rnase out  (Thermo Fisher)


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    Structured Review

    Thermo Fisher rnase out
    Complementary-template reverse-transcription (CT-RT) of single-stranded DNA primers reverse-transcribed from FFPE-RNA. ( a ) RNA extracted from FFPE tissue is reverse-transcribed, the mRNA/DNA duplex is filtered on an YM-50 column and the DNA is single-stranded with <t>RNase-H</t> and column purified. The 5′-NB-Oligo-dA (24) -cT7-3′ (complementary to the T7 promoter) is annealed to the FFPE-cDNA primers. ( b ) Total RNA from universal human reference (UHR, Stratagene) is amplified using the Sense-Amp <t>cRNA</t> amplification kit from Genisphere to provide RNA with the same orientation as messenger RNA ( 32 ). ( c ) Single-stranded DNA primers are hybridized to their sense-RNA template between 70 and 42°C for 90 min. The hybridized products are reverse-transcribed by a process described as CT-RT. The restored FFPE-cDNAs are doubled stranded and transcribed in vitro using T7 polymerase. (See Supplementary Data for technical description of points 1 through 6.)
    Rnase Out, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase out/product/Thermo Fisher
    Average 99 stars, based on 299 article reviews
    Price from $9.99 to $1999.99
    rnase out - by Bioz Stars, 2020-05
    99/100 stars

    Images

    1) Product Images from "Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)"

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm510

    Complementary-template reverse-transcription (CT-RT) of single-stranded DNA primers reverse-transcribed from FFPE-RNA. ( a ) RNA extracted from FFPE tissue is reverse-transcribed, the mRNA/DNA duplex is filtered on an YM-50 column and the DNA is single-stranded with RNase-H and column purified. The 5′-NB-Oligo-dA (24) -cT7-3′ (complementary to the T7 promoter) is annealed to the FFPE-cDNA primers. ( b ) Total RNA from universal human reference (UHR, Stratagene) is amplified using the Sense-Amp cRNA amplification kit from Genisphere to provide RNA with the same orientation as messenger RNA ( 32 ). ( c ) Single-stranded DNA primers are hybridized to their sense-RNA template between 70 and 42°C for 90 min. The hybridized products are reverse-transcribed by a process described as CT-RT. The restored FFPE-cDNAs are doubled stranded and transcribed in vitro using T7 polymerase. (See Supplementary Data for technical description of points 1 through 6.)
    Figure Legend Snippet: Complementary-template reverse-transcription (CT-RT) of single-stranded DNA primers reverse-transcribed from FFPE-RNA. ( a ) RNA extracted from FFPE tissue is reverse-transcribed, the mRNA/DNA duplex is filtered on an YM-50 column and the DNA is single-stranded with RNase-H and column purified. The 5′-NB-Oligo-dA (24) -cT7-3′ (complementary to the T7 promoter) is annealed to the FFPE-cDNA primers. ( b ) Total RNA from universal human reference (UHR, Stratagene) is amplified using the Sense-Amp cRNA amplification kit from Genisphere to provide RNA with the same orientation as messenger RNA ( 32 ). ( c ) Single-stranded DNA primers are hybridized to their sense-RNA template between 70 and 42°C for 90 min. The hybridized products are reverse-transcribed by a process described as CT-RT. The restored FFPE-cDNAs are doubled stranded and transcribed in vitro using T7 polymerase. (See Supplementary Data for technical description of points 1 through 6.)

    Techniques Used: Formalin-fixed Paraffin-Embedded, Purification, Amplification, In Vitro

    2) Product Images from "High-Pressure Inactivation of Rotaviruses: Role of Treatment Temperature and Strain Diversity in Virus Inactivation"

    Article Title: High-Pressure Inactivation of Rotaviruses: Role of Treatment Temperature and Strain Diversity in Virus Inactivation

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01853-15

    Effect of HPP on viral genomic RNA. (A) Effect of HPP on RV genomic RNA in the absence of RNase inhibitor. Purified RV strain Wa was treated at 400 and 600 MPa for 2 min at 4°C. After treatment, total viral RNA was extracted, and the VP7 gene
    Figure Legend Snippet: Effect of HPP on viral genomic RNA. (A) Effect of HPP on RV genomic RNA in the absence of RNase inhibitor. Purified RV strain Wa was treated at 400 and 600 MPa for 2 min at 4°C. After treatment, total viral RNA was extracted, and the VP7 gene

