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 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 "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

    3) 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:

    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

    Related Articles

    Clone Assay:

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Streptavidin-binding S1m DNA was synthesized and cloned into pcDNA5-CMV just ahead of the wild-type Chaer and truncations with roughly 500 bp intervals both from 5′ and 3′. .. All these constructs together with untagged wild-type Chaer and EGFP were transfected into MEFs for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 1.5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]).

    Article Title: The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation
    Article Snippet: Lysates were prepared in Lysis Buffer PD [20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5% NP-40, 10 mM DTT, protease inhibitors (Merck) and 0.5 U/μl RNase OUT (Thermo Scientific)]. .. Lysates were prepared in Lysis Buffer PD [20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5% NP-40, 10 mM DTT, protease inhibitors (Merck) and 0.5 U/μl RNase OUT (Thermo Scientific)].

    Centrifugation:

    Article Title: A high throughput experimental approach to identify miRNA targets in human cells
    Article Snippet: IP of ribonucleoprotein was performed as previously described ( ) in both HL cell lines (L1236 and L428) with and without transfection of anti-miR-17/20/93/106 and additionally L428 with transfection of anti-miR-220 as a negative control. .. Briefly, 10–20-million cells were lysed in 100 µl ice cold polysome lysis buffer (5 mM MgCl2 , 100 mM KCl, 10 mM Hepes, pH7.0 and 0.5% Nonidet P-40) with freshly added 1 mM DTT, 100 U/ml Rnase OUT (Invitrogen, Carlsbad, USA) and 1× complete mini EDTA free protease inhibitor cocktail (Roche, Basel, Switzerland) for 5 min. Centrifugation was carried out two times at 14 000g at 4°C for 10 min. Supernatant was mixed with 900 µl of ice-cold NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40) containing freshly added 200 U/ml Rnase OUT (Invitrogen, Carlsbad, USA), 0.5% vanadyl ribonucleoside (Invitrogen, Carlsbad, USA), 1 mM DTT, 15 mM EDTA and 50 µl mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated sepharose G beads (Abcam, Cambridge, UK). .. On the following day, beads were washed five times with ice-cold NT2 buffer and separated into two portions—one for RNA isolation to identify miRNA target genes and another portion for western blotting to check for successful IP of Ago2.

    Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response
    Article Snippet: HUVECs were cross-linked with 0.3% formaldehyde at room temperature for 10 min and neutralized with 0.125 M of glycine at room temperature for 5 min. Cross-linked cells were washed with 1× PBS, collected by scraping and lysed in polysome lysis buffer containing 100 mM KCl, 5 mM MgCl2 , 10 mM HEPES at pH 7.0, 0.5% Nonidet P-40, 1 mM dithiothreitol (DTT), 200 units/ml RNase OUT (Invitrogen Cat. No. 10777-019) and Complete Mini, EDTA-free Protease Inhibitor Tablet (11836170001, Roche) for 30 min on ice. .. Lysate was diluted to 1 ml in NT-2 buffer containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.5% NP-40, 20 mM EDTA at pH 8.0, 1 mM DTT, 200 units/ml RNase OUT and sonicated for 20 s at 20% power using Branson 250 Ultrasonic Sonifier.

    Amplification:

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
    Article Snippet: For each microarray experiment, 5 μg of cRNA synthesized from UHR RNA (reference) and 5 μg of cRNA from our samples were labeled. .. 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: Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques
    Article Snippet: In order to avoid the potential PCR biases, the amplification-free direct RNA sequencing (DRS) protocol (Version: DRS_9026_v1_revM_15Dec2016) from the ONTs was applied. .. XP Beads were handled before use with RNase OUT (40 U/μL; Life Technologies; 2 U enzyme/1 μL bead).

    Article Title: Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice
    Article Snippet: Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors. .. Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors.

    Article Title: Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice
    Article Snippet: Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors. .. Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors.

    Article Title: R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein
    Article Snippet: Reverse transcription was performed on 2.2 μg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs and ten units of RNAse OUT (Thermo Scientific) following the manufacturer's protocol in a total volume of 20 μl. .. Reverse transcription was performed on 2.2 μg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs and ten units of RNAse OUT (Thermo Scientific) following the manufacturer's protocol in a total volume of 20 μl.

    Synthesized:

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
    Article Snippet: For each microarray experiment, 5 μg of cRNA synthesized from UHR RNA (reference) and 5 μg of cRNA from our samples were labeled. .. 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.

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Streptavidin-binding S1m DNA was synthesized and cloned into pcDNA5-CMV just ahead of the wild-type Chaer and truncations with roughly 500 bp intervals both from 5′ and 3′. .. All these constructs together with untagged wild-type Chaer and EGFP were transfected into MEFs for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 1.5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]).

    Autoradiography:

    Article Title: CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo
    Article Snippet: To produce 5′-end-labeled sgRNAs, purified sgRNA transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BIoLabs) to 50 pmol of sgRNA in a final volume of 10 μL containing 50 mM Bis-Propane pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20U, Invitrogen). .. The reaction was stopped by adding formamide dye buffer (95% formamide, 10 mM EDTA, 0.025% bromophenol blue and 0.025% xylene cyanol), and the radiolabeled sgRNA purified by 8% polyacrylamide gel electrophoresis.

    Construct:

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Streptavidin-binding S1m DNA was synthesized and cloned into pcDNA5-CMV just ahead of the wild-type Chaer and truncations with roughly 500 bp intervals both from 5′ and 3′. .. All these constructs together with untagged wild-type Chaer and EGFP were transfected into MEFs for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 1.5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. Streptavidin sepharose beads were blocked with 500 ng/μl yeast tRNA and 1 mg/ml BSA in SA-RNP lysis buffer before added into cell lysates and incubated at 37°C for 2 h on a rotator.

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: All these constructs together with untagged wild-type Chaer and EGFP were transfected into MEFs for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 1.5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. The beads were then pelleted and washed for 5 times with SA-RNP washing buffer (20 mM Tris-HCl [pH7.5], 300 mM NaCl, 5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]).

    Article Title: The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation
    Article Snippet: Confluent monolayers of stable transformant cells HEK293-MS2-HB (10 × 106 ) were transfected with the constructs pCAP-luc, pCAP-H12-MS2h and pCAP-H12d-MS2h using lipofectamine (Thermo Scientific) according to the manufacturer's instructions. .. Lysates were prepared in Lysis Buffer PD [20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5% NP-40, 10 mM DTT, protease inhibitors (Merck) and 0.5 U/μl RNase OUT (Thermo Scientific)].

    SYBR Green Assay:

    Article Title: Mycobacterium abscessus subsp. massiliense mycma_0076 and mycma_0077 Genes Code for Ferritins That Are Modulated by Iron Concentration
    Article Snippet: Next, the system was transferred to ice and reverse transcriptase buffer, 200 U of M-MLV reverse transcriptase, and 40 U of RNAse OUT (Invitrogen) were added. .. The reaction was incubated at 37°C for 1.5 h. Then the synthesized cDNA was stored at -20°C until its use for Real Time PCR (RT-PCR).

