recombinant ribonuclease inhibitor rnaseout  (Thermo Fisher)


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    Name:
    RNaseOUT Recombinant Ribonuclease Inhibitor
    Description:
    RNaseOUT Recombinant Ribonuclease Inhibitor is a potent noncompetitive inhibitor of pancreatic type ribonucleases such as RNase A and is used to avoid RNA degradation in a variety of applications RNaseOUT Recombinant Ribonuclease Inhibitor is an acidic protein with a molecular weight of 52 kDa RNaseOUT inhibits RNase A RNase B and RNase C ApplicationscDNA synthesis RT PCR and in vitro transcription and translationSourcePurified by affinity chromatography from E coli expressing a cloned porcine genePerformance and quality testingSDS PAGE purity endodeoxyribonuclease assay protein concentration specific activity performance evaluated by RT PCRUnit definitionOne unit inhibits 5 ng of RNase A by 50 using cytidine 2 3 cyclic monophosphate cCMP as the substrateUnit reaction conditions100 mM Tris acetate pH 6 5 1 mM EDTA 0 2 mM cCMP 2 µg RNase A in 1 mL from 0 to 10 min at 25°C
    Catalog Number:
    10777019
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher recombinant ribonuclease inhibitor rnaseout
    Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of <t>RNaseOUT,</t> 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.
    RNaseOUT Recombinant Ribonuclease Inhibitor is a potent noncompetitive inhibitor of pancreatic type ribonucleases such as RNase A and is used to avoid RNA degradation in a variety of applications RNaseOUT Recombinant Ribonuclease Inhibitor is an acidic protein with a molecular weight of 52 kDa RNaseOUT inhibits RNase A RNase B and RNase C ApplicationscDNA synthesis RT PCR and in vitro transcription and translationSourcePurified by affinity chromatography from E coli expressing a cloned porcine genePerformance and quality testingSDS PAGE purity endodeoxyribonuclease assay protein concentration specific activity performance evaluated by RT PCRUnit definitionOne unit inhibits 5 ng of RNase A by 50 using cytidine 2 3 cyclic monophosphate cCMP as the substrateUnit reaction conditions100 mM Tris acetate pH 6 5 1 mM EDTA 0 2 mM cCMP 2 µg RNase A in 1 mL from 0 to 10 min at 25°C
    https://www.bioz.com/result/recombinant ribonuclease inhibitor rnaseout/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    recombinant ribonuclease inhibitor rnaseout - by Bioz Stars, 2020-04
    99/100 stars

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    1) Product Images from "Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers"

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku074

    Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.
    Figure Legend Snippet: Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.

    Techniques Used: Purification, Amplification, Concentration Assay, Hybridization, Fluorescence

    Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.
    Figure Legend Snippet: Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.

    Techniques Used: Generated, Purification, Incubation, Sequencing, Binding Assay, Synthesized, Amplification

    Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.
    Figure Legend Snippet: Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.

    Techniques Used: Polymerase Chain Reaction, Generated, Electrophoresis, Purification, Incubation

    Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.
    Figure Legend Snippet: Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.

    Techniques Used: Generated, Sequencing, Amplification, Incubation, Purification, Negative Control, Transduction

    Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.
    Figure Legend Snippet: Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.

    Techniques Used: Filtration

    Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.
    Figure Legend Snippet: Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.

    Techniques Used: Gel Purification, Amplification, Clone Assay, Plasmid Preparation, Incubation

    An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).
    Figure Legend Snippet: An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification, Clone Assay, Plasmid Preparation, Sequencing, Incubation, Fluorescence, Purification, Concentration Assay

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    Picogreen Assay:

    Article Title: FACS purification of Drosophila larval Neuroblasts for next generation sequencing
    Article Snippet: .. Chloroform/Trichlormethan (Roth, cat. no. 3313.4) Isopropanol (Roth, cat. no. CP41) RNase free water (Life technologies, cat. no. AM9932) Diethylpyrocarbonate (DEPC) treated water (Life technologies, cat. no. 750023) Glycoblue (Life technologies, cat. no. AM9515) Agilent RNA 6000 Nano Kit (Agilent Technologies, cat. no. 5067-1511) Agilent RNA 6000 Pico Kit (Agilent Technologies, cat. no. 5067-1513) Dynabeads mRNA purification Kit (Life technologies, cat. no. 61006) Fragmentation buffer 5× (see REAGENT SETUP) Tris base, adjusted to pH 8.2 (Sigma-Aldrich, cat. no. T1503) Potassium acetate (Sigma-Aldrich, cat. no. P1190) Magensium acetate (Sigma-Aldrich, cat. no. M5661) Sodium Acetate 3 M, pH 5.5 (Life technologies, cat. no. AM9740) Random hexamer primers 3 μg/μL (Life technologies, cat. no. 48190-011) dNTP Mix, 10 mM (Life technologies, cat. no. 18427-013) SuperScript® II Reverse Transcriptase (Life technologies, cat. no. 18064-014) SuperScript® III Reverse Transcriptase, in a set containing 5× first-strand buffer and 0.1 M DTT (Life technologies, cat. no. 18080-044) iQ SYBR Green Supermix (Bio-Rad, cat. no. 170-8882) RNaseOUT™ (Life technologies, cat. no. 10777-019) Second-Strand Buffer (Life technologies, cat. no. 10812-014) dUTP (Fermentas, cat. no. R0133) dATP (Fermentas, cat. no. R0141) dCTP (Fermentas, cat. no. R0151) dGTP (Fermentas, cat. no. R0161) DNA Polymerase I ( Escherichia coli ) (Life technologies, cat. no. 18010-017) Ribonuclease H (Life technologies, cat. no. 18021-014) E. coli DNA Ligase (Life technologies, cat. no. 18052-019) MinElute Reaction Cleanup Kit (QIAGEN, cat. no. 28204) Quant-iT Picogreen dsDNA Assay Kit (Life technologies, cat. no. P7589) NEBNext DNA sample Prep Reagent kits (New England Biolabs, cat. no. E6040) .. Foreceps Dumont Nr.5 (Fine Science Tools, cat. no. 11252-23) Stereomicrosope SteREO Discovery.V8 (Zeiss) Glass-bottom dishes, 35mm (MatTek Corporation, cat. no. P35G-0-14-C) Steriflip (Millipore, cat. no. SCGP00525) Thermo mixer compact (Eppendorf) Cell strainer, 35 μm (BD Bioscience Falcon, cat. no. 352235) FACSAriaIII, cell sorter (BD Bioscience) Low-binding reagent tubes, 1.5 mL (Life technologies, cat. no. AM12450) Phase-lock heavy gel tubes, 2 mL (5 PRIME, cat. no. 2302830) Agilent 2100 Bioanalyzer (Agilent Technologies) Magnetic rack (for example DynaMag™-2, Life technologies, cat. no. 12321D) NanoDrop 3300 fluorospectrophotometer (Thermo Scientific) Genome Analyzer IIX (GAIIX; Illumina) HiSeq 2000 sequence analyzer (Illumina)

    Quantitative RT-PCR:

    Article Title: The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells
    Article Snippet: Total RNA was then extracted and converted to cDNA using MMLV reverse transcriptase (Invitrogen) and TaqMan real-time RT-PCR was performed and gene expression levels were calculated as described previously ( ). .. Nuclear and cytoplasmic RNA fractions were separated using the CelLytic NuCLEAR Extraction Kit (Sigma) supplemented with RNaseOUT ribonuclease inhibitor (Life Technologies).

    SYBR Green Assay:

    Article Title: Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors
    Article Snippet: .. Quantitative PCR The Ambion PureLink RNA mini kit was used to extract total cellular RNA. cDNA reverse transcription was then conducted using Superscript III reverse transcriptase and RNAseOUT recombinant ribonuclease inhibitor (Invitrogen). qPCR was performed using BioRad SsoAdvanced SYBR green supermix and the Biorad CFX Connect real-time system. .. Relative gene expression was normalized to the housekeeping gene TBP, and fold-change calculated based on a comparison to the expression level of the adult pancreas.

