at 37°C for 10 min. RNAs of interest were refolded in reticulocyte lysis buffer (1× RRL: 20 mM HEPES-KOH, pH 7.0, 100 mM KCl, 0.5 mM MgCl2 ) ( ). .. Briefly, RNA dissolved in water was heated to 95°C for 1 min, snap cooled on ice, adjusted to 1× RRL buffer by addition of an appropriate amount of 10× RRL, and refolded at 37°C for 15 min. Refolded RNA was subsequently added to RNase-treated Gag and incubated at 37°C for 30 min; 1× RRL buffer was added to negative control and RNase-treated control reactions.

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: Thirty microliters of Protein G Dynabeads (Invitrogen) were added and incubated for 3 h at 4 °C with gentle rotation. .. The beads were washed thrice with wash buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.05% Nonidet P-40) containing RNase Inhibitor (Promega) and then twice with PBS containing RNase Inhibitor (Promega).

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: The supernatants were incubated with anti-FLAG M2 Magnetic Beads (SIGMA) for 2 h at 4 °C with gentle rotation. .. The beads were washed thrice with wash buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.05% Nonidet P-40) containing protease inhibitors and RNase Inhibitor (Promega) and then twice with PBS containing protease inhibitors and RNase Inhibitor (Promega).

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: Cells were centrifuged at 16,400 × g for 15 min at 4 °C, and the supernatants were incubated with anti-FLAG M2 Magnetic Beads (SIGMA) for 2 h at 4 °C with gentle rotation. .. The beads were washed thrice with Ubi Wash buffer (1% Tritone X-100, 50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.05% SDS) containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega) and then twice with PBS containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega).

    Activity Assay:

    Article Title: Anti-prion activity of an RNA aptamer and its structural basis
    Article Snippet: Paragraph title: Evaluation of anti-prion activity ... For the conditions with an RNase inhibitor, 800 U of RNasin (Promega) was added to the medium 5 min before the addition of the R12 solution.

    Expressing:

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: Indicated expression plasmids were transfected into HCT116 or 293FT cells that had been treated with or without MG132. .. The beads were washed thrice with wash buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.05% Nonidet P-40) containing protease inhibitors and RNase Inhibitor (Promega) and then twice with PBS containing protease inhibitors and RNase Inhibitor (Promega).

    Modification:

    Article Title: A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43
    Article Snippet: We have also prepared (UG)4 and A3 (GG)4 A3 RNAs covalently modified with fluorescein at 5′-end (FASMAC) and examined the interactions between the RRMs and those RNAs by fluorescence anisotropy experiments. .. Experimental conditions for RNA were the same with those for ssDNA as described above, but RNase inhibitor, RNasin (Promega), was further added in the sample solution for preventing adventitious degradation of RNA.

    Article Title: Anti-prion activity of an RNA aptamer and its structural basis
    Article Snippet: The cells were grown and maintained at 37°C under 5% of CO2 in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% of fetal bovine serum (Equitech-bio), 50 U/ml of penicillin G sodium and 50 μg/ml of streptomycin sulphate (Invitrogen). .. For the conditions with an RNase inhibitor, 800 U of RNasin (Promega) was added to the medium 5 min before the addition of the R12 solution.

    Western Blot:

    Article Title: Anti-prion activity of an RNA aptamer and its structural basis
    Article Snippet: For the conditions with an RNase inhibitor, 800 U of RNasin (Promega) was added to the medium 5 min before the addition of the R12 solution. .. For the conditions with an RNase inhibitor, 800 U of RNasin (Promega) was added to the medium 5 min before the addition of the R12 solution.

    Electron Microscopy:

    Article Title: Characterization of the Ebola virus nucleoprotein-RNA complex
    Article Snippet: To examine whether the NP-associated RNA in the NP helix is protected from RNase digestion, as occurs with paramyxoviruses , the recombinant NP–RNA complex was treated with a high concentration of RNase A (10 ng μl−1 ) in PBS at 37 °C for 30 min. After excess RNase inhibitor (160 U; Promega) was added, the RNA was phenol/chloroform extracted from the complex as described above. .. To determine whether the recombinant NP–RNA complex possesses the kind of conformational flexibility that has been observed with paramyxo- and rhabdoviruses , we next examined the effect of salt concentration on the conformation of the helix.

    Transfection:

    Article Title: SOX9 has distinct regulatory roles in alternative splicing and transcription
    Article Snippet: HEK293T cells were transfected with wt SOX9 or its mutants together with the ZDHHC16 minigene reporter constructs. .. Nuclear extracts were then prepared using, successively, a lysis buffer (Tris 50 mM pH8, NaCl 100 mM, MgCl2 5 mM, 0.5% NP-40, Protease inhibitor (Roche) and RNAsin 50U/ml (Promega)) and FA lysis buffer (Tris 50 mM pH8, NaCl 140 mM, ethylenediaminetetraacetic acid (EDTA) 1 mM, 1% Triton X-100, 0.1% Na-deoxycholate, Protease inhibitor (Roche) and RNAsin 50U/ml (Promega)).