    Microarray:

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
    Article Snippet: Paragraph title: Cy3/Cy5 labeling of cRNA and microarray hybridization ... 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.

    Incubation:

    Article Title: A high throughput experimental approach to identify miRNA targets in human cells
    Article Snippet: Briefly, 10–20-million cells were lysed in 100 µl ice cold polysome lysis buffer (5 mM MgCl2 , 100 mM KCl, 10 mM Hepes, pH7.0 and 0.5% Nonidet P-40) with freshly added 1 mM DTT, 100 U/ml Rnase OUT (Invitrogen, Carlsbad, USA) and 1× complete mini EDTA free protease inhibitor cocktail (Roche, Basel, Switzerland) for 5 min. Centrifugation was carried out two times at 14 000g at 4°C for 10 min. Supernatant was mixed with 900 µl of ice-cold NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40) containing freshly added 200 U/ml Rnase OUT (Invitrogen, Carlsbad, USA), 0.5% vanadyl ribonucleoside (Invitrogen, Carlsbad, USA), 1 mM DTT, 15 mM EDTA and 50 µl mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated sepharose G beads (Abcam, Cambridge, UK). .. The mouse anti-Ago2 used in the IP was also used for western blotting at a dilution of 1 : 1000 while secondary antibody was rabbit anti mouse conjugated with horse radish peroxidase (Dako, Glostrup, Denmark), also at a dilution of 1 : 1000.

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
    Article Snippet: For each microarray experiment, 5 μg of cRNA synthesized from UHR RNA (reference) and 5 μg of cRNA from our samples were labeled. .. 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: 0.25, 0.5, 1, 2 μM) were mixed in Tris-HCl potassium chloride buffer. .. 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). .. 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).

    Article Title: Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques
    Article Snippet: Following a 10-minute incubation, the first-strand cDNAs were generated with SuperScript III Reverse Transcriptase (Life Technologies), according to the DRS protocol, at 50°C for 50 minutes, then at 70°C for 10 minutes in a Veriti Thermal Cycler. .. XP Beads were handled before use with RNase OUT (40 U/μL; Life Technologies; 2 U enzyme/1 μL bead).

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Mouse hearts or cells were homogenized in adequate volumes of polysome lysis buffer (10 mM HEPES-KOH [pH 7.0], 100 mM KCl, 5 mM MgCl2 , 25 mM EDTA, 0.5% IGEPAL, 2 mM dithiothreitol [DTT], 0.2 mg/ml Heparin, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. Lysates containing 1 mg protein were incubated with 500 ng normal IgG (Cell Signaling Technologies, MA, USA; #2729, 1:200), anti-SUZ12 (Cell Signaling Technologies, MA, USA; #3737, 1:200), anti-Ezh2 (Cell Signaling Technologies, MA, USA; #5246, 1:200), anti-WDR5 (Abcam, Cambridge, UK; #56919, 1:200), or anti-LSD1(Cell Signaling Technologies, MA, USA; #2139, 1:200) at 4°C over night on an inverse rotator.

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Streptavidin sepharose beads were blocked with 500 ng/μl yeast tRNA and 1 mg/ml BSA in SA-RNP lysis buffer before added into cell lysates and incubated at 37°C for 2 h on a rotator. .. The beads were then pelleted and washed for 5 times with SA-RNP washing buffer (20 mM Tris-HCl [pH7.5], 300 mM NaCl, 5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]).

    Article Title: Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing
    Article Snippet: Protein A-Dynabeads (Invitrogen) were incubated with either rabbit IgG or MATR3 antibody (Bethyl Labs). .. Beads coated in antibody were resuspended in NT2 buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40] supplemented with RNase OUT (Invitrogen) and 1 mM DTT.

    Article Title: Mycobacterium abscessus subsp. massiliense mycma_0076 and mycma_0077 Genes Code for Ferritins That Are Modulated by Iron Concentration
    Article Snippet: The reaction consisted of 200 ηg of total RNA, 0.5 mM of dNTPS and 0.63 μM of random hexamer primers (Gibco/Thermo Fisher Scientific) and was incubated for 5 min at 65°C. .. Next, the system was transferred to ice and reverse transcriptase buffer, 200 U of M-MLV reverse transcriptase, and 40 U of RNAse OUT (Invitrogen) were added.

    Article Title: The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation
    Article Snippet: Lysates were prepared in Lysis Buffer PD [20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5% NP-40, 10 mM DTT, protease inhibitors (Merck) and 0.5 U/μl RNase OUT (Thermo Scientific)]. .. After incubation on ice 10 min, cell debris was discarded by spinning twice at 16 000 g 10 min 4 °C, and the protein concentration in the supernatant was measured by Bradford assay.

    Article Title: The mechanisms of a mammalian splicing enhancer
    Article Snippet: Splicing reactions were prepared with 50% nuclear extract, 3.2 mM MgCl2 , 50 mM monopotassium glutamate, 20 mM phosphocreatine, 1.5 mM ATP, 20 mM Hepes pH 7.5 and 3 units RNase OUT (Invitrogen). .. A 2′-O -methyl oligonucleotide complementary to U6 snRNA was added at 1 μM to block splicing at complex A ( , ).

    Expressing:

    Article Title: Mycobacterium abscessus subsp. massiliense mycma_0076 and mycma_0077 Genes Code for Ferritins That Are Modulated by Iron Concentration
    Article Snippet: Paragraph title: Gene Expression Evaluation of M. abscessus subsp. massiliense ... Next, the system was transferred to ice and reverse transcriptase buffer, 200 U of M-MLV reverse transcriptase, and 40 U of RNAse OUT (Invitrogen) were added.

    Article Title: The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation
    Article Snippet: After monitoring the expression of MS2-HB protein, cells were frozen until needed. .. Lysates were prepared in Lysis Buffer PD [20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5% NP-40, 10 mM DTT, protease inhibitors (Merck) and 0.5 U/μl RNase OUT (Thermo Scientific)].

    Western Blot:

    Article Title: A high throughput experimental approach to identify miRNA targets in human cells
    Article Snippet: Paragraph title: RIP-Chip: IP and western blotting ... Briefly, 10–20-million cells were lysed in 100 µl ice cold polysome lysis buffer (5 mM MgCl2 , 100 mM KCl, 10 mM Hepes, pH7.0 and 0.5% Nonidet P-40) with freshly added 1 mM DTT, 100 U/ml Rnase OUT (Invitrogen, Carlsbad, USA) and 1× complete mini EDTA free protease inhibitor cocktail (Roche, Basel, Switzerland) for 5 min. Centrifugation was carried out two times at 14 000g at 4°C for 10 min. Supernatant was mixed with 900 µl of ice-cold NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40) containing freshly added 200 U/ml Rnase OUT (Invitrogen, Carlsbad, USA), 0.5% vanadyl ribonucleoside (Invitrogen, Carlsbad, USA), 1 mM DTT, 15 mM EDTA and 50 µl mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated sepharose G beads (Abcam, Cambridge, UK).