    Article Title: FACS purification of Drosophila larval Neuroblasts for next generation sequencing
    Article Snippet: .. Chloroform/Trichlormethan (Roth, cat. no. 3313.4) Isopropanol (Roth, cat. no. CP41) RNase free water (Life technologies, cat. no. AM9932) Diethylpyrocarbonate (DEPC) treated water (Life technologies, cat. no. 750023) Glycoblue (Life technologies, cat. no. AM9515) Agilent RNA 6000 Nano Kit (Agilent Technologies, cat. no. 5067-1511) Agilent RNA 6000 Pico Kit (Agilent Technologies, cat. no. 5067-1513) Dynabeads mRNA purification Kit (Life technologies, cat. no. 61006) Fragmentation buffer 5× (see REAGENT SETUP) Tris base, adjusted to pH 8.2 (Sigma-Aldrich, cat. no. T1503) Potassium acetate (Sigma-Aldrich, cat. no. P1190) Magensium acetate (Sigma-Aldrich, cat. no. M5661) Sodium Acetate 3 M, pH 5.5 (Life technologies, cat. no. AM9740) Random hexamer primers 3 μg/μL (Life technologies, cat. no. 48190-011) dNTP Mix, 10 mM (Life technologies, cat. no. 18427-013) SuperScript® II Reverse Transcriptase (Life technologies, cat. no. 18064-014) SuperScript® III Reverse Transcriptase, in a set containing 5× first-strand buffer and 0.1 M DTT (Life technologies, cat. no. 18080-044) iQ SYBR Green Supermix (Bio-Rad, cat. no. 170-8882) RNaseOUT™ (Life technologies, cat. no. 10777-019) Second-Strand Buffer (Life technologies, cat. no. 10812-014) dUTP (Fermentas, cat. no. R0133) dATP (Fermentas, cat. no. R0141) dCTP (Fermentas, cat. no. R0151) dGTP (Fermentas, cat. no. R0161) DNA Polymerase I ( Escherichia coli ) (Life technologies, cat. no. 18010-017) Ribonuclease H (Life technologies, cat. no. 18021-014) E. coli DNA Ligase (Life technologies, cat. no. 18052-019) MinElute Reaction Cleanup Kit (QIAGEN, cat. no. 28204) Quant-iT Picogreen dsDNA Assay Kit (Life technologies, cat. no. P7589) NEBNext DNA sample Prep Reagent kits (New England Biolabs, cat. no. E6040) .. Foreceps Dumont Nr.5 (Fine Science Tools, cat. no. 11252-23) Stereomicrosope SteREO Discovery.V8 (Zeiss) Glass-bottom dishes, 35mm (MatTek Corporation, cat. no. P35G-0-14-C) Steriflip (Millipore, cat. no. SCGP00525) Thermo mixer compact (Eppendorf) Cell strainer, 35 μm (BD Bioscience Falcon, cat. no. 352235) FACSAriaIII, cell sorter (BD Bioscience) Low-binding reagent tubes, 1.5 mL (Life technologies, cat. no. AM12450) Phase-lock heavy gel tubes, 2 mL (5 PRIME, cat. no. 2302830) Agilent 2100 Bioanalyzer (Agilent Technologies) Magnetic rack (for example DynaMag™-2, Life technologies, cat. no. 12321D) NanoDrop 3300 fluorospectrophotometer (Thermo Scientific) Genome Analyzer IIX (GAIIX; Illumina) HiSeq 2000 sequence analyzer (Illumina)

    Microarray:

    Article Title: Repression of Salmonella entericaphoP Expression by Small Molecules from Physiological Bile
    Article Snippet: Paragraph title: Microarray experiments. ... The entire RNA sample (≥3 μg) was mixed with 6 μg of random primers (Invitrogen, Burlington, Canada) and 40 U of RNaseOUT recombinant RNase inhibitor (Invitrogen) in a total volume of 18.5 μl and incubated at 70°C for 10 min.

    Random Hexamer Labeling:

    Article Title: FACS purification of Drosophila larval Neuroblasts for next generation sequencing
    Article Snippet: .. Chloroform/Trichlormethan (Roth, cat. no. 3313.4) Isopropanol (Roth, cat. no. CP41) RNase free water (Life technologies, cat. no. AM9932) Diethylpyrocarbonate (DEPC) treated water (Life technologies, cat. no. 750023) Glycoblue (Life technologies, cat. no. AM9515) Agilent RNA 6000 Nano Kit (Agilent Technologies, cat. no. 5067-1511) Agilent RNA 6000 Pico Kit (Agilent Technologies, cat. no. 5067-1513) Dynabeads mRNA purification Kit (Life technologies, cat. no. 61006) Fragmentation buffer 5× (see REAGENT SETUP) Tris base, adjusted to pH 8.2 (Sigma-Aldrich, cat. no. T1503) Potassium acetate (Sigma-Aldrich, cat. no. P1190) Magensium acetate (Sigma-Aldrich, cat. no. M5661) Sodium Acetate 3 M, pH 5.5 (Life technologies, cat. no. AM9740) Random hexamer primers 3 μg/μL (Life technologies, cat. no. 48190-011) dNTP Mix, 10 mM (Life technologies, cat. no. 18427-013) SuperScript® II Reverse Transcriptase (Life technologies, cat. no. 18064-014) SuperScript® III Reverse Transcriptase, in a set containing 5× first-strand buffer and 0.1 M DTT (Life technologies, cat. no. 18080-044) iQ SYBR Green Supermix (Bio-Rad, cat. no. 170-8882) RNaseOUT™ (Life technologies, cat. no. 10777-019) Second-Strand Buffer (Life technologies, cat. no. 10812-014) dUTP (Fermentas, cat. no. R0133) dATP (Fermentas, cat. no. R0141) dCTP (Fermentas, cat. no. R0151) dGTP (Fermentas, cat. no. R0161) DNA Polymerase I ( Escherichia coli ) (Life technologies, cat. no. 18010-017) Ribonuclease H (Life technologies, cat. no. 18021-014) E. coli DNA Ligase (Life technologies, cat. no. 18052-019) MinElute Reaction Cleanup Kit (QIAGEN, cat. no. 28204) Quant-iT Picogreen dsDNA Assay Kit (Life technologies, cat. no. P7589) NEBNext DNA sample Prep Reagent kits (New England Biolabs, cat. no. E6040) .. Foreceps Dumont Nr.5 (Fine Science Tools, cat. no. 11252-23) Stereomicrosope SteREO Discovery.V8 (Zeiss) Glass-bottom dishes, 35mm (MatTek Corporation, cat. no. P35G-0-14-C) Steriflip (Millipore, cat. no. SCGP00525) Thermo mixer compact (Eppendorf) Cell strainer, 35 μm (BD Bioscience Falcon, cat. no. 352235) FACSAriaIII, cell sorter (BD Bioscience) Low-binding reagent tubes, 1.5 mL (Life technologies, cat. no. AM12450) Phase-lock heavy gel tubes, 2 mL (5 PRIME, cat. no. 2302830) Agilent 2100 Bioanalyzer (Agilent Technologies) Magnetic rack (for example DynaMag™-2, Life technologies, cat. no. 12321D) NanoDrop 3300 fluorospectrophotometer (Thermo Scientific) Genome Analyzer IIX (GAIIX; Illumina) HiSeq 2000 sequence analyzer (Illumina)

    Expressing:

    Article Title: Pharmacokinetics, toxicity, and cytochrome P450 modulatory activity of plumbagin
    Article Snippet: Paragraph title: Effect of plumbagin on CYP1A2 and CYP3A11 mRNA expression ... The reaction was inactivated by heating at 70 °C for 15 min and chilled on ice for 2 min. cDNA was synthesized in a total volume of 20 μL containing 5× First-Strand Buffer (250 mM Tris–HCl (pH 8.3), 375 mM KCl, and 15 mM MgCl2 ), 0.1 M DTT, 40 U RNaseOUT™ Recombinant RNase Inhibitor, 10 mM dNTPs, and 200 U Superscript III Reverse Transcriptase Rnase H (Invitrogen, Thermo Fisher Scienyific, NY, USA).

    Article Title: Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors
    Article Snippet: Quantitative PCR The Ambion PureLink RNA mini kit was used to extract total cellular RNA. cDNA reverse transcription was then conducted using Superscript III reverse transcriptase and RNAseOUT recombinant ribonuclease inhibitor (Invitrogen). qPCR was performed using BioRad SsoAdvanced SYBR green supermix and the Biorad CFX Connect real-time system. .. Relative gene expression was normalized to the housekeeping gene TBP, and fold-change calculated based on a comparison to the expression level of the adult pancreas.

    Article Title: The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells
    Article Snippet: Total RNA was then extracted and converted to cDNA using MMLV reverse transcriptase (Invitrogen) and TaqMan real-time RT-PCR was performed and gene expression levels were calculated as described previously ( ). .. Nuclear and cytoplasmic RNA fractions were separated using the CelLytic NuCLEAR Extraction Kit (Sigma) supplemented with RNaseOUT ribonuclease inhibitor (Life Technologies).