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: Paragraph title: RNA Transfection and RNA ChIP Assay. ... After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega).

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: Indicated expression plasmids were transfected into HCT116 or 293FT cells that had been treated with or without MG132. .. The beads were washed thrice with wash buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.05% Nonidet P-40) containing protease inhibitors and RNase Inhibitor (Promega) and then twice with PBS containing protease inhibitors and RNase Inhibitor (Promega).

    Immunoprecipitation:

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: Paragraph title: Immunoprecipitation. ... The beads were washed thrice with wash buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.05% Nonidet P-40) containing protease inhibitors and RNase Inhibitor (Promega) and then twice with PBS containing protease inhibitors and RNase Inhibitor (Promega).

    Protease Inhibitor:

    Article Title: SOX9 has distinct regulatory roles in alternative splicing and transcription
    Article Snippet: Twenty-four hours later, cells were harvested and cross-linked for 10 min in 1% formaldehyde and the reaction was blocked with 10 mM final Glycine. .. Nuclear extracts were then prepared using, successively, a lysis buffer (Tris 50 mM pH8, NaCl 100 mM, MgCl2 5 mM, 0.5% NP-40, Protease inhibitor (Roche) and RNAsin 50U/ml (Promega)) and FA lysis buffer (Tris 50 mM pH8, NaCl 140 mM, ethylenediaminetetraacetic acid (EDTA) 1 mM, 1% Triton X-100, 0.1% Na-deoxycholate, Protease inhibitor (Roche) and RNAsin 50U/ml (Promega)). .. Extracts were then sonicated for 8 min (30s on/30s off) in a Bioruptor® twin sonicator (Diagenode).

    Hemagglutination Assay:

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: 293FT cells were cotransfected with HA-tagged ubiquitin, Myc-tagged β-TrCP1, Myc-tagged β-TrCP2, FLAG-tagged UHRF1, and/or UPAT . .. The beads were washed thrice with Ubi Wash buffer (1% Tritone X-100, 50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.05% SDS) containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega) and then twice with PBS containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega).

    Polymerase Chain Reaction:

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: WT and mutant RNAs started at the transcription initiation site from the iraD P2 promoter and were synthesized using an RNAMaxx kit (Agilent Technologies) and PCR-generated DNA templates. .. Binding reaction mixtures (10 µl) contained 0.1 nM labeled RNA, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 100 mM KCl, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithiothreitol, 4 U of RNase inhibitor (Promega), 0.1 mg/ml xylene cyanol, and various concentrations of purified CsrA-H6.

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega). .. After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega).

    Binding Assay:

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: Labeled RNAs were renatured by heating for 1 min at 85°C followed by slow cooling to room temperature. .. Binding reaction mixtures (10 µl) contained 0.1 nM labeled RNA, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 100 mM KCl, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithiothreitol, 4 U of RNase inhibitor (Promega), 0.1 mg/ml xylene cyanol, and various concentrations of purified CsrA-H6. .. Previously frozen CsrA was thawed and activated by incubation for 15 min at 37°C.

    In Vivo:

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: Paragraph title: In Vivo Ubiquitination Assay. ... The beads were washed thrice with Ubi Wash buffer (1% Tritone X-100, 50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.05% SDS) containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega) and then twice with PBS containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega).

    Fluorescence:

    Article Title: A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43
    Article Snippet: Paragraph title: Fluorescence anisotropy measurements ... Experimental conditions for RNA were the same with those for ssDNA as described above, but RNase inhibitor, RNasin (Promega), was further added in the sample solution for preventing adventitious degradation of RNA.

    Magnetic Beads:

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: The supernatants were incubated with anti-FLAG M2 Magnetic Beads (SIGMA) for 2 h at 4 °C with gentle rotation. .. The beads were washed thrice with wash buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.05% Nonidet P-40) containing protease inhibitors and RNase Inhibitor (Promega) and then twice with PBS containing protease inhibitors and RNase Inhibitor (Promega).

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: Cells were centrifuged at 16,400 × g for 15 min at 4 °C, and the supernatants were incubated with anti-FLAG M2 Magnetic Beads (SIGMA) for 2 h at 4 °C with gentle rotation. .. The beads were washed thrice with Ubi Wash buffer (1% Tritone X-100, 50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.05% SDS) containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega) and then twice with PBS containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega).

    Mutagenesis:

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: Plasmid pT7-P1(BS2)iraD '- 'lacZ is identical to pT7-P1iraD '- 'lacZ except that it contains a GGA-to-CGA mutation in BS2. .. Reaction mixtures contained a 20 nM concentration of plasmid DNA template and various concentrations of purified CsrA-His6 with 10 U of RNase inhibitor (Promega).