    Hybridization:

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
    Article Snippet: Paragraph title: Cy3/Cy5 labeling of cRNA and microarray hybridization ... 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.

    Article Title: A Pair of Maternal Chromosomes Derived from Meiotic Nondisjunction in Trisomy 21 Affects Nuclear Architecture and Transcriptional Regulation
    Article Snippet: DNA FISH was carried out as previously described , . .. In nascent RNA FISH, cellular DNA was not denatured and hybridisation was performed with 4 U/µl RNase OUT (Invitrogen) to avoid the elimination of RNA. .. APP and DYRK1A were probed with BACs from BACPAC Resources (APP , RP11-910G8; DYRK1A , Rp11-105O24), while GART and MRPL39 were probed with PCR products (39 Kb and 22 Kb, respectively).

    Transfection:

    Article Title: A high throughput experimental approach to identify miRNA targets in human cells
    Article Snippet: IP of ribonucleoprotein was performed as previously described ( ) in both HL cell lines (L1236 and L428) with and without transfection of anti-miR-17/20/93/106 and additionally L428 with transfection of anti-miR-220 as a negative control. .. Briefly, 10–20-million cells were lysed in 100 µl ice cold polysome lysis buffer (5 mM MgCl2 , 100 mM KCl, 10 mM Hepes, pH7.0 and 0.5% Nonidet P-40) with freshly added 1 mM DTT, 100 U/ml Rnase OUT (Invitrogen, Carlsbad, USA) and 1× complete mini EDTA free protease inhibitor cocktail (Roche, Basel, Switzerland) for 5 min. Centrifugation was carried out two times at 14 000g at 4°C for 10 min. Supernatant was mixed with 900 µl of ice-cold NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40) containing freshly added 200 U/ml Rnase OUT (Invitrogen, Carlsbad, USA), 0.5% vanadyl ribonucleoside (Invitrogen, Carlsbad, USA), 1 mM DTT, 15 mM EDTA and 50 µl mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated sepharose G beads (Abcam, Cambridge, UK).

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Streptavidin-binding S1m DNA was synthesized and cloned into pcDNA5-CMV just ahead of the wild-type Chaer and truncations with roughly 500 bp intervals both from 5′ and 3′. .. All these constructs together with untagged wild-type Chaer and EGFP were transfected into MEFs for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 1.5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. Streptavidin sepharose beads were blocked with 500 ng/μl yeast tRNA and 1 mg/ml BSA in SA-RNP lysis buffer before added into cell lysates and incubated at 37°C for 2 h on a rotator.

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: All these constructs together with untagged wild-type Chaer and EGFP were transfected into MEFs for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 1.5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. The beads were then pelleted and washed for 5 times with SA-RNP washing buffer (20 mM Tris-HCl [pH7.5], 300 mM NaCl, 5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]).

    Article Title: The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation
    Article Snippet: Confluent monolayers of stable transformant cells HEK293-MS2-HB (10 × 106 ) were transfected with the constructs pCAP-luc, pCAP-H12-MS2h and pCAP-H12d-MS2h using lipofectamine (Thermo Scientific) according to the manufacturer's instructions. .. Lysates were prepared in Lysis Buffer PD [20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5% NP-40, 10 mM DTT, protease inhibitors (Merck) and 0.5 U/μl RNase OUT (Thermo Scientific)].

    Ligation:

    Article Title: Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques
    Article Snippet: XP Beads were handled before use with RNase OUT (40 U/μL; Life Technologies; 2 U enzyme/1 μL bead). .. XP Beads were handled before use with RNase OUT (40 U/μL; Life Technologies; 2 U enzyme/1 μL bead).

    Protease Inhibitor:

    Article Title: A high throughput experimental approach to identify miRNA targets in human cells
    Article Snippet: IP of ribonucleoprotein was performed as previously described ( ) in both HL cell lines (L1236 and L428) with and without transfection of anti-miR-17/20/93/106 and additionally L428 with transfection of anti-miR-220 as a negative control. .. Briefly, 10–20-million cells were lysed in 100 µl ice cold polysome lysis buffer (5 mM MgCl2 , 100 mM KCl, 10 mM Hepes, pH7.0 and 0.5% Nonidet P-40) with freshly added 1 mM DTT, 100 U/ml Rnase OUT (Invitrogen, Carlsbad, USA) and 1× complete mini EDTA free protease inhibitor cocktail (Roche, Basel, Switzerland) for 5 min. Centrifugation was carried out two times at 14 000g at 4°C for 10 min. Supernatant was mixed with 900 µl of ice-cold NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40) containing freshly added 200 U/ml Rnase OUT (Invitrogen, Carlsbad, USA), 0.5% vanadyl ribonucleoside (Invitrogen, Carlsbad, USA), 1 mM DTT, 15 mM EDTA and 50 µl mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated sepharose G beads (Abcam, Cambridge, UK). .. On the following day, beads were washed five times with ice-cold NT2 buffer and separated into two portions—one for RNA isolation to identify miRNA target genes and another portion for western blotting to check for successful IP of Ago2.

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Streptavidin-binding S1m DNA was synthesized and cloned into pcDNA5-CMV just ahead of the wild-type Chaer and truncations with roughly 500 bp intervals both from 5′ and 3′. .. All these constructs together with untagged wild-type Chaer and EGFP were transfected into MEFs for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 1.5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. Streptavidin sepharose beads were blocked with 500 ng/μl yeast tRNA and 1 mg/ml BSA in SA-RNP lysis buffer before added into cell lysates and incubated at 37°C for 2 h on a rotator.

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: RNA-protein interactions were validated using RNA immunoprecipitation (RIP) essentially as described . .. Mouse hearts or cells were homogenized in adequate volumes of polysome lysis buffer (10 mM HEPES-KOH [pH 7.0], 100 mM KCl, 5 mM MgCl2 , 25 mM EDTA, 0.5% IGEPAL, 2 mM dithiothreitol [DTT], 0.2 mg/ml Heparin, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. Lysates containing 1 mg protein were incubated with 500 ng normal IgG (Cell Signaling Technologies, MA, USA; #2729, 1:200), anti-SUZ12 (Cell Signaling Technologies, MA, USA; #3737, 1:200), anti-Ezh2 (Cell Signaling Technologies, MA, USA; #5246, 1:200), anti-WDR5 (Abcam, Cambridge, UK; #56919, 1:200), or anti-LSD1(Cell Signaling Technologies, MA, USA; #2139, 1:200) at 4°C over night on an inverse rotator.

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Streptavidin sepharose beads were blocked with 500 ng/μl yeast tRNA and 1 mg/ml BSA in SA-RNP lysis buffer before added into cell lysates and incubated at 37°C for 2 h on a rotator. .. The beads were then pelleted and washed for 5 times with SA-RNP washing buffer (20 mM Tris-HCl [pH7.5], 300 mM NaCl, 5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. After the last wash, RNA-bound proteins were eluted by addition of 5% RNase A (New England Biolabs, MA, USA) in low salt buffer (20 mM Tris-HCl [pH7.5], 30 mM NaCl, 5 mM MgCl2, 2 mM DTT, 1× complete protease inhibitor tablet [Roche]) for 30 min at 4°C.