    Gel Purification:

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers
    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies). .. Transcripts intended for purification were either filtered through Sephadex G25 using the Illustra MicroSpin G-25 columns, according to the manufacturer’s instructions (GE Healthcare, Piscataway, NJ, USA), or run through RNA gel purification.

    Flow Cytometry:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The Cap-selected mRNA sample was subjected to end repair and adapter ligation steps – as was described above – before loading on the Flow Cells. .. The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific).

    Immunoprecipitation:

    Article Title: Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis
    Article Snippet: Paragraph title: RNA-Interacting Protein Immunoprecipitation ... Cell pellets from approximately 50 million cells were homogenized, lysed in ice-cold lysis buffer (10 mM HEPES (pH 7.3, ThermoFisher Scientific), 100 mM KCl (1049360250, Merck), 0.5 % NP40 (NP40S, Sigma-Aldrich), 5 mM MgCl2 , 0.5 mM dithiothreitol (DTT, DTT-RO, Sigma-Aldrich), protease inhibitors (118735, Sigma-Aldrich), recombinant RNase inhibitors (10777019, ThermoFisher Schientific), 1 mM PMSF (36978, ThermoFisher Scientific) using TissueLyser LT (50 Hz, 2 min, Qiagen).

    DNA HS Assay:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific). .. Sample concentration was determined using a Qubit 2.0 Fluorometer and Qubit DNA HS Assay Kit (Thermo Fisher Scientific).

    Imaging:

    Article Title: Replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence
    Article Snippet: Nuclear staining using 0.5 μg/ml of Hoechst 33342 (Invitrogen) was performed afterwards and the coverslides were mounted in GLOX anti-fade media (10% glucose, 1 M Tris-HCl pH8.0, glucose oxidase, catalase, diluted in 2× SSC; all from Sigma) before imaging. .. For RNA FISH coupled with immunofluorescence (IF) (RNA FISH-IF), fixed cells were permeabilized with 70% ethanol for 1 h. Permeabilized cells were then incubated with anti-human active caspase-3 antibody (1:100 dilution; Cell Signaling, cat. no. 9661) or anti-human cleaved PARP-1 antibody (1:100 dilution; Cell Signaling, cat. no. 5625) followed by Alexa Fluor 488-labeled goat anti-rabbit IgG (1:500 dilution; Invitrogen, cat. no. R37116) diluted in 1% BSA in the presence of 40 U/ml RNase inhibitor (Invitrogen, cat. no. 10777-019).

    Sequencing:

    Article Title: Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli
    Article Snippet: Purification of NTH-RNase E and discontinuous cleavage assays Recombinant, N-terminally hexahistidine-tagged polypeptides corresponding to the NTH of RNase E (residues 1–529) with WT sequence or the T170V substitution were purified as described previously ( , ). .. Discontinuous cleavage assays were performed in a buffer containing 25 mM bis -Tris propane (pH 8.3), 100 mM NaCl, 15 mM MgCl2 , 0.1% (v/v) Triton X-100, 1 mM dithiothreitol (DTT) and 32 U RNaseOUT™ ribonuclease inhibitor (Invitrogen).

    Article Title: Munc13-Independent Vesicle Priming at Mouse Photoreceptor Ribbon Synapses
    Article Snippet: The content of the tubes was collected by brief centrifugation and the following components were added: 4 μl of 5× First-Strand Buffer, 1 μl of 0.1 m DTT, 1 μl of RNaseOUT Recombinant RNase Inhibitor (Invitrogen), and 1 μl of SuperScript III RT (200 U/μl). .. The reaction was inactivated by heating at 70°C for 15 min. RNA was removed by RNaseH treatment at 37°C for 20 min. ubMunc13-2-specific primers ( ) were selected on the basis of the published mouse cDNA sequence (GenBank accession no. ).

    Article Title: The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells
    Article Snippet: Nuclear and cytoplasmic RNA fractions were separated using the CelLytic NuCLEAR Extraction Kit (Sigma) supplemented with RNaseOUT ribonuclease inhibitor (Life Technologies). .. All values are presented as the mean±s.e.m. of replicates in pooled experiments. lncRNA real-time RT-PCR amplification products were sequence verified by cloning into the pCR4-TOPO vector (Life Technologies).

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: Paragraph title: Oxford Nanopore MinION Sequencing ... The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific).

    Recombinant:

    Article Title: Pharmacokinetics, toxicity, and cytochrome P450 modulatory activity of plumbagin
    Article Snippet: .. The reaction was inactivated by heating at 70 °C for 15 min and chilled on ice for 2 min. cDNA was synthesized in a total volume of 20 μL containing 5× First-Strand Buffer (250 mM Tris–HCl (pH 8.3), 375 mM KCl, and 15 mM MgCl2 ), 0.1 M DTT, 40 U RNaseOUT™ Recombinant RNase Inhibitor, 10 mM dNTPs, and 200 U Superscript III Reverse Transcriptase Rnase H (Invitrogen, Thermo Fisher Scienyific, NY, USA). .. The platinum SYBRTM Green qPCR Supermix-UDG (Invitrgen, Carsbad, CA, USA) was used for real-time PCR (RT-PCR) analysis using iCycler IQ machine (BioRad Laboratories Inc., Hercules, CA, USA).

    Article Title: Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors
    Article Snippet: .. Quantitative PCR The Ambion PureLink RNA mini kit was used to extract total cellular RNA. cDNA reverse transcription was then conducted using Superscript III reverse transcriptase and RNAseOUT recombinant ribonuclease inhibitor (Invitrogen). qPCR was performed using BioRad SsoAdvanced SYBR green supermix and the Biorad CFX Connect real-time system. .. Relative gene expression was normalized to the housekeeping gene TBP, and fold-change calculated based on a comparison to the expression level of the adult pancreas.

    Article Title: Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli
    Article Snippet: Purification of NTH-RNase E and discontinuous cleavage assays Recombinant, N-terminally hexahistidine-tagged polypeptides corresponding to the NTH of RNase E (residues 1–529) with WT sequence or the T170V substitution were purified as described previously ( , ). .. Discontinuous cleavage assays were performed in a buffer containing 25 mM bis -Tris propane (pH 8.3), 100 mM NaCl, 15 mM MgCl2 , 0.1% (v/v) Triton X-100, 1 mM dithiothreitol (DTT) and 32 U RNaseOUT™ ribonuclease inhibitor (Invitrogen).

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers
    Article Snippet: .. In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies). .. Transcription reactions were incubated at 42°C for 1–2 h. After this, transcripts of the circuit components were either (i) used directly for assembly or (ii) subjected to purification before assembly.

    Article Title: Postmortem diagnosis of infectious heart diseases: A mystifying cause of Sudden Infant Death
    Article Snippet: .. Enterovirus PCR from VP1- capsid protein region was conducted in two stages as follows: (1) first stage- 10 μl of previously extracted RNA were reverse-transcribed into cDNA at 42°C for 45 min using 200 units of Super-ScriptIII reverse transcriptase and 2.5 ng/μl of random primers (Invitrogen, Cergy Pontoise, France) in the presence of 10 units of RnaseOUT recombinant RNase inhibitor (Invitrogen, Cergy Pontoise, France) and (2) second stage- 5 μl of cDNA were amplified using 50 pmol of the 292 and 222 primers directed to the enteroviral VP1-capsid-protein coding region ( ) and 1.25 units of Platinum Taq DNA polymerase (Invitrogen, Cergy Pontoise, France) in 50 μl of reaction mixture according to the protocol [ ]. ..

    Article Title: Munc13-Independent Vesicle Priming at Mouse Photoreceptor Ribbon Synapses
    Article Snippet: .. The content of the tubes was collected by brief centrifugation and the following components were added: 4 μl of 5× First-Strand Buffer, 1 μl of 0.1 m DTT, 1 μl of RNaseOUT Recombinant RNase Inhibitor (Invitrogen), and 1 μl of SuperScript III RT (200 U/μl). .. The reaction was inactivated by heating at 70°C for 15 min. RNA was removed by RNaseH treatment at 37°C for 20 min. ubMunc13-2-specific primers ( ) were selected on the basis of the published mouse cDNA sequence (GenBank accession no. ).

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: .. The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific). .. Sample concentration was determined using a Qubit 2.0 Fluorometer and Qubit DNA HS Assay Kit (Thermo Fisher Scientific).