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: WT and mutant RNAs started at the transcription initiation site from the iraD P2 promoter and were synthesized using an RNAMaxx kit (Agilent Technologies) and PCR-generated DNA templates. .. Binding reaction mixtures (10 µl) contained 0.1 nM labeled RNA, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 100 mM KCl, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithiothreitol, 4 U of RNase inhibitor (Promega), 0.1 mg/ml xylene cyanol, and various concentrations of purified CsrA-H6.

    RNA Extraction:

    Article Title: Characterization of the Ebola virus nucleoprotein-RNA complex
    Article Snippet: To examine whether the NP-associated RNA in the NP helix is protected from RNase digestion, as occurs with paramyxoviruses , the recombinant NP–RNA complex was treated with a high concentration of RNase A (10 ng μl−1 ) in PBS at 37 °C for 30 min. After excess RNase inhibitor (160 U; Promega) was added, the RNA was phenol/chloroform extracted from the complex as described above. .. Unlike the recombinant NP–RNA complexes of paramyxoviruses , but as has been reported for some rhabdoviruses ( ; ), the NP-associated RNA was sensitive to RNase A and was digested (Fig. ).

    Labeling:

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: Labeled RNAs were renatured by heating for 1 min at 85°C followed by slow cooling to room temperature. .. Binding reaction mixtures (10 µl) contained 0.1 nM labeled RNA, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 100 mM KCl, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithiothreitol, 4 U of RNase inhibitor (Promega), 0.1 mg/ml xylene cyanol, and various concentrations of purified CsrA-H6. .. Previously frozen CsrA was thawed and activated by incubation for 15 min at 37°C.

    Article Title: Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells
    Article Snippet: In vitro transcription (IVT) using whole cell extract (WCE), uniform labeling with [α-32 P]-UTP and the linear or circular (coligo) templates was performed as follows. .. A typical 20 µl reaction mixture contained 25 µg total WCE protein, 20 units RNase inhibitor (Promega), 1.25 mM each adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), 0.2 mM uridine triphosphate (UTP), [except B, which contained 1.25 mM each nucleotide triphosphate (NTP)], ∼2 μCi [α-32 P]-UTP, 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 10 mM dithiothreitol (DTT), 2 mM spermidine, 100 µM NaCl, 100 nM coligo template unless otherwise indicated.

    Purification:

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: These four plasmids were used as templates for coupled transcription-translation reactions using the PURExpress in vitro protein synthesis kit according to the manufacturer’s instructions. .. Reaction mixtures contained a 20 nM concentration of plasmid DNA template and various concentrations of purified CsrA-His6 with 10 U of RNase inhibitor (Promega). .. The mixtures were incubated for 2 h at 37°C, and β-galactosidase activity was determined according to the manufacturer’s instructions.

    Article Title: Characterization of the Ebola virus nucleoprotein-RNA complex
    Article Snippet: To examine whether the NP-associated RNA in the NP helix is protected from RNase digestion, as occurs with paramyxoviruses , the recombinant NP–RNA complex was treated with a high concentration of RNase A (10 ng μl−1 ) in PBS at 37 °C for 30 min. After excess RNase inhibitor (160 U; Promega) was added, the RNA was phenol/chloroform extracted from the complex as described above. .. To determine whether the recombinant NP–RNA complex possesses the kind of conformational flexibility that has been observed with paramyxo- and rhabdoviruses , we next examined the effect of salt concentration on the conformation of the helix.

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: Labeled RNAs were renatured by heating for 1 min at 85°C followed by slow cooling to room temperature. .. Binding reaction mixtures (10 µl) contained 0.1 nM labeled RNA, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 100 mM KCl, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithiothreitol, 4 U of RNase inhibitor (Promega), 0.1 mg/ml xylene cyanol, and various concentrations of purified CsrA-H6. .. Previously frozen CsrA was thawed and activated by incubation for 15 min at 37°C.

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega). .. After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega).

    Article Title: Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells
    Article Snippet: A typical 20 µl reaction mixture contained 25 µg total WCE protein, 20 units RNase inhibitor (Promega), 1.25 mM each adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), 0.2 mM uridine triphosphate (UTP), [except B, which contained 1.25 mM each nucleotide triphosphate (NTP)], ∼2 μCi [α-32 P]-UTP, 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 10 mM dithiothreitol (DTT), 2 mM spermidine, 100 µM NaCl, 100 nM coligo template unless otherwise indicated. .. A typical 20 µl reaction mixture contained 25 µg total WCE protein, 20 units RNase inhibitor (Promega), 1.25 mM each adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), 0.2 mM uridine triphosphate (UTP), [except B, which contained 1.25 mM each nucleotide triphosphate (NTP)], ∼2 μCi [α-32 P]-UTP, 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 10 mM dithiothreitol (DTT), 2 mM spermidine, 100 µM NaCl, 100 nM coligo template unless otherwise indicated.