    Article Title: Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing
    Article Snippet: Briefly, myoblasts grown in 100 mm collagen coated dishes, rinsed twice with ice-cold PBS, crosslinked with UV (254 nm) and lysed with an equal pellet volume of RIPA-2 buffer [50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% Na-deoxycholate, 1 mM EDTA, RNase OUT (Invitrogen), and 1 cOmplete protease inhibitor tablet (Sigma)]. .. Beads coated in antibody were resuspended in NT2 buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40] supplemented with RNase OUT (Invitrogen) and 1 mM DTT.

    Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response
    Article Snippet: Differentially expressed proteins were selected if the P -value was < 0.05 and the change > 20%. .. HUVECs were cross-linked with 0.3% formaldehyde at room temperature for 10 min and neutralized with 0.125 M of glycine at room temperature for 5 min. Cross-linked cells were washed with 1× PBS, collected by scraping and lysed in polysome lysis buffer containing 100 mM KCl, 5 mM MgCl2 , 10 mM HEPES at pH 7.0, 0.5% Nonidet P-40, 1 mM dithiothreitol (DTT), 200 units/ml RNase OUT (Invitrogen Cat. No. 10777-019) and Complete Mini, EDTA-free Protease Inhibitor Tablet (11836170001, Roche) for 30 min on ice. .. Lysate was diluted to 1 ml in NT-2 buffer containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.5% NP-40, 20 mM EDTA at pH 8.0, 1 mM DTT, 200 units/ml RNase OUT and sonicated for 20 s at 20% power using Branson 250 Ultrasonic Sonifier.

    Transferring:

    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.

    Nick Translation:

    Article Title: A Pair of Maternal Chromosomes Derived from Meiotic Nondisjunction in Trisomy 21 Affects Nuclear Architecture and Transcriptional Regulation
    Article Snippet: In nascent RNA FISH, cellular DNA was not denatured and hybridisation was performed with 4 U/µl RNase OUT (Invitrogen) to avoid the elimination of RNA. .. In nascent RNA FISH, cellular DNA was not denatured and hybridisation was performed with 4 U/µl RNase OUT (Invitrogen) to avoid the elimination of RNA.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: High-Pressure Inactivation of Rotaviruses: Role of Treatment Temperature and Strain Diversity in Virus Inactivation
    Article Snippet: Paragraph title: RT-PCR. ... Either viral RNA was directly subjected to HPP treatment, or 20 μl of viral RNA was treated with 1 μl RNase-out (Invitrogen) (diluted 1:10) prior to HPP treatment.

    Article Title: Mycobacterium abscessus subsp. massiliense mycma_0076 and mycma_0077 Genes Code for Ferritins That Are Modulated by Iron Concentration
    Article Snippet: Next, the system was transferred to ice and reverse transcriptase buffer, 200 U of M-MLV reverse transcriptase, and 40 U of RNAse OUT (Invitrogen) were added. .. The reaction was incubated at 37°C for 1.5 h. Then the synthesized cDNA was stored at -20°C until its use for Real Time PCR (RT-PCR).

    Generated:

    Article Title: Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques
    Article Snippet: Following a 10-minute incubation, the first-strand cDNAs were generated with SuperScript III Reverse Transcriptase (Life Technologies), according to the DRS protocol, at 50°C for 50 minutes, then at 70°C for 10 minutes in a Veriti Thermal Cycler. .. XP Beads were handled before use with RNase OUT (40 U/μL; Life Technologies; 2 U enzyme/1 μL bead).

    Inhibition:

    Article Title: Identification of RNA Oligonucleotides Binding to Several Proteins from Potential G-Quadruplex Forming Regions in Transcribed Pre-mRNA
    Article Snippet: 0.25, 0.5, 1, 2 μM) were mixed in Tris-HCl potassium chloride buffer. .. 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). .. 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).

    Sequencing:

    Article Title: Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques
    Article Snippet: Paragraph title: ONT MinION—dRNA sequencing ... XP Beads were handled before use with RNase OUT (40 U/μL; Life Technologies; 2 U enzyme/1 μL bead).

    Sonication:

    Article Title: The mechanisms of a mammalian splicing enhancer
    Article Snippet: Cover slips from Menzel-Gläser (22 mm × 50 mm, #1) were soaked in 1 M KOH for four hours before being washed with water, sonicated, dried under a nitrogen stream and cleaned in an argon plasma (MiniFlecto-PC-MFC, Gala Instruments) for 5 times 5 min. .. Splicing reactions were prepared with 50% nuclear extract, 3.2 mM MgCl2 , 50 mM monopotassium glutamate, 20 mM phosphocreatine, 1.5 mM ATP, 20 mM Hepes pH 7.5 and 3 units RNase OUT (Invitrogen).

    Quantitation Assay:

    Article Title: R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein
    Article Snippet: Reverse transcription was performed on 2.2 μg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs and ten units of RNAse OUT (Thermo Scientific) following the manufacturer's protocol in a total volume of 20 μl. .. PCR reactions are carried on thermocyclers GeneAmp PCR System 9700 (Thermo Scientific), and the amplified products were analysed by automated chip-based microcapillary electrophoresis on Caliper LC90 instruments (Perkin Elmer).

    Binding Assay:

    Article Title: Identification of RNA Oligonucleotides Binding to Several Proteins from Potential G-Quadruplex Forming Regions in Transcribed Pre-mRNA
    Article Snippet: Next EMSA was performed for evaluation of binding ability of RNAs. .. 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).

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: To identify the direct binding partner of Chaer , we employed the tagged RNA pull-down assay as previously described . .. All these constructs together with untagged wild-type Chaer and EGFP were transfected into MEFs for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 1.5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]).

    Article Title: The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation
    Article Snippet: Lysates were prepared in Lysis Buffer PD [20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5% NP-40, 10 mM DTT, protease inhibitors (Merck) and 0.5 U/μl RNase OUT (Thermo Scientific)]. .. After incubation on ice 10 min, cell debris was discarded by spinning twice at 16 000 g 10 min 4 °C, and the protein concentration in the supernatant was measured by Bradford assay.

    Nucleic Acid Electrophoresis:

    Article Title: High-Pressure Inactivation of Rotaviruses: Role of Treatment Temperature and Strain Diversity in Virus Inactivation
    Article Snippet: DNA bands were visualized using 1% gel electrophoresis. .. Either viral RNA was directly subjected to HPP treatment, or 20 μl of viral RNA was treated with 1 μl RNase-out (Invitrogen) (diluted 1:10) prior to HPP treatment.