    Techniques Used: Purification

    3) Product Images from "Identification of RNA Oligonucleotides Binding to Several Proteins from Potential G-Quadruplex Forming Regions in Transcribed Pre-mRNA"

    Article Title: Identification of RNA Oligonucleotides Binding to Several Proteins from Potential G-Quadruplex Forming Regions in Transcribed Pre-mRNA

    Journal: Molecules

    doi: 10.3390/molecules201119733

    Electromobility shift binding assay (EMSA) of VEGFA intronic G4_1 to VEGF165. In the presence or absence of targets, fluorescein-labeled VEGFA intronic G4_1 (or its mutant) were electrophoresed on 15% polyacrylamide gel in TBE buffer, and the fluorescence image (black band) was then detected. The white band was derived from loading dye (bromophenol blue). To inhibit RNA degradation, 1 U of RNase OUT (O) was mixed into the samples but we checked RNase OUT did not affect the binding of RNA and VEGF165 by silver staining. Arrows indicate bands of RNA-protein complex.
    Figure Legend Snippet: Electromobility shift binding assay (EMSA) of VEGFA intronic G4_1 to VEGF165. In the presence or absence of targets, fluorescein-labeled VEGFA intronic G4_1 (or its mutant) were electrophoresed on 15% polyacrylamide gel in TBE buffer, and the fluorescence image (black band) was then detected. The white band was derived from loading dye (bromophenol blue). To inhibit RNA degradation, 1 U of RNase OUT (O) was mixed into the samples but we checked RNase OUT did not affect the binding of RNA and VEGF165 by silver staining. Arrows indicate bands of RNA-protein complex.

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Fluorescence, Derivative Assay, Silver Staining

    Related Articles

    Labeling:

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
    Article Snippet: .. The aRNA was incubated in the presence of 2.67 μl of random primers (3 μg/μl) from Invitrogen in a final volume of 19 μl at 70°C for 10 min, spun down and put on ice for 5 min. Labeling reactions were performed by adding 8 μl of 5× first strand buffer, 4 μl of 0.1 M DTT, 4 μl of dNTP labeling mix (2.5 mM of each), 4 μl of 25 nM Cy3-labeled deoxyuridine triphosphate (Cy3-dUTP) for cRNA amplified from UHR (reference) or 4 μl of 25 nM Cy5-labeled deoxyuridine triphosphate (Cy5-dUTP; Amersham Pharmacia Biotech, NJ, USA) for cRNA from our samples, 1 μl of RNase-out at 40 U/μl (Invitrogen), 1.5 μl of Superscript II reverse-transcriptase 200 U/μl (Invitrogen) and incubated at 42°C for 1 h. After 1 h of incubation, 1.5 μl of Superscript II reverse-transcriptase was added for another 60 min at 42°C. .. The 40 μl reactions containing the labeled cDNAs were incubated in the presence of 44 μl of RNase-free water, 10 μl of 10× RNase-One buffer and 2 μl of RNase-One 10 U/μl (Promega) for 35 min at 37°C for removal of cRNA templates.

    Random Hexamer Labeling:

    Article Title: Expression of maternally derived KHDC3, NLRP5, OOEP and TLE6 is associated with oocyte developmental competence in the ovine species
    Article Snippet: .. Reverse-transcription was performed in a final volume of 20 μL, consisting of 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 , 5 mM DTT, 1 mM dNTPs, 2.5 μM random hexamer primers, 0.05 μg oligo (dT)18 primers, 20 U RNase OUT and 100 U SuperScript III RT (all purchased at Invitrogen Corporation, Carlsbad, CA). .. The reaction tubes were incubated at 25°C for 10 min, then at 42°C for 1 h and finally at 70°C for 15 min to inactivate the reaction.

    Amplification:

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
    Article Snippet: .. The aRNA was incubated in the presence of 2.67 μl of random primers (3 μg/μl) from Invitrogen in a final volume of 19 μl at 70°C for 10 min, spun down and put on ice for 5 min. Labeling reactions were performed by adding 8 μl of 5× first strand buffer, 4 μl of 0.1 M DTT, 4 μl of dNTP labeling mix (2.5 mM of each), 4 μl of 25 nM Cy3-labeled deoxyuridine triphosphate (Cy3-dUTP) for cRNA amplified from UHR (reference) or 4 μl of 25 nM Cy5-labeled deoxyuridine triphosphate (Cy5-dUTP; Amersham Pharmacia Biotech, NJ, USA) for cRNA from our samples, 1 μl of RNase-out at 40 U/μl (Invitrogen), 1.5 μl of Superscript II reverse-transcriptase 200 U/μl (Invitrogen) and incubated at 42°C for 1 h. After 1 h of incubation, 1.5 μl of Superscript II reverse-transcriptase was added for another 60 min at 42°C. .. The 40 μl reactions containing the labeled cDNAs were incubated in the presence of 44 μl of RNase-free water, 10 μl of 10× RNase-One buffer and 2 μl of RNase-One 10 U/μl (Promega) for 35 min at 37°C for removal of cRNA templates.