    Article Title: Repression of Salmonella entericaphoP Expression by Small Molecules from Physiological Bile
    Article Snippet: .. The entire RNA sample (≥3 μg) was mixed with 6 μg of random primers (Invitrogen, Burlington, Canada) and 40 U of RNaseOUT recombinant RNase inhibitor (Invitrogen) in a total volume of 18.5 μl and incubated at 70°C for 10 min. .. Samples were incubated on ice for 30 s, centrifuged to bring down condensation, and incubated with the following components (Invitrogen) at the final concentrations shown in parentheses: First Strand buffer (1×), dithiothreitol (10 mM), dNTPs (dATP, dCTP, dGTP, dTTP; 0.5 mM each) and 100 U of SuperScript II reverse transcriptase in a total volume of 30 μl.

    Article Title: Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis
    Article Snippet: .. Cell pellets from approximately 50 million cells were homogenized, lysed in ice-cold lysis buffer (10 mM HEPES (pH 7.3, ThermoFisher Scientific), 100 mM KCl (1049360250, Merck), 0.5 % NP40 (NP40S, Sigma-Aldrich), 5 mM MgCl2 , 0.5 mM dithiothreitol (DTT, DTT-RO, Sigma-Aldrich), protease inhibitors (118735, Sigma-Aldrich), recombinant RNase inhibitors (10777019, ThermoFisher Schientific), 1 mM PMSF (36978, ThermoFisher Scientific) using TissueLyser LT (50 Hz, 2 min, Qiagen). ..

    Immunofluorescence:

    Article Title: Replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence
    Article Snippet: .. For RNA FISH coupled with immunofluorescence (IF) (RNA FISH-IF), fixed cells were permeabilized with 70% ethanol for 1 h. Permeabilized cells were then incubated with anti-human active caspase-3 antibody (1:100 dilution; Cell Signaling, cat. no. 9661) or anti-human cleaved PARP-1 antibody (1:100 dilution; Cell Signaling, cat. no. 5625) followed by Alexa Fluor 488-labeled goat anti-rabbit IgG (1:500 dilution; Invitrogen, cat. no. R37116) diluted in 1% BSA in the presence of 40 U/ml RNase inhibitor (Invitrogen, cat. no. 10777-019). .. Stained cells were incubated in 4% formaldehyde for 10 min prior to RNA FISH, washed with PBS, and then equilibrated in wash buffer.

    RNA Sequencing Assay:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The direct RNA sequencing approach Libraries were prepared using the Direct RNA Sequencing Kit (SQK-RNA001; Oxford Nanopore Technologies) The first strand cDNA was synthesized by SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) using an RT adapter with T10 nts. .. The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific).

    Magnetic Beads:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: .. The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific). .. Sample concentration was determined using a Qubit 2.0 Fluorometer and Qubit DNA HS Assay Kit (Thermo Fisher Scientific).

    Isolation:

    Article Title: Repression of Salmonella entericaphoP Expression by Small Molecules from Physiological Bile
    Article Snippet: Cells were pelleted by centrifugation, and RNA was isolated using the RNeasy minikit (Qiagen) with on-column DNase digestion, according to the manufacturer's recommendations. .. The entire RNA sample (≥3 μg) was mixed with 6 μg of random primers (Invitrogen, Burlington, Canada) and 40 U of RNaseOUT recombinant RNase inhibitor (Invitrogen) in a total volume of 18.5 μl and incubated at 70°C for 10 min.

    Polymerase Chain Reaction:

    Article Title: Pharmacokinetics, toxicity, and cytochrome P450 modulatory activity of plumbagin
    Article Snippet: The reaction was inactivated by heating at 70 °C for 15 min and chilled on ice for 2 min. cDNA was synthesized in a total volume of 20 μL containing 5× First-Strand Buffer (250 mM Tris–HCl (pH 8.3), 375 mM KCl, and 15 mM MgCl2 ), 0.1 M DTT, 40 U RNaseOUT™ Recombinant RNase Inhibitor, 10 mM dNTPs, and 200 U Superscript III Reverse Transcriptase Rnase H (Invitrogen, Thermo Fisher Scienyific, NY, USA). .. The PCR cycles for CYP1A2 , CYP3A11 and GAPDH were as follows: denaturation at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 s, and annealing at 60 °C for 1 min. Each RT-PCR was performed in duplicate.

    Article Title: Postmortem diagnosis of infectious heart diseases: A mystifying cause of Sudden Infant Death
    Article Snippet: .. Enterovirus PCR from VP1- capsid protein region was conducted in two stages as follows: (1) first stage- 10 μl of previously extracted RNA were reverse-transcribed into cDNA at 42°C for 45 min using 200 units of Super-ScriptIII reverse transcriptase and 2.5 ng/μl of random primers (Invitrogen, Cergy Pontoise, France) in the presence of 10 units of RnaseOUT recombinant RNase inhibitor (Invitrogen, Cergy Pontoise, France) and (2) second stage- 5 μl of cDNA were amplified using 50 pmol of the 292 and 222 primers directed to the enteroviral VP1-capsid-protein coding region ( ) and 1.25 units of Platinum Taq DNA polymerase (Invitrogen, Cergy Pontoise, France) in 50 μl of reaction mixture according to the protocol [ ]. ..

    Article Title: Munc13-Independent Vesicle Priming at Mouse Photoreceptor Ribbon Synapses
    Article Snippet: The content of the tubes was collected by brief centrifugation and the following components were added: 4 μl of 5× First-Strand Buffer, 1 μl of 0.1 m DTT, 1 μl of RNaseOUT Recombinant RNase Inhibitor (Invitrogen), and 1 μl of SuperScript III RT (200 U/μl). .. All PCR products were cloned into the pCR-XL-TOPO vector (Invitrogen); clones containing inserts were purified using the PureLink Quick Plasmid Miniprep Kit (Invitrogen) and sequenced.

    Article Title: The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells
    Article Snippet: Paragraph title: RNA extraction and real-time reverse transcription PCR ... Nuclear and cytoplasmic RNA fractions were separated using the CelLytic NuCLEAR Extraction Kit (Sigma) supplemented with RNaseOUT ribonuclease inhibitor (Life Technologies).

    Article Title: Repression of Salmonella entericaphoP Expression by Small Molecules from Physiological Bile
    Article Snippet: The entire RNA sample (≥3 μg) was mixed with 6 μg of random primers (Invitrogen, Burlington, Canada) and 40 U of RNaseOUT recombinant RNase inhibitor (Invitrogen) in a total volume of 18.5 μl and incubated at 70°C for 10 min. .. The mixture was incubated at 42°C overnight, and the reaction was stopped and the RNA degraded through the addition of 0.5 M EDTA and 1 M sodium hydroxide (10 μl each) and incubation at 65°C for 15 min. Then, 25 μl of 1 M Tris (pH 7) was added to neutralize the pH of the cDNA solution. cDNA was purified using the MinElute PCR purification kit (Qiagen) according to the manufacturer's recommendations, except that the Qiagen wash and elution buffers were substituted for by phosphate buffers, according to the PFGRC microarray protocol. cDNA was then precipitated using ammonium acetate and ethanol, as described elsewhere , and labeled with Cy3 (LB cultures) and Cy5 (bile cultures) using the ULS aRNA fluorescent labeling kit (Kreatech Biotechnology, Amsterdam, The Netherlands) according to the manufacturer's instructions.

    Size-exclusion Chromatography:

    Article Title: Postmortem diagnosis of infectious heart diseases: A mystifying cause of Sudden Infant Death
    Article Snippet: The reaction was conducted with an initial reverse transcription step at 42°C for 30 min, followed by PCR activation at 94°C for 5 min, 30 amplification cycles (94°C, 30 sec; 42°C, 1 min; 72°C, 2 min) and a final 10-min extension at 72°C in an Eppendorf Mastercycler Thermal Cycler. .. Enterovirus PCR from VP1- capsid protein region was conducted in two stages as follows: (1) first stage- 10 μl of previously extracted RNA were reverse-transcribed into cDNA at 42°C for 45 min using 200 units of Super-ScriptIII reverse transcriptase and 2.5 ng/μl of random primers (Invitrogen, Cergy Pontoise, France) in the presence of 10 units of RnaseOUT recombinant RNase inhibitor (Invitrogen, Cergy Pontoise, France) and (2) second stage- 5 μl of cDNA were amplified using 50 pmol of the 292 and 222 primers directed to the enteroviral VP1-capsid-protein coding region ( ) and 1.25 units of Platinum Taq DNA polymerase (Invitrogen, Cergy Pontoise, France) in 50 μl of reaction mixture according to the protocol [ ].