    RNA Sequencing Assay:

    Article Title: Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques
    Article Snippet: RNA was mixed with the RT (oligo(dT)-containing T10) adapter (provided by the ONT Direct RNA Sequencing Kit; SQK-RNA001) and T4 DNA ligase (2M U/mL; New England BioLabs). .. XP Beads were handled before use with RNase OUT (40 U/μL; Life Technologies; 2 U enzyme/1 μL bead).

    Pull Down Assay:

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: To identify the direct binding partner of Chaer , we employed the tagged RNA pull-down assay as previously described . .. All these constructs together with untagged wild-type Chaer and EGFP were transfected into MEFs for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 1.5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]).

    Fluorescence:

    Article Title: Mycobacterium abscessus subsp. massiliense mycma_0076 and mycma_0077 Genes Code for Ferritins That Are Modulated by Iron Concentration
    Article Snippet: Next, the system was transferred to ice and reverse transcriptase buffer, 200 U of M-MLV reverse transcriptase, and 40 U of RNAse OUT (Invitrogen) were added. .. Next, the system was transferred to ice and reverse transcriptase buffer, 200 U of M-MLV reverse transcriptase, and 40 U of RNAse OUT (Invitrogen) were added.

    Isolation:

    Article Title: Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing
    Article Snippet: Beads coated in antibody were resuspended in NT2 buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40] supplemented with RNase OUT (Invitrogen) and 1 mM DTT. .. After incubation, beads were magnetized and washed five times with cold NT2 buffer.

    Labeling:

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
    Article Snippet: For each microarray experiment, 5 μg of cRNA synthesized from UHR RNA (reference) and 5 μg of cRNA from our samples were labeled. .. 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: Fluorescein labeled RNAs were diluted into 2 μM in Tris-HCl potassium chloride buffer (50 mM Tris-HCl, with 100 mM KCl, pH 7.5) and folded at 65 °C for five min and then allowed to cool to room temperature for 30 min. Heat treated RNAs (f.c. .. 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).

    Article Title: CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo
    Article Snippet: Paragraph title: sgRNA labeling and in-line probing ... To produce 5′-end-labeled sgRNAs, purified sgRNA transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BIoLabs) to 50 pmol of sgRNA in a final volume of 10 μL containing 50 mM Bis-Propane pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20U, Invitrogen).

    Purification:

    Article Title: Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques
    Article Snippet: Samples were purified by using Agencourt AMPure XP Beads (Beckman Coulter). .. XP Beads were handled before use with RNase OUT (40 U/μL; Life Technologies; 2 U enzyme/1 μL bead).

    Article Title: Mycobacterium abscessus subsp. massiliense mycma_0076 and mycma_0077 Genes Code for Ferritins That Are Modulated by Iron Concentration
    Article Snippet: The solution was then applied to an RNA purification column (Phenol-Free Total RNA Purification, Amresco) for purification according manufacture’s instructions. .. Next, the system was transferred to ice and reverse transcriptase buffer, 200 U of M-MLV reverse transcriptase, and 40 U of RNAse OUT (Invitrogen) were added.

    Article Title: CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo
    Article Snippet: DNA was then eluted, ethanol precipitated, dissolved in water and PCR amplified as mentioned above. .. To produce 5′-end-labeled sgRNAs, purified sgRNA transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BIoLabs) to 50 pmol of sgRNA in a final volume of 10 μL containing 50 mM Bis-Propane pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20U, Invitrogen). .. Dephosphorylated sgRNAs (5 pmol) were 5′-end-rediolabeled using 3 U of T4 polynucleotide kinase (New England Biolabs) for 1 h at 37°C in the presence of 3.2 pmol of [α-32 P]ATP (6000 Ci/mmol; Perkin Elmer).

    Polymerase Chain Reaction:

    Article Title: Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques
    Article Snippet: In order to avoid the potential PCR biases, the amplification-free direct RNA sequencing (DRS) protocol (Version: DRS_9026_v1_revM_15Dec2016) from the ONTs was applied. .. XP Beads were handled before use with RNase OUT (40 U/μL; Life Technologies; 2 U enzyme/1 μL bead).

    Article Title: R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein
    Article Snippet: Reverse transcription was performed on 2.2 μg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs and ten units of RNAse OUT (Thermo Scientific) following the manufacturer's protocol in a total volume of 20 μl. .. End-point PCR reactions were done on 10 ng cDNA in 10 μl final volume containing 0.2 mmol l−1 each dNTP, 1.5 mmol l−1 MgCl2 , 0.6 μmol l−1 each primer, and 0.2 units of Platinum Taq DNA polymerase (Thermo Scientific).

    Immunoprecipitation:

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Paragraph title: RNA immunoprecipitation ... Mouse hearts or cells were homogenized in adequate volumes of polysome lysis buffer (10 mM HEPES-KOH [pH 7.0], 100 mM KCl, 5 mM MgCl2 , 25 mM EDTA, 0.5% IGEPAL, 2 mM dithiothreitol [DTT], 0.2 mg/ml Heparin, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]).

    Article Title: Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing
    Article Snippet: Paragraph title: Crosslinking and immunoprecipitation of RNA (CLIP) ... Beads coated in antibody were resuspended in NT2 buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40] supplemented with RNase OUT (Invitrogen) and 1 mM DTT.

    Article Title: Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice
    Article Snippet: Paragraph title: Immunoprecipitation ... Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors.

    Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response
    Article Snippet: Paragraph title: RNA immunoprecipitation ... HUVECs were cross-linked with 0.3% formaldehyde at room temperature for 10 min and neutralized with 0.125 M of glycine at room temperature for 5 min. Cross-linked cells were washed with 1× PBS, collected by scraping and lysed in polysome lysis buffer containing 100 mM KCl, 5 mM MgCl2 , 10 mM HEPES at pH 7.0, 0.5% Nonidet P-40, 1 mM dithiothreitol (DTT), 200 units/ml RNase OUT (Invitrogen Cat. No. 10777-019) and Complete Mini, EDTA-free Protease Inhibitor Tablet (11836170001, Roche) for 30 min on ice.

    Quantitative RT-PCR:

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Mouse hearts or cells were homogenized in adequate volumes of polysome lysis buffer (10 mM HEPES-KOH [pH 7.0], 100 mM KCl, 5 mM MgCl2 , 25 mM EDTA, 0.5% IGEPAL, 2 mM dithiothreitol [DTT], 0.2 mg/ml Heparin, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. Mouse hearts or cells were homogenized in adequate volumes of polysome lysis buffer (10 mM HEPES-KOH [pH 7.0], 100 mM KCl, 5 mM MgCl2 , 25 mM EDTA, 0.5% IGEPAL, 2 mM dithiothreitol [DTT], 0.2 mg/ml Heparin, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]).

    Article Title: Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice
    Article Snippet: Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors. .. Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors.

    Article Title: Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice
    Article Snippet: Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors. .. Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors.