    Inhibition:

    Article Title: Identification of RNA Oligonucleotides Binding to Several Proteins from Potential G-Quadruplex Forming Regions in Transcribed Pre-mRNA
    Article Snippet: .. For inhibition of RNA degradation, 1 U of RNase OUT (Invitrogen, Carlsbad, CA, USA) was mixed into the samples then the mixtures were incubated at room temperature for 30 min. After incubation, ten microliters of the mixtures were electrophoresed on 15% polyacrylamide gel in TBE buffer, followed by fluorescein scanning the gel using Typhoon8600 (GE Healthcare, Little Chalfont, Buckinghamshire, UK). .. Surface Plasmon Resonance (SPR) Assay The binding affinities of the G4-forming RNAs for VEGF165, PDGF-AA, and PDGF-BB were analyzed at 25 °C on a Biacore T200 instrument (GE Healthcare).

    Incubation:

    Article Title: Is There Additive Therapeutic Effect When GCSF Combined with Adipose-Derived Stem Cell in a Rat Model of Acute Spinal Cord Injury?
    Article Snippet: .. The cDNA mixture was incubated at 65°C for 5 minutes, placed on ice for 1 minute, and then incubated at 50°C for 50 minutes in the following cDNA synthesis mixture: 2 μL 10×reverse transcriptase buffer, 4 μL 25 mM MgCl2 , 2 μL 0.1 M DTT, 1 μL RNase OUT™ (40 U/μL; Invitrogen), and 1 μL Superscript™ III RT (200 U/μL; Invitrogen). .. Following the addition of 1 μL of RNase H, the sample was incubated for 20 minutes at 37°C.

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
    Article Snippet: .. The aRNA was incubated in the presence of 2.67 μl of random primers (3 μg/μl) from Invitrogen in a final volume of 19 μl at 70°C for 10 min, spun down and put on ice for 5 min. Labeling reactions were performed by adding 8 μl of 5× first strand buffer, 4 μl of 0.1 M DTT, 4 μl of dNTP labeling mix (2.5 mM of each), 4 μl of 25 nM Cy3-labeled deoxyuridine triphosphate (Cy3-dUTP) for cRNA amplified from UHR (reference) or 4 μl of 25 nM Cy5-labeled deoxyuridine triphosphate (Cy5-dUTP; Amersham Pharmacia Biotech, NJ, USA) for cRNA from our samples, 1 μl of RNase-out at 40 U/μl (Invitrogen), 1.5 μl of Superscript II reverse-transcriptase 200 U/μl (Invitrogen) and incubated at 42°C for 1 h. After 1 h of incubation, 1.5 μl of Superscript II reverse-transcriptase was added for another 60 min at 42°C. .. The 40 μl reactions containing the labeled cDNAs were incubated in the presence of 44 μl of RNase-free water, 10 μl of 10× RNase-One buffer and 2 μl of RNase-One 10 U/μl (Promega) for 35 min at 37°C for removal of cRNA templates.

    Article Title: Identification of RNA Oligonucleotides Binding to Several Proteins from Potential G-Quadruplex Forming Regions in Transcribed Pre-mRNA
    Article Snippet: .. For inhibition of RNA degradation, 1 U of RNase OUT (Invitrogen, Carlsbad, CA, USA) was mixed into the samples then the mixtures were incubated at room temperature for 30 min. After incubation, ten microliters of the mixtures were electrophoresed on 15% polyacrylamide gel in TBE buffer, followed by fluorescein scanning the gel using Typhoon8600 (GE Healthcare, Little Chalfont, Buckinghamshire, UK). .. Surface Plasmon Resonance (SPR) Assay The binding affinities of the G4-forming RNAs for VEGF165, PDGF-AA, and PDGF-BB were analyzed at 25 °C on a Biacore T200 instrument (GE Healthcare).

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    Thermo Fisher rnase out
    Rnase Out, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase out/product/Thermo Fisher
    Average 99 stars, based on 299 article reviews
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