    Labeling:

    Article Title: Repression of Salmonella entericaphoP Expression by Small Molecules from Physiological Bile
    Article Snippet: The entire RNA sample (≥3 μg) was mixed with 6 μg of random primers (Invitrogen, Burlington, Canada) and 40 U of RNaseOUT recombinant RNase inhibitor (Invitrogen) in a total volume of 18.5 μl and incubated at 70°C for 10 min. .. The mixture was incubated at 42°C overnight, and the reaction was stopped and the RNA degraded through the addition of 0.5 M EDTA and 1 M sodium hydroxide (10 μl each) and incubation at 65°C for 15 min. Then, 25 μl of 1 M Tris (pH 7) was added to neutralize the pH of the cDNA solution. cDNA was purified using the MinElute PCR purification kit (Qiagen) according to the manufacturer's recommendations, except that the Qiagen wash and elution buffers were substituted for by phosphate buffers, according to the PFGRC microarray protocol. cDNA was then precipitated using ammonium acetate and ethanol, as described elsewhere , and labeled with Cy3 (LB cultures) and Cy5 (bile cultures) using the ULS aRNA fluorescent labeling kit (Kreatech Biotechnology, Amsterdam, The Netherlands) according to the manufacturer's instructions.

    Purification:

    Article Title: Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli
    Article Snippet: Paragraph title: Purification of NTH-RNase E and discontinuous cleavage assays ... Discontinuous cleavage assays were performed in a buffer containing 25 mM bis -Tris propane (pH 8.3), 100 mM NaCl, 15 mM MgCl2 , 0.1% (v/v) Triton X-100, 1 mM dithiothreitol (DTT) and 32 U RNaseOUT™ ribonuclease inhibitor (Invitrogen).

    Article Title: FACS purification of Drosophila larval Neuroblasts for next generation sequencing
    Article Snippet: .. Chloroform/Trichlormethan (Roth, cat. no. 3313.4) Isopropanol (Roth, cat. no. CP41) RNase free water (Life technologies, cat. no. AM9932) Diethylpyrocarbonate (DEPC) treated water (Life technologies, cat. no. 750023) Glycoblue (Life technologies, cat. no. AM9515) Agilent RNA 6000 Nano Kit (Agilent Technologies, cat. no. 5067-1511) Agilent RNA 6000 Pico Kit (Agilent Technologies, cat. no. 5067-1513) Dynabeads mRNA purification Kit (Life technologies, cat. no. 61006) Fragmentation buffer 5× (see REAGENT SETUP) Tris base, adjusted to pH 8.2 (Sigma-Aldrich, cat. no. T1503) Potassium acetate (Sigma-Aldrich, cat. no. P1190) Magensium acetate (Sigma-Aldrich, cat. no. M5661) Sodium Acetate 3 M, pH 5.5 (Life technologies, cat. no. AM9740) Random hexamer primers 3 μg/μL (Life technologies, cat. no. 48190-011) dNTP Mix, 10 mM (Life technologies, cat. no. 18427-013) SuperScript® II Reverse Transcriptase (Life technologies, cat. no. 18064-014) SuperScript® III Reverse Transcriptase, in a set containing 5× first-strand buffer and 0.1 M DTT (Life technologies, cat. no. 18080-044) iQ SYBR Green Supermix (Bio-Rad, cat. no. 170-8882) RNaseOUT™ (Life technologies, cat. no. 10777-019) Second-Strand Buffer (Life technologies, cat. no. 10812-014) dUTP (Fermentas, cat. no. R0133) dATP (Fermentas, cat. no. R0141) dCTP (Fermentas, cat. no. R0151) dGTP (Fermentas, cat. no. R0161) DNA Polymerase I ( Escherichia coli ) (Life technologies, cat. no. 18010-017) Ribonuclease H (Life technologies, cat. no. 18021-014) E. coli DNA Ligase (Life technologies, cat. no. 18052-019) MinElute Reaction Cleanup Kit (QIAGEN, cat. no. 28204) Quant-iT Picogreen dsDNA Assay Kit (Life technologies, cat. no. P7589) NEBNext DNA sample Prep Reagent kits (New England Biolabs, cat. no. E6040) .. Foreceps Dumont Nr.5 (Fine Science Tools, cat. no. 11252-23) Stereomicrosope SteREO Discovery.V8 (Zeiss) Glass-bottom dishes, 35mm (MatTek Corporation, cat. no. P35G-0-14-C) Steriflip (Millipore, cat. no. SCGP00525) Thermo mixer compact (Eppendorf) Cell strainer, 35 μm (BD Bioscience Falcon, cat. no. 352235) FACSAriaIII, cell sorter (BD Bioscience) Low-binding reagent tubes, 1.5 mL (Life technologies, cat. no. AM12450) Phase-lock heavy gel tubes, 2 mL (5 PRIME, cat. no. 2302830) Agilent 2100 Bioanalyzer (Agilent Technologies) Magnetic rack (for example DynaMag™-2, Life technologies, cat. no. 12321D) NanoDrop 3300 fluorospectrophotometer (Thermo Scientific) Genome Analyzer IIX (GAIIX; Illumina) HiSeq 2000 sequence analyzer (Illumina)

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers
    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies). .. Transcription reactions were incubated at 42°C for 1–2 h. After this, transcripts of the circuit components were either (i) used directly for assembly or (ii) subjected to purification before assembly.

    Article Title: Munc13-Independent Vesicle Priming at Mouse Photoreceptor Ribbon Synapses
    Article Snippet: The concentration of purified RNA was measured photometrically. .. The content of the tubes was collected by brief centrifugation and the following components were added: 4 μl of 5× First-Strand Buffer, 1 μl of 0.1 m DTT, 1 μl of RNaseOUT Recombinant RNase Inhibitor (Invitrogen), and 1 μl of SuperScript III RT (200 U/μl).

    Article Title: The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells
    Article Snippet: RNA extraction and real-time reverse transcription PCR Cartilage, synovium and fat pad samples were ground into powder and homogenised using Invitrogen TRIzol Reagent (Life Technologies) prior to RNA purification using the Qiagen RNeasy mini kit (Qiagen) essentially as previously described ( ). .. Nuclear and cytoplasmic RNA fractions were separated using the CelLytic NuCLEAR Extraction Kit (Sigma) supplemented with RNaseOUT ribonuclease inhibitor (Life Technologies).

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: .. The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific). .. Sample concentration was determined using a Qubit 2.0 Fluorometer and Qubit DNA HS Assay Kit (Thermo Fisher Scientific).

    Article Title: Repression of Salmonella entericaphoP Expression by Small Molecules from Physiological Bile
    Article Snippet: The entire RNA sample (≥3 μg) was mixed with 6 μg of random primers (Invitrogen, Burlington, Canada) and 40 U of RNaseOUT recombinant RNase inhibitor (Invitrogen) in a total volume of 18.5 μl and incubated at 70°C for 10 min. .. The mixture was incubated at 42°C overnight, and the reaction was stopped and the RNA degraded through the addition of 0.5 M EDTA and 1 M sodium hydroxide (10 μl each) and incubation at 65°C for 15 min. Then, 25 μl of 1 M Tris (pH 7) was added to neutralize the pH of the cDNA solution. cDNA was purified using the MinElute PCR purification kit (Qiagen) according to the manufacturer's recommendations, except that the Qiagen wash and elution buffers were substituted for by phosphate buffers, according to the PFGRC microarray protocol. cDNA was then precipitated using ammonium acetate and ethanol, as described elsewhere , and labeled with Cy3 (LB cultures) and Cy5 (bile cultures) using the ULS aRNA fluorescent labeling kit (Kreatech Biotechnology, Amsterdam, The Netherlands) according to the manufacturer's instructions.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Pharmacokinetics, toxicity, and cytochrome P450 modulatory activity of plumbagin
    Article Snippet: The reaction was inactivated by heating at 70 °C for 15 min and chilled on ice for 2 min. cDNA was synthesized in a total volume of 20 μL containing 5× First-Strand Buffer (250 mM Tris–HCl (pH 8.3), 375 mM KCl, and 15 mM MgCl2 ), 0.1 M DTT, 40 U RNaseOUT™ Recombinant RNase Inhibitor, 10 mM dNTPs, and 200 U Superscript III Reverse Transcriptase Rnase H (Invitrogen, Thermo Fisher Scienyific, NY, USA). .. The platinum SYBRTM Green qPCR Supermix-UDG (Invitrgen, Carsbad, CA, USA) was used for real-time PCR (RT-PCR) analysis using iCycler IQ machine (BioRad Laboratories Inc., Hercules, CA, USA).