    Polyacrylamide Gel Electrophoresis:

    Article Title: CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo
    Article Snippet: To produce 5′-end-labeled sgRNAs, purified sgRNA transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BIoLabs) to 50 pmol of sgRNA in a final volume of 10 μL containing 50 mM Bis-Propane pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20U, Invitrogen). .. To produce 5′-end-labeled sgRNAs, purified sgRNA transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BIoLabs) to 50 pmol of sgRNA in a final volume of 10 μL containing 50 mM Bis-Propane pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20U, Invitrogen).

    Electro Mobility Shift Assay:

    Article Title: Identification of RNA Oligonucleotides Binding to Several Proteins from Potential G-Quadruplex Forming Regions in Transcribed Pre-mRNA
    Article Snippet: Paragraph title: 3.3. Electromobility Shift Assay (EMSA) ... 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).

    Mouse Assay:

    Article Title: Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice
    Article Snippet: PCR results were normalized to the expression of actin. .. Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors. .. The lysate was pre-cleared with 15 ug of rabbit Ig and 50 µl protein G/A sepharose, then the protein concentration was measured.

    Article Title: Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice
    Article Snippet: PCR results were normalized to the expression of actin. .. Hippocampi of wildtype or Pum2 –/– mice were extracted with PLB buffer (100 mM KCL, 5 mM MgCl2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM Dithiothrectol (DTT), 100 units/ml RNase OUT (Invitrogen-cat# 10777-019), supplemented with RNAse inhibitors and protease inhibitors. .. The lysate was pre-cleared with 15 ug of rabbit Ig and 50 µl protein G/A sepharose, then the protein concentration was measured.

    Chromatin Immunoprecipitation:

    Article Title: R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein
    Article Snippet: Reverse transcription was performed on 2.2 μg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs and ten units of RNAse OUT (Thermo Scientific) following the manufacturer's protocol in a total volume of 20 μl. .. End-point PCR reactions were done on 10 ng cDNA in 10 μl final volume containing 0.2 mmol l−1 each dNTP, 1.5 mmol l−1 MgCl2 , 0.6 μmol l−1 each primer, and 0.2 units of Platinum Taq DNA polymerase (Thermo Scientific).

    Software:

    Article Title: R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein
    Article Snippet: Reverse transcription was performed on 2.2 μg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs and ten units of RNAse OUT (Thermo Scientific) following the manufacturer's protocol in a total volume of 20 μl. .. PCR reactions are carried on thermocyclers GeneAmp PCR System 9700 (Thermo Scientific), and the amplified products were analysed by automated chip-based microcapillary electrophoresis on Caliper LC90 instruments (Perkin Elmer).

    Irradiation:

    Article Title: The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation
    Article Snippet: Monolayers were washed 24 h post-transfection with ice-cold PBS prior to UV irradiation (400 mJ/cm2 at 254 nm). .. Lysates were prepared in Lysis Buffer PD [20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5% NP-40, 10 mM DTT, protease inhibitors (Merck) and 0.5 U/μl RNase OUT (Thermo Scientific)].

    Negative Control:

    Article Title: A high throughput experimental approach to identify miRNA targets in human cells
    Article Snippet: IP of ribonucleoprotein was performed as previously described ( ) in both HL cell lines (L1236 and L428) with and without transfection of anti-miR-17/20/93/106 and additionally L428 with transfection of anti-miR-220 as a negative control. .. Briefly, 10–20-million cells were lysed in 100 µl ice cold polysome lysis buffer (5 mM MgCl2 , 100 mM KCl, 10 mM Hepes, pH7.0 and 0.5% Nonidet P-40) with freshly added 1 mM DTT, 100 U/ml Rnase OUT (Invitrogen, Carlsbad, USA) and 1× complete mini EDTA free protease inhibitor cocktail (Roche, Basel, Switzerland) for 5 min. Centrifugation was carried out two times at 14 000g at 4°C for 10 min. Supernatant was mixed with 900 µl of ice-cold NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40) containing freshly added 200 U/ml Rnase OUT (Invitrogen, Carlsbad, USA), 0.5% vanadyl ribonucleoside (Invitrogen, Carlsbad, USA), 1 mM DTT, 15 mM EDTA and 50 µl mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated sepharose G beads (Abcam, Cambridge, UK).

    Sample Prep:

    Article Title: The mechanisms of a mammalian splicing enhancer
    Article Snippet: Paragraph title: Sample preparation ... Splicing reactions were prepared with 50% nuclear extract, 3.2 mM MgCl2 , 50 mM monopotassium glutamate, 20 mM phosphocreatine, 1.5 mM ATP, 20 mM Hepes pH 7.5 and 3 units RNase OUT (Invitrogen).

    Electrophoresis:

    Article Title: R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein
    Article Snippet: Reverse transcription was performed on 2.2 μg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs and ten units of RNAse OUT (Thermo Scientific) following the manufacturer's protocol in a total volume of 20 μl. .. End-point PCR reactions were done on 10 ng cDNA in 10 μl final volume containing 0.2 mmol l−1 each dNTP, 1.5 mmol l−1 MgCl2 , 0.6 μmol l−1 each primer, and 0.2 units of Platinum Taq DNA polymerase (Thermo Scientific).

    Cross-linking Immunoprecipitation:

    Article Title: Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing
    Article Snippet: Paragraph title: Crosslinking and immunoprecipitation of RNA (CLIP) ... Beads coated in antibody were resuspended in NT2 buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40] supplemented with RNase OUT (Invitrogen) and 1 mM DTT.

    Random Hexamer Labeling:

    Article Title: Mycobacterium abscessus subsp. massiliense mycma_0076 and mycma_0077 Genes Code for Ferritins That Are Modulated by Iron Concentration
    Article Snippet: The reaction consisted of 200 ηg of total RNA, 0.5 mM of dNTPS and 0.63 μM of random hexamer primers (Gibco/Thermo Fisher Scientific) and was incubated for 5 min at 65°C. .. Next, the system was transferred to ice and reverse transcriptase buffer, 200 U of M-MLV reverse transcriptase, and 40 U of RNAse OUT (Invitrogen) were added.

    Produced:

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
    Article Snippet: The cRNA produced from each amplification was used to make fluorescent probes by RT. .. 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.

    Concentration Assay:

    Article Title: The mechanisms of a mammalian splicing enhancer
    Article Snippet: Splicing reactions were prepared with 50% nuclear extract, 3.2 mM MgCl2 , 50 mM monopotassium glutamate, 20 mM phosphocreatine, 1.5 mM ATP, 20 mM Hepes pH 7.5 and 3 units RNase OUT (Invitrogen). .. A 2′-O -methyl oligonucleotide complementary to U6 snRNA was added at 1 μM to block splicing at complex A ( , ).