    Quantitative RT-PCR:

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega). .. After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega).

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: The beads were washed three times with RIP wash buffer [50 mM Hepes (pH 7.5), 150 mM KCl, 0.05% Nonidet P-40] containing RNase inhibitor (Promega) and then were washed twice with PBS containing RNase inhibitor (Promega). .. RNA was extracted using the Total RNA Isolation kit (MACHEREY-NAGEL). qRT-PCR was performed as described above.

    Article Title: Chicken Cells Sense Influenza A Virus Infection through MDA5 and CARDIF Signaling Involving LGP2
    Article Snippet: For silencing, DF-1 or HD-11 cells were transfected with siRNAs at a 100 nM final concentration using TransIT-TKO (Mirus) according to the manufacturer's protocol. .. RNA was extracted using TRIzol (Invitrogen), DNase I (Ambion), and RNase inhibitor (RNasin Plus; Promega) or with the Nucleospin RNA II kit (Macherey-Nagel) and amplified with the SuperScript III platinum one-step quantitative RT-PCR system (Invitrogen) using oligonucleotide primers and probes ( ) purchased from Microsynth. .. Primers and probes for chIFN-β were published earlier ( ).

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: The beads were washed thrice with wash buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.05% Nonidet P-40) containing RNase Inhibitor (Promega) and then twice with PBS containing RNase Inhibitor (Promega). .. RNA was extracted using the Total RNA Isolation kit (MACHEREY-NAGEL), and qRT-PCR was performed as described above.

    Chromatin Immunoprecipitation:

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: Paragraph title: RNA Transfection and RNA ChIP Assay. ... After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega).

    SDS Page:

    Article Title: Inhibition of HIV-1 Gag–membrane interactions by specific RNAs
    Article Snippet: Following incubation at 37°C for 20 min, RNase A was inactivated by addition of 8 μL of RNasin (Promega) per reaction and incubated at 37°C for 10 min. RNAs of interest were refolded in reticulocyte lysis buffer (1× RRL: 20 mM HEPES-KOH, pH 7.0, 100 mM KCl, 0.5 mM MgCl2 ) ( ). .. Briefly, RNA dissolved in water was heated to 95°C for 1 min, snap cooled on ice, adjusted to 1× RRL buffer by addition of an appropriate amount of 10× RRL, and refolded at 37°C for 15 min. Refolded RNA was subsequently added to RNase-treated Gag and incubated at 37°C for 30 min; 1× RRL buffer was added to negative control and RNase-treated control reactions.

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: The beads were washed thrice with wash buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.05% Nonidet P-40) containing protease inhibitors and RNase Inhibitor (Promega) and then twice with PBS containing protease inhibitors and RNase Inhibitor (Promega). .. The beads were washed thrice with wash buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.05% Nonidet P-40) containing protease inhibitors and RNase Inhibitor (Promega) and then twice with PBS containing protease inhibitors and RNase Inhibitor (Promega).

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: The beads were washed thrice with Ubi Wash buffer (1% Tritone X-100, 50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.05% SDS) containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega) and then twice with PBS containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega). .. The beads were washed thrice with Ubi Wash buffer (1% Tritone X-100, 50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.05% SDS) containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega) and then twice with PBS containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega).

    Plasmid Preparation:

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: These four plasmids were used as templates for coupled transcription-translation reactions using the PURExpress in vitro protein synthesis kit according to the manufacturer’s instructions. .. Reaction mixtures contained a 20 nM concentration of plasmid DNA template and various concentrations of purified CsrA-His6 with 10 U of RNase inhibitor (Promega). .. The mixtures were incubated for 2 h at 37°C, and β-galactosidase activity was determined according to the manufacturer’s instructions.

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: Biotin-labeled RNA was synthesized using the mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Ambion), with template plasmid (1 μg) and biotin-CTP (Invitrogen). .. After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega).

    Software:

    Article Title: Inhibition of HIV-1 Gag–membrane interactions by specific RNAs
    Article Snippet: Following incubation at 37°C for 20 min, RNase A was inactivated by addition of 8 μL of RNasin (Promega) per reaction and incubated at 37°C for 10 min. RNAs of interest were refolded in reticulocyte lysis buffer (1× RRL: 20 mM HEPES-KOH, pH 7.0, 100 mM KCl, 0.5 mM MgCl2 ) ( ). .. One hundred microliters of well-vortexed fractions were added to 50 μL of 3× SDS loading buffer (188 mM Tris–HCl, pH 6.8, 30% [v/v] glycerol, 15.2% [v/v] β-mercaptoethanol, 9.4% [w/v] SDS, 0.02% bromophenol blue), boiled for 5 min and then subjected to SDS-PAGE.