    Article Title: Postmortem diagnosis of infectious heart diseases: A mystifying cause of Sudden Infant Death
    Article Snippet: The RT-PCR products were run on a 2% agarose gel stained with ethidium bromide and visualized under UV light. .. Enterovirus PCR from VP1- capsid protein region was conducted in two stages as follows: (1) first stage- 10 μl of previously extracted RNA were reverse-transcribed into cDNA at 42°C for 45 min using 200 units of Super-ScriptIII reverse transcriptase and 2.5 ng/μl of random primers (Invitrogen, Cergy Pontoise, France) in the presence of 10 units of RnaseOUT recombinant RNase inhibitor (Invitrogen, Cergy Pontoise, France) and (2) second stage- 5 μl of cDNA were amplified using 50 pmol of the 292 and 222 primers directed to the enteroviral VP1-capsid-protein coding region ( ) and 1.25 units of Platinum Taq DNA polymerase (Invitrogen, Cergy Pontoise, France) in 50 μl of reaction mixture according to the protocol [ ].

    Blocking Assay:

    Article Title: Repression of Salmonella entericaphoP Expression by Small Molecules from Physiological Bile
    Article Snippet: The entire RNA sample (≥3 μg) was mixed with 6 μg of random primers (Invitrogen, Burlington, Canada) and 40 U of RNaseOUT recombinant RNase inhibitor (Invitrogen) in a total volume of 18.5 μl and incubated at 70°C for 10 min. .. Prehybridization, hybridization, and washing steps were performed essentially as described in the PFGRC protocols, except that the hybridization buffer contained KREAblock blocking buffer (25%; Kreatech Biotechnology).

    Staining:

    Article Title: Replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence
    Article Snippet: Paragraph title: RNA FISH and immunofluorescence staining ... For RNA FISH coupled with immunofluorescence (IF) (RNA FISH-IF), fixed cells were permeabilized with 70% ethanol for 1 h. Permeabilized cells were then incubated with anti-human active caspase-3 antibody (1:100 dilution; Cell Signaling, cat. no. 9661) or anti-human cleaved PARP-1 antibody (1:100 dilution; Cell Signaling, cat. no. 5625) followed by Alexa Fluor 488-labeled goat anti-rabbit IgG (1:500 dilution; Invitrogen, cat. no. R37116) diluted in 1% BSA in the presence of 40 U/ml RNase inhibitor (Invitrogen, cat. no. 10777-019).

    Article Title: Postmortem diagnosis of infectious heart diseases: A mystifying cause of Sudden Infant Death
    Article Snippet: The RT-PCR products were run on a 2% agarose gel stained with ethidium bromide and visualized under UV light. .. Enterovirus PCR from VP1- capsid protein region was conducted in two stages as follows: (1) first stage- 10 μl of previously extracted RNA were reverse-transcribed into cDNA at 42°C for 45 min using 200 units of Super-ScriptIII reverse transcriptase and 2.5 ng/μl of random primers (Invitrogen, Cergy Pontoise, France) in the presence of 10 units of RnaseOUT recombinant RNase inhibitor (Invitrogen, Cergy Pontoise, France) and (2) second stage- 5 μl of cDNA were amplified using 50 pmol of the 292 and 222 primers directed to the enteroviral VP1-capsid-protein coding region ( ) and 1.25 units of Platinum Taq DNA polymerase (Invitrogen, Cergy Pontoise, France) in 50 μl of reaction mixture according to the protocol [ ].

    Sample Prep:

    Article Title: FACS purification of Drosophila larval Neuroblasts for next generation sequencing
    Article Snippet: .. Chloroform/Trichlormethan (Roth, cat. no. 3313.4) Isopropanol (Roth, cat. no. CP41) RNase free water (Life technologies, cat. no. AM9932) Diethylpyrocarbonate (DEPC) treated water (Life technologies, cat. no. 750023) Glycoblue (Life technologies, cat. no. AM9515) Agilent RNA 6000 Nano Kit (Agilent Technologies, cat. no. 5067-1511) Agilent RNA 6000 Pico Kit (Agilent Technologies, cat. no. 5067-1513) Dynabeads mRNA purification Kit (Life technologies, cat. no. 61006) Fragmentation buffer 5× (see REAGENT SETUP) Tris base, adjusted to pH 8.2 (Sigma-Aldrich, cat. no. T1503) Potassium acetate (Sigma-Aldrich, cat. no. P1190) Magensium acetate (Sigma-Aldrich, cat. no. M5661) Sodium Acetate 3 M, pH 5.5 (Life technologies, cat. no. AM9740) Random hexamer primers 3 μg/μL (Life technologies, cat. no. 48190-011) dNTP Mix, 10 mM (Life technologies, cat. no. 18427-013) SuperScript® II Reverse Transcriptase (Life technologies, cat. no. 18064-014) SuperScript® III Reverse Transcriptase, in a set containing 5× first-strand buffer and 0.1 M DTT (Life technologies, cat. no. 18080-044) iQ SYBR Green Supermix (Bio-Rad, cat. no. 170-8882) RNaseOUT™ (Life technologies, cat. no. 10777-019) Second-Strand Buffer (Life technologies, cat. no. 10812-014) dUTP (Fermentas, cat. no. R0133) dATP (Fermentas, cat. no. R0141) dCTP (Fermentas, cat. no. R0151) dGTP (Fermentas, cat. no. R0161) DNA Polymerase I ( Escherichia coli ) (Life technologies, cat. no. 18010-017) Ribonuclease H (Life technologies, cat. no. 18021-014) E. coli DNA Ligase (Life technologies, cat. no. 18052-019) MinElute Reaction Cleanup Kit (QIAGEN, cat. no. 28204) Quant-iT Picogreen dsDNA Assay Kit (Life technologies, cat. no. P7589) NEBNext DNA sample Prep Reagent kits (New England Biolabs, cat. no. E6040) .. Foreceps Dumont Nr.5 (Fine Science Tools, cat. no. 11252-23) Stereomicrosope SteREO Discovery.V8 (Zeiss) Glass-bottom dishes, 35mm (MatTek Corporation, cat. no. P35G-0-14-C) Steriflip (Millipore, cat. no. SCGP00525) Thermo mixer compact (Eppendorf) Cell strainer, 35 μm (BD Bioscience Falcon, cat. no. 352235) FACSAriaIII, cell sorter (BD Bioscience) Low-binding reagent tubes, 1.5 mL (Life technologies, cat. no. AM12450) Phase-lock heavy gel tubes, 2 mL (5 PRIME, cat. no. 2302830) Agilent 2100 Bioanalyzer (Agilent Technologies) Magnetic rack (for example DynaMag™-2, Life technologies, cat. no. 12321D) NanoDrop 3300 fluorospectrophotometer (Thermo Scientific) Genome Analyzer IIX (GAIIX; Illumina) HiSeq 2000 sequence analyzer (Illumina)

    Plasmid Preparation:

    Article Title: Munc13-Independent Vesicle Priming at Mouse Photoreceptor Ribbon Synapses
    Article Snippet: The content of the tubes was collected by brief centrifugation and the following components were added: 4 μl of 5× First-Strand Buffer, 1 μl of 0.1 m DTT, 1 μl of RNaseOUT Recombinant RNase Inhibitor (Invitrogen), and 1 μl of SuperScript III RT (200 U/μl). .. All PCR products were cloned into the pCR-XL-TOPO vector (Invitrogen); clones containing inserts were purified using the PureLink Quick Plasmid Miniprep Kit (Invitrogen) and sequenced.

    Article Title: The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells
    Article Snippet: Nuclear and cytoplasmic RNA fractions were separated using the CelLytic NuCLEAR Extraction Kit (Sigma) supplemented with RNaseOUT ribonuclease inhibitor (Life Technologies). .. All values are presented as the mean±s.e.m. of replicates in pooled experiments. lncRNA real-time RT-PCR amplification products were sequence verified by cloning into the pCR4-TOPO vector (Life Technologies).