    Lysis:

    Article Title: A high throughput experimental approach to identify miRNA targets in human cells
    Article Snippet: IP of ribonucleoprotein was performed as previously described ( ) in both HL cell lines (L1236 and L428) with and without transfection of anti-miR-17/20/93/106 and additionally L428 with transfection of anti-miR-220 as a negative control. .. Briefly, 10–20-million cells were lysed in 100 µl ice cold polysome lysis buffer (5 mM MgCl2 , 100 mM KCl, 10 mM Hepes, pH7.0 and 0.5% Nonidet P-40) with freshly added 1 mM DTT, 100 U/ml Rnase OUT (Invitrogen, Carlsbad, USA) and 1× complete mini EDTA free protease inhibitor cocktail (Roche, Basel, Switzerland) for 5 min. Centrifugation was carried out two times at 14 000g at 4°C for 10 min. Supernatant was mixed with 900 µl of ice-cold NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.05% Nonidet P-40) containing freshly added 200 U/ml Rnase OUT (Invitrogen, Carlsbad, USA), 0.5% vanadyl ribonucleoside (Invitrogen, Carlsbad, USA), 1 mM DTT, 15 mM EDTA and 50 µl mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated sepharose G beads (Abcam, Cambridge, UK). .. On the following day, beads were washed five times with ice-cold NT2 buffer and separated into two portions—one for RNA isolation to identify miRNA target genes and another portion for western blotting to check for successful IP of Ago2.

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Streptavidin-binding S1m DNA was synthesized and cloned into pcDNA5-CMV just ahead of the wild-type Chaer and truncations with roughly 500 bp intervals both from 5′ and 3′. .. All these constructs together with untagged wild-type Chaer and EGFP were transfected into MEFs for 48 h. Cells were then harvested in SA-RNP lysis buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 1.5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. Streptavidin sepharose beads were blocked with 500 ng/μl yeast tRNA and 1 mg/ml BSA in SA-RNP lysis buffer before added into cell lysates and incubated at 37°C for 2 h on a rotator.

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: RNA-protein interactions were validated using RNA immunoprecipitation (RIP) essentially as described . .. Mouse hearts or cells were homogenized in adequate volumes of polysome lysis buffer (10 mM HEPES-KOH [pH 7.0], 100 mM KCl, 5 mM MgCl2 , 25 mM EDTA, 0.5% IGEPAL, 2 mM dithiothreitol [DTT], 0.2 mg/ml Heparin, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]). .. Lysates containing 1 mg protein were incubated with 500 ng normal IgG (Cell Signaling Technologies, MA, USA; #2729, 1:200), anti-SUZ12 (Cell Signaling Technologies, MA, USA; #3737, 1:200), anti-Ezh2 (Cell Signaling Technologies, MA, USA; #5246, 1:200), anti-WDR5 (Abcam, Cambridge, UK; #56919, 1:200), or anti-LSD1(Cell Signaling Technologies, MA, USA; #2139, 1:200) at 4°C over night on an inverse rotator.

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Streptavidin sepharose beads were blocked with 500 ng/μl yeast tRNA and 1 mg/ml BSA in SA-RNP lysis buffer before added into cell lysates and incubated at 37°C for 2 h on a rotator. .. The beads were then pelleted and washed for 5 times with SA-RNP washing buffer (20 mM Tris-HCl [pH7.5], 300 mM NaCl, 5 mM MgCl2 , 2 mM DTT, 50 U/ml RNase OUT [Life Technologies, NY, USA], 50 U/ml Superase IN [Ambion], 1× complete protease inhibitor tablet [Roche]).

    Article Title: LncRNA Meg3 protects endothelial function by regulating the DNA damage response
    Article Snippet: Differentially expressed proteins were selected if the P -value was < 0.05 and the change > 20%. .. HUVECs were cross-linked with 0.3% formaldehyde at room temperature for 10 min and neutralized with 0.125 M of glycine at room temperature for 5 min. Cross-linked cells were washed with 1× PBS, collected by scraping and lysed in polysome lysis buffer containing 100 mM KCl, 5 mM MgCl2 , 10 mM HEPES at pH 7.0, 0.5% Nonidet P-40, 1 mM dithiothreitol (DTT), 200 units/ml RNase OUT (Invitrogen Cat. No. 10777-019) and Complete Mini, EDTA-free Protease Inhibitor Tablet (11836170001, Roche) for 30 min on ice. .. Lysate was diluted to 1 ml in NT-2 buffer containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.5% NP-40, 20 mM EDTA at pH 8.0, 1 mM DTT, 200 units/ml RNase OUT and sonicated for 20 s at 20% power using Branson 250 Ultrasonic Sonifier.

    Article Title: The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation
    Article Snippet: Monolayers were washed 24 h post-transfection with ice-cold PBS prior to UV irradiation (400 mJ/cm2 at 254 nm). .. Lysates were prepared in Lysis Buffer PD [20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5% NP-40, 10 mM DTT, protease inhibitors (Merck) and 0.5 U/μl RNase OUT (Thermo Scientific)]. .. After incubation on ice 10 min, cell debris was discarded by spinning twice at 16 000 g 10 min 4 °C, and the protein concentration in the supernatant was measured by Bradford assay.

    Fluorescence In Situ Hybridization:

    Article Title: A Pair of Maternal Chromosomes Derived from Meiotic Nondisjunction in Trisomy 21 Affects Nuclear Architecture and Transcriptional Regulation
    Article Snippet: DNA FISH was carried out as previously described , . .. In nascent RNA FISH, cellular DNA was not denatured and hybridisation was performed with 4 U/µl RNase OUT (Invitrogen) to avoid the elimination of RNA. .. APP and DYRK1A were probed with BACs from BACPAC Resources (APP , RP11-910G8; DYRK1A , Rp11-105O24), while GART and MRPL39 were probed with PCR products (39 Kb and 22 Kb, respectively).

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    Thermo Fisher depc treated water rnase out recombinant ribonuclease inhibitor
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    Thermo Fisher rac1
    RHOG activates <t>RAC1</t> leading to tube formation in ECV cells. ( A ) Cells were transfected with either luciferase or RHOG siRNA. Cells were then lysed and incubated with GST-CRIB (CDC42 and RAC interactive binding domain) to pull down the active RAC1. Samples from the pull-down as well as the total lysates were blotted against RAC1. The lower 2 gels are Western blots for RHOG for the knockdown control and actin for the loading control. ( B ) Quantitation of GTP-RAC1 from (A) normalized to total RAC1 and expressed as a fold decrease from the luciferase control. Data are the mean ± SEM of three independent experiments. * p
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    Image Search Results


    RHOG activates RAC1 leading to tube formation in ECV cells. ( A ) Cells were transfected with either luciferase or RHOG siRNA. Cells were then lysed and incubated with GST-CRIB (CDC42 and RAC interactive binding domain) to pull down the active RAC1. Samples from the pull-down as well as the total lysates were blotted against RAC1. The lower 2 gels are Western blots for RHOG for the knockdown control and actin for the loading control. ( B ) Quantitation of GTP-RAC1 from (A) normalized to total RAC1 and expressed as a fold decrease from the luciferase control. Data are the mean ± SEM of three independent experiments. * p