    Negative Control:

    Article Title: Inhibition of HIV-1 Gag–membrane interactions by specific RNAs
    Article Snippet: HB (1.5 μL) was added to the negative control while RNase A (Thermo Scientific, diluted to 1 μg/μL in HB) was added to the pool, 1.5 μL per reaction. .. Following incubation at 37°C for 20 min, RNase A was inactivated by addition of 8 μL of RNasin (Promega) per reaction and incubated at 37°C for 10 min. RNAs of interest were refolded in reticulocyte lysis buffer (1× RRL: 20 mM HEPES-KOH, pH 7.0, 100 mM KCl, 0.5 mM MgCl2 ) ( ).

    Recombinant:

    Article Title: Characterization of the Ebola virus nucleoprotein-RNA complex
    Article Snippet: The nucleic acid with mock treatment ran as a smear with bands around 500–1000 nt (Fig. ) and was unaffected by DNase I treatment (Fig. ), but was digested by RNase A treatment (Fig. ), demonstrating that the recombinant NP helix binds to non-viral RNAs, similar to recombinant paramyxo- and rhabdovirus NPs. .. To examine whether the NP-associated RNA in the NP helix is protected from RNase digestion, as occurs with paramyxoviruses , the recombinant NP–RNA complex was treated with a high concentration of RNase A (10 ng μl−1 ) in PBS at 37 °C for 30 min. After excess RNase inhibitor (160 U; Promega) was added, the RNA was phenol/chloroform extracted from the complex as described above. .. Unlike the recombinant NP–RNA complexes of paramyxoviruses , but as has been reported for some rhabdoviruses ( ; ), the NP-associated RNA was sensitive to RNase A and was digested (Fig. ).

    Agarose Gel Electrophoresis:

    Article Title: Characterization of the Ebola virus nucleoprotein-RNA complex
    Article Snippet: To identify the nucleic acid in the recombinant NP helices, it was phenol/chloroform extracted from the recombinant NP helix, treated with 10 ng RNaseA μl−1 , 0.2 Kunitz U DNase I μl−1 or mock treated at 37 °C for 30 min, and then analysed on a 1 % agarose gel. .. To examine whether the NP-associated RNA in the NP helix is protected from RNase digestion, as occurs with paramyxoviruses , the recombinant NP–RNA complex was treated with a high concentration of RNase A (10 ng μl−1 ) in PBS at 37 °C for 30 min. After excess RNase inhibitor (160 U; Promega) was added, the RNA was phenol/chloroform extracted from the complex as described above.

    In Vitro:

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: These four plasmids were used as templates for coupled transcription-translation reactions using the PURExpress in vitro protein synthesis kit according to the manufacturer’s instructions. .. Reaction mixtures contained a 20 nM concentration of plasmid DNA template and various concentrations of purified CsrA-His6 with 10 U of RNase inhibitor (Promega).

    Article Title: Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells
    Article Snippet: Paragraph title: In vitro transcription ... A typical 20 µl reaction mixture contained 25 µg total WCE protein, 20 units RNase inhibitor (Promega), 1.25 mM each adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), 0.2 mM uridine triphosphate (UTP), [except B, which contained 1.25 mM each nucleotide triphosphate (NTP)], ∼2 μCi [α-32 P]-UTP, 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 10 mM dithiothreitol (DTT), 2 mM spermidine, 100 µM NaCl, 100 nM coligo template unless otherwise indicated.

    Article Title: Inhibition of HIV-1 Gag–membrane interactions by specific RNAs
    Article Snippet: Gag was prepared by in vitro transcription/translation (TNT) in a rabbit reticulocyte lysate (Promega, L4600) as per manufacturer's instructions except that a homemade stock of amino acid (-Met) was used (Sigma, LAA21-1KT, 1 mM each amino acid in 10 mM Tris pH 7.0) in some experiments. .. Following incubation at 37°C for 20 min, RNase A was inactivated by addition of 8 μL of RNasin (Promega) per reaction and incubated at 37°C for 10 min. RNAs of interest were refolded in reticulocyte lysis buffer (1× RRL: 20 mM HEPES-KOH, pH 7.0, 100 mM KCl, 0.5 mM MgCl2 ) ( ).

    Ethanol Precipitation:

    Article Title: Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells
    Article Snippet: A typical 20 µl reaction mixture contained 25 µg total WCE protein, 20 units RNase inhibitor (Promega), 1.25 mM each adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), 0.2 mM uridine triphosphate (UTP), [except B, which contained 1.25 mM each nucleotide triphosphate (NTP)], ∼2 μCi [α-32 P]-UTP, 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 10 mM dithiothreitol (DTT), 2 mM spermidine, 100 µM NaCl, 100 nM coligo template unless otherwise indicated. .. Transcription reaction mixtures were incubated for 90 min at 37°C, after which time the RNA was extracted with 150 μl TriReagent (Invitrogen) per 20 µl reaction volume, according to the manufacturer’s instructions, with 10 µg glycogen added.