    Hybridization:

    Article Title: Replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence
    Article Snippet: RNA FISH was performed by hybridizing fixed cells with probes (125 nM for SeV and 250 nM for RSV) diluted in 50 μl hybridization buffer consisting of 10 % formamide, 2× SSC, and 10 % (wt/vol) dextran sulfate. .. For RNA FISH coupled with immunofluorescence (IF) (RNA FISH-IF), fixed cells were permeabilized with 70% ethanol for 1 h. Permeabilized cells were then incubated with anti-human active caspase-3 antibody (1:100 dilution; Cell Signaling, cat. no. 9661) or anti-human cleaved PARP-1 antibody (1:100 dilution; Cell Signaling, cat. no. 5625) followed by Alexa Fluor 488-labeled goat anti-rabbit IgG (1:500 dilution; Invitrogen, cat. no. R37116) diluted in 1% BSA in the presence of 40 U/ml RNase inhibitor (Invitrogen, cat. no. 10777-019).

    Article Title: Repression of Salmonella entericaphoP Expression by Small Molecules from Physiological Bile
    Article Snippet: The entire RNA sample (≥3 μg) was mixed with 6 μg of random primers (Invitrogen, Burlington, Canada) and 40 U of RNaseOUT recombinant RNase inhibitor (Invitrogen) in a total volume of 18.5 μl and incubated at 70°C for 10 min. .. Prehybridization, hybridization, and washing steps were performed essentially as described in the PFGRC protocols, except that the hybridization buffer contained KREAblock blocking buffer (25%; Kreatech Biotechnology).

    Real-time Polymerase Chain Reaction:

    Article Title: Pharmacokinetics, toxicity, and cytochrome P450 modulatory activity of plumbagin
    Article Snippet: The reaction was inactivated by heating at 70 °C for 15 min and chilled on ice for 2 min. cDNA was synthesized in a total volume of 20 μL containing 5× First-Strand Buffer (250 mM Tris–HCl (pH 8.3), 375 mM KCl, and 15 mM MgCl2 ), 0.1 M DTT, 40 U RNaseOUT™ Recombinant RNase Inhibitor, 10 mM dNTPs, and 200 U Superscript III Reverse Transcriptase Rnase H (Invitrogen, Thermo Fisher Scienyific, NY, USA). .. The platinum SYBRTM Green qPCR Supermix-UDG (Invitrgen, Carsbad, CA, USA) was used for real-time PCR (RT-PCR) analysis using iCycler IQ machine (BioRad Laboratories Inc., Hercules, CA, USA).

    Article Title: Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors
    Article Snippet: .. Quantitative PCR The Ambion PureLink RNA mini kit was used to extract total cellular RNA. cDNA reverse transcription was then conducted using Superscript III reverse transcriptase and RNAseOUT recombinant ribonuclease inhibitor (Invitrogen). qPCR was performed using BioRad SsoAdvanced SYBR green supermix and the Biorad CFX Connect real-time system. .. Relative gene expression was normalized to the housekeeping gene TBP, and fold-change calculated based on a comparison to the expression level of the adult pancreas.

    RNA Extraction:

    Article Title: The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells
    Article Snippet: Paragraph title: RNA extraction and real-time reverse transcription PCR ... Nuclear and cytoplasmic RNA fractions were separated using the CelLytic NuCLEAR Extraction Kit (Sigma) supplemented with RNaseOUT ribonuclease inhibitor (Life Technologies).

    Agarose Gel Electrophoresis:

    Article Title: Postmortem diagnosis of infectious heart diseases: A mystifying cause of Sudden Infant Death
    Article Snippet: The RT-PCR products were run on a 2% agarose gel stained with ethidium bromide and visualized under UV light. .. Enterovirus PCR from VP1- capsid protein region was conducted in two stages as follows: (1) first stage- 10 μl of previously extracted RNA were reverse-transcribed into cDNA at 42°C for 45 min using 200 units of Super-ScriptIII reverse transcriptase and 2.5 ng/μl of random primers (Invitrogen, Cergy Pontoise, France) in the presence of 10 units of RnaseOUT recombinant RNase inhibitor (Invitrogen, Cergy Pontoise, France) and (2) second stage- 5 μl of cDNA were amplified using 50 pmol of the 292 and 222 primers directed to the enteroviral VP1-capsid-protein coding region ( ) and 1.25 units of Platinum Taq DNA polymerase (Invitrogen, Cergy Pontoise, France) in 50 μl of reaction mixture according to the protocol [ ].

    In Vitro:

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers
    Article Snippet: .. In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies). .. Transcription reactions were incubated at 42°C for 1–2 h. After this, transcripts of the circuit components were either (i) used directly for assembly or (ii) subjected to purification before assembly.

    Ligation:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The Cap-selected mRNA sample was subjected to end repair and adapter ligation steps – as was described above – before loading on the Flow Cells. .. The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific).

    Incubation:

    Article Title: Pharmacokinetics, toxicity, and cytochrome P450 modulatory activity of plumbagin
    Article Snippet: Briefly, total RNA (10 pg–5 μg) and 1 μL of 50 μM oligo(dT)20 were incubated at 55 °C for 50 min. .. The reaction was inactivated by heating at 70 °C for 15 min and chilled on ice for 2 min. cDNA was synthesized in a total volume of 20 μL containing 5× First-Strand Buffer (250 mM Tris–HCl (pH 8.3), 375 mM KCl, and 15 mM MgCl2 ), 0.1 M DTT, 40 U RNaseOUT™ Recombinant RNase Inhibitor, 10 mM dNTPs, and 200 U Superscript III Reverse Transcriptase Rnase H (Invitrogen, Thermo Fisher Scienyific, NY, USA).

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers
    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies). .. Transcription reactions were incubated at 42°C for 1–2 h. After this, transcripts of the circuit components were either (i) used directly for assembly or (ii) subjected to purification before assembly.

    Article Title: Replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence
    Article Snippet: .. For RNA FISH coupled with immunofluorescence (IF) (RNA FISH-IF), fixed cells were permeabilized with 70% ethanol for 1 h. Permeabilized cells were then incubated with anti-human active caspase-3 antibody (1:100 dilution; Cell Signaling, cat. no. 9661) or anti-human cleaved PARP-1 antibody (1:100 dilution; Cell Signaling, cat. no. 5625) followed by Alexa Fluor 488-labeled goat anti-rabbit IgG (1:500 dilution; Invitrogen, cat. no. R37116) diluted in 1% BSA in the presence of 40 U/ml RNase inhibitor (Invitrogen, cat. no. 10777-019). .. Stained cells were incubated in 4% formaldehyde for 10 min prior to RNA FISH, washed with PBS, and then equilibrated in wash buffer.

    Article Title: Repression of Salmonella entericaphoP Expression by Small Molecules from Physiological Bile
    Article Snippet: .. The entire RNA sample (≥3 μg) was mixed with 6 μg of random primers (Invitrogen, Burlington, Canada) and 40 U of RNaseOUT recombinant RNase inhibitor (Invitrogen) in a total volume of 18.5 μl and incubated at 70°C for 10 min. .. Samples were incubated on ice for 30 s, centrifuged to bring down condensation, and incubated with the following components (Invitrogen) at the final concentrations shown in parentheses: First Strand buffer (1×), dithiothreitol (10 mM), dNTPs (dATP, dCTP, dGTP, dTTP; 0.5 mM each) and 100 U of SuperScript II reverse transcriptase in a total volume of 30 μl.

    Article Title: Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis
    Article Snippet: Cell pellets from approximately 50 million cells were homogenized, lysed in ice-cold lysis buffer (10 mM HEPES (pH 7.3, ThermoFisher Scientific), 100 mM KCl (1049360250, Merck), 0.5 % NP40 (NP40S, Sigma-Aldrich), 5 mM MgCl2 , 0.5 mM dithiothreitol (DTT, DTT-RO, Sigma-Aldrich), protease inhibitors (118735, Sigma-Aldrich), recombinant RNase inhibitors (10777019, ThermoFisher Schientific), 1 mM PMSF (36978, ThermoFisher Scientific) using TissueLyser LT (50 Hz, 2 min, Qiagen). .. The RIP samples were incubated with Dynabeads Protein G beads (10009D, ThermoFisher Scientific) coated with anti-hAGO2 antibody (a gift from JA Nelson) ( ) for 24h at 4°C with end-over-end rotation.

    Spectrophotometry:

    Article Title: Pharmacokinetics, toxicity, and cytochrome P450 modulatory activity of plumbagin
    Article Snippet: The RNA pellets were dried and dissolved in 20 μL DEPC-treated water and the concentrations were determined using NanoDrop Spectrophotometry (NanoDrop Technologies, Wilmington, DE, USA). .. The reaction was inactivated by heating at 70 °C for 15 min and chilled on ice for 2 min. cDNA was synthesized in a total volume of 20 μL containing 5× First-Strand Buffer (250 mM Tris–HCl (pH 8.3), 375 mM KCl, and 15 mM MgCl2 ), 0.1 M DTT, 40 U RNaseOUT™ Recombinant RNase Inhibitor, 10 mM dNTPs, and 200 U Superscript III Reverse Transcriptase Rnase H (Invitrogen, Thermo Fisher Scienyific, NY, USA).