    Concentration Assay:

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame
    Article Snippet: These four plasmids were used as templates for coupled transcription-translation reactions using the PURExpress in vitro protein synthesis kit according to the manufacturer’s instructions. .. Reaction mixtures contained a 20 nM concentration of plasmid DNA template and various concentrations of purified CsrA-His6 with 10 U of RNase inhibitor (Promega). .. The mixtures were incubated for 2 h at 37°C, and β-galactosidase activity was determined according to the manufacturer’s instructions.

    Article Title: Characterization of the Ebola virus nucleoprotein-RNA complex
    Article Snippet: The nucleic acid with mock treatment ran as a smear with bands around 500–1000 nt (Fig. ) and was unaffected by DNase I treatment (Fig. ), but was digested by RNase A treatment (Fig. ), demonstrating that the recombinant NP helix binds to non-viral RNAs, similar to recombinant paramyxo- and rhabdovirus NPs. .. To examine whether the NP-associated RNA in the NP helix is protected from RNase digestion, as occurs with paramyxoviruses , the recombinant NP–RNA complex was treated with a high concentration of RNase A (10 ng μl−1 ) in PBS at 37 °C for 30 min. After excess RNase inhibitor (160 U; Promega) was added, the RNA was phenol/chloroform extracted from the complex as described above. .. Unlike the recombinant NP–RNA complexes of paramyxoviruses , but as has been reported for some rhabdoviruses ( ; ), the NP-associated RNA was sensitive to RNase A and was digested (Fig. ).

    Article Title: A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43
    Article Snippet: Fluorescein-modified ssDNA (0.1 μM) was prepared in a TN-low buffer with RRM proteins, the final concentration of which was in the range from 0 to 30 μM. .. Experimental conditions for RNA were the same with those for ssDNA as described above, but RNase inhibitor, RNasin (Promega), was further added in the sample solution for preventing adventitious degradation of RNA.

    Article Title: Anti-prion activity of an RNA aptamer and its structural basis
    Article Snippet: Either 80 μl of the R12 solution or 57 μl of the D12 solution was added to 2 ml of the medium, with the final concentration of both R12 and D12 being 10 μM. .. For the conditions with an RNase inhibitor, 800 U of RNasin (Promega) was added to the medium 5 min before the addition of the R12 solution.

    Lysis:

    Article Title: SOX9 has distinct regulatory roles in alternative splicing and transcription
    Article Snippet: Twenty-four hours later, cells were harvested and cross-linked for 10 min in 1% formaldehyde and the reaction was blocked with 10 mM final Glycine. .. Nuclear extracts were then prepared using, successively, a lysis buffer (Tris 50 mM pH8, NaCl 100 mM, MgCl2 5 mM, 0.5% NP-40, Protease inhibitor (Roche) and RNAsin 50U/ml (Promega)) and FA lysis buffer (Tris 50 mM pH8, NaCl 140 mM, ethylenediaminetetraacetic acid (EDTA) 1 mM, 1% Triton X-100, 0.1% Na-deoxycholate, Protease inhibitor (Roche) and RNAsin 50U/ml (Promega)). .. Extracts were then sonicated for 8 min (30s on/30s off) in a Bioruptor® twin sonicator (Diagenode).

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: HCT116 cells were transfected with biotin-labeled ASBEL or anti- ASBEL using the TransIT transfection reagent (Mirus). .. After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega). .. Chromatin was sheered by sonication and centrifuged, and the supernatant was incubated for 1 h at 4 °C with streptavidin beads.

    Article Title: ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells
    Article Snippet: Cells growing in six-well dishes were lysed in 0.5 mL of lysis buffer [0 mM Hepes (pH 7.5), 150 mM KCl, 0.5% Nonidet P-40, 2 mM EDTA, 1 mM NaF] containing protease inhibitors and RNase inhibitor (Promega) and were centrifuged at 16,400 × g for 10 min. .. The beads were washed three times with RIP wash buffer [50 mM Hepes (pH 7.5), 150 mM KCl, 0.05% Nonidet P-40] containing RNase inhibitor (Promega) and then were washed twice with PBS containing RNase inhibitor (Promega).

    Article Title: Inhibition of HIV-1 Gag–membrane interactions by specific RNAs
    Article Snippet: HB (1.5 μL) was added to the negative control while RNase A (Thermo Scientific, diluted to 1 μg/μL in HB) was added to the pool, 1.5 μL per reaction. .. Following incubation at 37°C for 20 min, RNase A was inactivated by addition of 8 μL of RNasin (Promega) per reaction and incubated at 37°C for 10 min. RNAs of interest were refolded in reticulocyte lysis buffer (1× RRL: 20 mM HEPES-KOH, pH 7.0, 100 mM KCl, 0.5 mM MgCl2 ) ( ). .. Briefly, RNA dissolved in water was heated to 95°C for 1 min, snap cooled on ice, adjusted to 1× RRL buffer by addition of an appropriate amount of 10× RRL, and refolded at 37°C for 15 min. Refolded RNA was subsequently added to RNase-treated Gag and incubated at 37°C for 30 min; 1× RRL buffer was added to negative control and RNase-treated control reactions.

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: Cells growing in six-well dishes were lysed in 0.5 mL of lysis buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.5% Nonidet P-40, 2 mM EDTA, 1 mM NaF) containing protease inhibitors and RNase Inhibitor (Promega) and centrifuged at 16,400 × g for 10 min. .. The beads were washed thrice with wash buffer (50 mM Hepes pH 7.5, 150 mM KCl, 0.05% Nonidet P-40) containing RNase Inhibitor (Promega) and then twice with PBS containing RNase Inhibitor (Promega).

    Article Title: Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1
    Article Snippet: After treatment with 10 μM MG132 for 3 h, cells were lysed in Ubi Lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris·HCl, pH 8) containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega) and were disrupted by 10 passages through a 25-gauge needle. .. The beads were washed thrice with Ubi Wash buffer (1% Tritone X-100, 50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.05% SDS) containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega) and then twice with PBS containing 10 mM N -ethyl maleimide, protease inhibitors, and RNase Inhibitor (Promega).

    Article Title: Anti-prion activity of an RNA aptamer and its structural basis
    Article Snippet: For the conditions with an RNase inhibitor, 800 U of RNasin (Promega) was added to the medium 5 min before the addition of the R12 solution. .. As a reference nucleic acid, U12 was added to the medium to the final concentration of 10 μM after the addition of the RNase inhibitor.

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    Promega rnase inhibitor
    Regulatory circuitry of the Csr, stringent response, and general stress response systems. ppGpp activates transcription of csrB , csrC , iraD , and rpoS . <t>CsrA</t> represses IraD synthesis via coupling with ORF27. IraD stabilizes RpoS by inhibiting RssB-mediated degradation of RpoS. RpoS activates transcription of genes involved in stationary-phase processes. RpoS activates transcription of csrA , while CsrA represses stationary-phase processes. CsrB/C sRNAs bind to and sequester CsrA from its mRNA targets, while CsrA indirectly activates csrB / C expression. CsrD targets CsrB/C for degradation by <t>RNase</t> E, and CsrA indirectly represses csrD expression. See text for additional details.
    Rnase Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 74/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Regulatory circuitry of the Csr, stringent response, and general stress response systems. ppGpp activates transcription of csrB , csrC , iraD , and rpoS . CsrA represses IraD synthesis via coupling with ORF27. IraD stabilizes RpoS by inhibiting RssB-mediated degradation of RpoS. RpoS activates transcription of genes involved in stationary-phase processes. RpoS activates transcription of csrA , while CsrA represses stationary-phase processes. CsrB/C sRNAs bind to and sequester CsrA from its mRNA targets, while CsrA indirectly activates csrB / C expression. CsrD targets CsrB/C for degradation by RNase E, and CsrA indirectly represses csrD expression. See text for additional details.

    Journal: mBio

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame

    doi: 10.1128/mBio.01355-17

    Figure Lengend Snippet: Regulatory circuitry of the Csr, stringent response, and general stress response systems. ppGpp activates transcription of csrB , csrC , iraD , and rpoS . CsrA represses IraD synthesis via coupling with ORF27. IraD stabilizes RpoS by inhibiting RssB-mediated degradation of RpoS. RpoS activates transcription of genes involved in stationary-phase processes. RpoS activates transcription of csrA , while CsrA represses stationary-phase processes. CsrB/C sRNAs bind to and sequester CsrA from its mRNA targets, while CsrA indirectly activates csrB / C expression. CsrD targets CsrB/C for degradation by RNase E, and CsrA indirectly represses csrD expression. See text for additional details.

    Article Snippet: Reaction mixtures contained a 20 nM concentration of plasmid DNA template and various concentrations of purified CsrA-His6 with 10 U of RNase inhibitor (Promega).

    Techniques: Expressing

    CsrA- iraD RNA footprint and toeprint analyses. (A) CsrA- iraD RNA footprint. Labeled iraD RNA was treated with RNase T1 in the presence of the CsrA concentration shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase T1 treatment (C), are marked. Positions of BS2, BS3, BS4, the iraD start codon (Met), and the Shine-Dalgarno (SD) sequence are shown. Residues that were protected by bound CsrA from RNase T1 cleavage are marked (–). Numbering is with respect to the start of iraD translation. (B) CsrA- iraD RNA toeprint. The concentration of CsrA used is shown at the top of the lane. Positions of BS2, BS3, BS4, and the CsrA toeprint (carat) are marked. Lanes corresponding to results of sequencing to reveal A, C, G, and U residues are labeled.