    Activation Assay:

    Article Title: Postmortem diagnosis of infectious heart diseases: A mystifying cause of Sudden Infant Death
    Article Snippet: The reaction was conducted with an initial reverse transcription step at 42°C for 30 min, followed by PCR activation at 94°C for 5 min, 30 amplification cycles (94°C, 30 sec; 42°C, 1 min; 72°C, 2 min) and a final 10-min extension at 72°C in an Eppendorf Mastercycler Thermal Cycler. .. Enterovirus PCR from VP1- capsid protein region was conducted in two stages as follows: (1) first stage- 10 μl of previously extracted RNA were reverse-transcribed into cDNA at 42°C for 45 min using 200 units of Super-ScriptIII reverse transcriptase and 2.5 ng/μl of random primers (Invitrogen, Cergy Pontoise, France) in the presence of 10 units of RnaseOUT recombinant RNase inhibitor (Invitrogen, Cergy Pontoise, France) and (2) second stage- 5 μl of cDNA were amplified using 50 pmol of the 292 and 222 primers directed to the enteroviral VP1-capsid-protein coding region ( ) and 1.25 units of Platinum Taq DNA polymerase (Invitrogen, Cergy Pontoise, France) in 50 μl of reaction mixture according to the protocol [ ].

    Concentration Assay:

    Article Title: Munc13-Independent Vesicle Priming at Mouse Photoreceptor Ribbon Synapses
    Article Snippet: The concentration of purified RNA was measured photometrically. .. The content of the tubes was collected by brief centrifugation and the following components were added: 4 μl of 5× First-Strand Buffer, 1 μl of 0.1 m DTT, 1 μl of RNaseOUT Recombinant RNase Inhibitor (Invitrogen), and 1 μl of SuperScript III RT (200 U/μl).

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The cDNA sample was purified between each step using Agencourt AMPure XP magnetic beads (Beckman Coulter) and the library concentration was determined using a Qubit 2.0 Fluorometer through use of the Qubit (ds)DNA HS Assay Kit (Thermo Fisher Scientific). .. The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific).

    Lysis:

    Article Title: The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells
    Article Snippet: MSC chondrogenesis pellets were disrupted in Ambion Cells-to-cDNA II Cell Lysis buffer (Life Technologies). .. Nuclear and cytoplasmic RNA fractions were separated using the CelLytic NuCLEAR Extraction Kit (Sigma) supplemented with RNaseOUT ribonuclease inhibitor (Life Technologies).

    Article Title: Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis
    Article Snippet: .. Cell pellets from approximately 50 million cells were homogenized, lysed in ice-cold lysis buffer (10 mM HEPES (pH 7.3, ThermoFisher Scientific), 100 mM KCl (1049360250, Merck), 0.5 % NP40 (NP40S, Sigma-Aldrich), 5 mM MgCl2 , 0.5 mM dithiothreitol (DTT, DTT-RO, Sigma-Aldrich), protease inhibitors (118735, Sigma-Aldrich), recombinant RNase inhibitors (10777019, ThermoFisher Schientific), 1 mM PMSF (36978, ThermoFisher Scientific) using TissueLyser LT (50 Hz, 2 min, Qiagen). ..

    Fluorescence In Situ Hybridization:

    Article Title: Replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence
    Article Snippet: .. For RNA FISH coupled with immunofluorescence (IF) (RNA FISH-IF), fixed cells were permeabilized with 70% ethanol for 1 h. Permeabilized cells were then incubated with anti-human active caspase-3 antibody (1:100 dilution; Cell Signaling, cat. no. 9661) or anti-human cleaved PARP-1 antibody (1:100 dilution; Cell Signaling, cat. no. 5625) followed by Alexa Fluor 488-labeled goat anti-rabbit IgG (1:500 dilution; Invitrogen, cat. no. R37116) diluted in 1% BSA in the presence of 40 U/ml RNase inhibitor (Invitrogen, cat. no. 10777-019). .. Stained cells were incubated in 4% formaldehyde for 10 min prior to RNA FISH, washed with PBS, and then equilibrated in wash buffer.

    Hood:

    Article Title: FACS purification of Drosophila larval Neuroblasts for next generation sequencing
    Article Snippet: D. melanogaster larvae, 5 d old, reared at 25 °C, e.g., asense -Gal4 (ref. ) PBS buffer (see REAGENT SETUP) Ethanol (Roth, cat. no. P076) Rinaldini’s solution (see REAGENT SETUP) NaCl (Sigma-Aldrich, cat. no. S3014) KCl (Sigma-Aldrich, cat. no. P9541) NaH2 PO4 (Sigma-Aldrich, cat. no. S9638) Na2 HPO4 (Sigma-Aldrich, cat. no. S3264) NaHCO3 (Sigma-Aldrich, cat. no. S5761) Glucose (Sigma-Aldrich, cat. no. G0350500) Schneider’s culture medium (Gibco, cat. no. 21720024) Penicillin-Streptomycin solution (Sigma-Aldrich, cat. no. P4458) Insulin solution from bovine pancreas (Sigma-Aldrich, cat. no. I0516) L-Glutamine (Sigma-Aldrich, cat. no. G7513) L-Glutathione (Sigma-Aldrich, cat. no. G6013) FBS, heat inactivated (Sigma-Aldrich, cat. no. F4135) Collagenase Type I (Sigma-Aldrich, cat. no. C2674) Papain (Sigma-Aldrich, cat. no. P4762) FACSFlow sheath fluid (BD Bioscience, cat. no. 342003) Poly-D-lysin-hydobromide (Sigma-Aldrich, cat. no. P6282) TRIzol LS reagent (Life technologies, cat. no. 10296-028) !CAUTION TRIzol is highly toxic and should only be used in the fume hood. .. Chloroform/Trichlormethan (Roth, cat. no. 3313.4) Isopropanol (Roth, cat. no. CP41) RNase free water (Life technologies, cat. no. AM9932) Diethylpyrocarbonate (DEPC) treated water (Life technologies, cat. no. 750023) Glycoblue (Life technologies, cat. no. AM9515) Agilent RNA 6000 Nano Kit (Agilent Technologies, cat. no. 5067-1511) Agilent RNA 6000 Pico Kit (Agilent Technologies, cat. no. 5067-1513) Dynabeads mRNA purification Kit (Life technologies, cat. no. 61006) Fragmentation buffer 5× (see REAGENT SETUP) Tris base, adjusted to pH 8.2 (Sigma-Aldrich, cat. no. T1503) Potassium acetate (Sigma-Aldrich, cat. no. P1190) Magensium acetate (Sigma-Aldrich, cat. no. M5661) Sodium Acetate 3 M, pH 5.5 (Life technologies, cat. no. AM9740) Random hexamer primers 3 μg/μL (Life technologies, cat. no. 48190-011) dNTP Mix, 10 mM (Life technologies, cat. no. 18427-013) SuperScript® II Reverse Transcriptase (Life technologies, cat. no. 18064-014) SuperScript® III Reverse Transcriptase, in a set containing 5× first-strand buffer and 0.1 M DTT (Life technologies, cat. no. 18080-044) iQ SYBR Green Supermix (Bio-Rad, cat. no. 170-8882) RNaseOUT™ (Life technologies, cat. no. 10777-019) Second-Strand Buffer (Life technologies, cat. no. 10812-014) dUTP (Fermentas, cat. no. R0133) dATP (Fermentas, cat. no. R0141) dCTP (Fermentas, cat. no. R0151) dGTP (Fermentas, cat. no. R0161) DNA Polymerase I ( Escherichia coli ) (Life technologies, cat. no. 18010-017) Ribonuclease H (Life technologies, cat. no. 18021-014) E. coli DNA Ligase (Life technologies, cat. no. 18052-019) MinElute Reaction Cleanup Kit (QIAGEN, cat. no. 28204) Quant-iT Picogreen dsDNA Assay Kit (Life technologies, cat. no. P7589) NEBNext DNA sample Prep Reagent kits (New England Biolabs, cat. no. E6040)

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    Thermo Fisher rnaseout recombinant ribonuclease inhibitor
    Rnaseout Recombinant Ribonuclease Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 17 article reviews
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    rnaseout recombinant ribonuclease inhibitor - by Bioz Stars, 2020-04
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