    Journal: mBio

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame

    doi: 10.1128/mBio.01355-17

    Figure Lengend Snippet: CsrA- iraD RNA footprint and toeprint analyses. (A) CsrA- iraD RNA footprint. Labeled iraD RNA was treated with RNase T1 in the presence of the CsrA concentration shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase T1 treatment (C), are marked. Positions of BS2, BS3, BS4, the iraD start codon (Met), and the Shine-Dalgarno (SD) sequence are shown. Residues that were protected by bound CsrA from RNase T1 cleavage are marked (–). Numbering is with respect to the start of iraD translation. (B) CsrA- iraD RNA toeprint. The concentration of CsrA used is shown at the top of the lane. Positions of BS2, BS3, BS4, and the CsrA toeprint (carat) are marked. Lanes corresponding to results of sequencing to reveal A, C, G, and U residues are labeled.

    Article Snippet: Reaction mixtures contained a 20 nM concentration of plasmid DNA template and various concentrations of purified CsrA-His6 with 10 U of RNase inhibitor (Promega).

    Techniques: Labeling, Concentration Assay, Sequencing

    Characterization of the purified NP helix. (a) SDS-PAGE of the visible band isolated from the CsCl gradient. NP(Δ601–739) lacks C-terminal aa 601–739 of NP. NP(Δ451–739) lacks C-terminal aa 451–739 of NP. M, Molecular mass marker. (b) EM of negatively stained NP helices composed of wild-type NP, NP(Δ601–739) and NP(Δ451–739). Bars, 100 nm. A magnified area of the black rectangle is shown in each micrograph (b, insets). (c) Agarose gel electrophoresis of nucleic acids extracted from wild-type NP helices. The extracted nucleic acids were treated with mock, RNase A or DNase I. (d) Agarose gel electrophoresis of the RNA fraction extracted from wild-type NP helices after RNase A treatment.

    Journal: The Journal of General Virology

    Article Title: Characterization of the Ebola virus nucleoprotein-RNA complex

    doi: 10.1099/vir.0.019794-0

    Figure Lengend Snippet: Characterization of the purified NP helix. (a) SDS-PAGE of the visible band isolated from the CsCl gradient. NP(Δ601–739) lacks C-terminal aa 601–739 of NP. NP(Δ451–739) lacks C-terminal aa 451–739 of NP. M, Molecular mass marker. (b) EM of negatively stained NP helices composed of wild-type NP, NP(Δ601–739) and NP(Δ451–739). Bars, 100 nm. A magnified area of the black rectangle is shown in each micrograph (b, insets). (c) Agarose gel electrophoresis of nucleic acids extracted from wild-type NP helices. The extracted nucleic acids were treated with mock, RNase A or DNase I. (d) Agarose gel electrophoresis of the RNA fraction extracted from wild-type NP helices after RNase A treatment.

    Article Snippet: To examine whether the NP-associated RNA in the NP helix is protected from RNase digestion, as occurs with paramyxoviruses , the recombinant NP–RNA complex was treated with a high concentration of RNase A (10 ng μl−1 ) in PBS at 37 °C for 30 min. After excess RNase inhibitor (160 U; Promega) was added, the RNA was phenol/chloroform extracted from the complex as described above.

    Techniques: Purification, SDS Page, Isolation, Marker, Electron Microscopy, Staining, Agarose Gel Electrophoresis

    EM of NP–RNA complex treated with RNase A. (a) NP–RNA complex in 150 mM NaCl in PB was treated with RNase A. (b) Sample (a) was then dialysed against 0 mM NaCl in PB. (c) NP–RNA complex in 0 mM NaCl in PB was treated with RNase A. (d) Sample (c) was then dialysed against 150 mM NaCl in PB. Bars, 100 nm.

    Journal: The Journal of General Virology

    Article Title: Characterization of the Ebola virus nucleoprotein-RNA complex

    doi: 10.1099/vir.0.019794-0

    Figure Lengend Snippet: EM of NP–RNA complex treated with RNase A. (a) NP–RNA complex in 150 mM NaCl in PB was treated with RNase A. (b) Sample (a) was then dialysed against 0 mM NaCl in PB. (c) NP–RNA complex in 0 mM NaCl in PB was treated with RNase A. (d) Sample (c) was then dialysed against 150 mM NaCl in PB. Bars, 100 nm.

    Article Snippet: To examine whether the NP-associated RNA in the NP helix is protected from RNase digestion, as occurs with paramyxoviruses , the recombinant NP–RNA complex was treated with a high concentration of RNase A (10 ng μl−1 ) in PBS at 37 °C for 30 min. After excess RNase inhibitor (160 U; Promega) was added, the RNA was phenol/chloroform extracted from the complex as described above.

    Techniques: Electron Microscopy