    Journal: Cells

    Article Title: RHOG Activates RAC1 through CDC42 Leading to Tube Formation in Vascular Endothelial Cells

    doi: 10.3390/cells8020171

    Figure Lengend Snippet: RHOG activates RAC1 leading to tube formation in ECV cells. ( A ) Cells were transfected with either luciferase or RHOG siRNA. Cells were then lysed and incubated with GST-CRIB (CDC42 and RAC interactive binding domain) to pull down the active RAC1. Samples from the pull-down as well as the total lysates were blotted against RAC1. The lower 2 gels are Western blots for RHOG for the knockdown control and actin for the loading control. ( B ) Quantitation of GTP-RAC1 from (A) normalized to total RAC1 and expressed as a fold decrease from the luciferase control. Data are the mean ± SEM of three independent experiments. * p

    Article Snippet: Human FlexiTube siRNA for each of RHOG [oligo 1, 2 and 3 out of the NM_001665 panel), RAC1 (oligo 5 and 6 out of the NM_006908, NM_018890 and NM_198829 panels), CDC42 (oligo 4 and 7 out of the NM_001039802, NM_001791 and NM_044472 panels), STARD13 (oligo 4 out of the NM_001243466 panel], RHOA (oligo 1 and 6 out of the NM_001664 panel), RHOC (oligo 5 and 6 out of the NM_001042678, NM_00104269 and NM_175744 panels), ROCK1 (oligo 9 and 10 out of the NM_005406 panel) and ROCK2 (oligo 5 and 6 out of the NM_004850 panel) were brought from Qiagen (Qiagen, Hilden, Germany).

    Techniques: Transfection, Luciferase, Incubation, Binding Assay, Western Blot, Quantitation Assay

    RHOG activates CDC42 which activates RAC1 in ECV cells. ( A ) Cells were transfected with either the luciferase control or CDC42 siRNA (2 oligos). The cells were then lysed, and the samples blotted with anti-CDC42 antibody. ( B ) Quantitation of (A) (knockdown efficiency) normalized to actin and expressed as a fold decrease compared to the luciferase control. Data are the mean ± SEM of three independent experiments. * p

    Journal: Cells

    Article Title: RHOG Activates RAC1 through CDC42 Leading to Tube Formation in Vascular Endothelial Cells

    doi: 10.3390/cells8020171

    Figure Lengend Snippet: RHOG activates CDC42 which activates RAC1 in ECV cells. ( A ) Cells were transfected with either the luciferase control or CDC42 siRNA (2 oligos). The cells were then lysed, and the samples blotted with anti-CDC42 antibody. ( B ) Quantitation of (A) (knockdown efficiency) normalized to actin and expressed as a fold decrease compared to the luciferase control. Data are the mean ± SEM of three independent experiments. * p

    Article Snippet: Human FlexiTube siRNA for each of RHOG [oligo 1, 2 and 3 out of the NM_001665 panel), RAC1 (oligo 5 and 6 out of the NM_006908, NM_018890 and NM_198829 panels), CDC42 (oligo 4 and 7 out of the NM_001039802, NM_001791 and NM_044472 panels), STARD13 (oligo 4 out of the NM_001243466 panel], RHOA (oligo 1 and 6 out of the NM_001664 panel), RHOC (oligo 5 and 6 out of the NM_001042678, NM_00104269 and NM_175744 panels), ROCK1 (oligo 9 and 10 out of the NM_005406 panel) and ROCK2 (oligo 5 and 6 out of the NM_004850 panel) were brought from Qiagen (Qiagen, Hilden, Germany).

    Techniques: Transfection, Luciferase, Quantitation Assay

    RAC1 positively regulates tube formation in ECV cells. ECV cells were transfected with the luciferase control siRNA or with RAC1 siRNA. Two different siRNA oligos against RAC1 were used in each experiment. ( A ) The cells were lysed and immunoblotted using Western blot analysis for RAC1 (upper gel) or for actin (lower gel) for the loading control. ( B ) Western blot bands were quantified using imageJ and normalized to the number of total proteins and expressed as fold decreases from the luciferase control. Data are the mean ± SEM of three independent experiments. * p

    Journal: Cells

    Article Title: RHOG Activates RAC1 through CDC42 Leading to Tube Formation in Vascular Endothelial Cells

    doi: 10.3390/cells8020171

    Figure Lengend Snippet: RAC1 positively regulates tube formation in ECV cells. ECV cells were transfected with the luciferase control siRNA or with RAC1 siRNA. Two different siRNA oligos against RAC1 were used in each experiment. ( A ) The cells were lysed and immunoblotted using Western blot analysis for RAC1 (upper gel) or for actin (lower gel) for the loading control. ( B ) Western blot bands were quantified using imageJ and normalized to the number of total proteins and expressed as fold decreases from the luciferase control. Data are the mean ± SEM of three independent experiments. * p

    Article Snippet: Human FlexiTube siRNA for each of RHOG [oligo 1, 2 and 3 out of the NM_001665 panel), RAC1 (oligo 5 and 6 out of the NM_006908, NM_018890 and NM_198829 panels), CDC42 (oligo 4 and 7 out of the NM_001039802, NM_001791 and NM_044472 panels), STARD13 (oligo 4 out of the NM_001243466 panel], RHOA (oligo 1 and 6 out of the NM_001664 panel), RHOC (oligo 5 and 6 out of the NM_001042678, NM_00104269 and NM_175744 panels), ROCK1 (oligo 9 and 10 out of the NM_005406 panel) and ROCK2 (oligo 5 and 6 out of the NM_004850 panel) were brought from Qiagen (Qiagen, Hilden, Germany).

    Techniques: Transfection, Luciferase, Western Blot

    RHOG stimulates tube formation through CDC42 and RAC1. ECV-cells were transfected with the luciferase control, CDC42 (2 oligos) or STARD13 siRNA and plated on growth factor-reduced Matrigel by ECV for 24, 48, or 72 h. ( A ) Representative images of tube formation assay for the luciferase control and the CDC42 knockdown cells at 24, 48, and 72 h after plating. The scale bar is 100 μm. ( B – D ) Quantitation of (A) for the total tube length, total tube number, and the number of branching points, respectively. Data are the mean ± SEM of three independent experiments. * p

    Journal: Cells

    Article Title: RHOG Activates RAC1 through CDC42 Leading to Tube Formation in Vascular Endothelial Cells

    doi: 10.3390/cells8020171

    Figure Lengend Snippet: RHOG stimulates tube formation through CDC42 and RAC1. ECV-cells were transfected with the luciferase control, CDC42 (2 oligos) or STARD13 siRNA and plated on growth factor-reduced Matrigel by ECV for 24, 48, or 72 h. ( A ) Representative images of tube formation assay for the luciferase control and the CDC42 knockdown cells at 24, 48, and 72 h after plating. The scale bar is 100 μm. ( B – D ) Quantitation of (A) for the total tube length, total tube number, and the number of branching points, respectively. Data are the mean ± SEM of three independent experiments. * p

    Article Snippet: Human FlexiTube siRNA for each of RHOG [oligo 1, 2 and 3 out of the NM_001665 panel), RAC1 (oligo 5 and 6 out of the NM_006908, NM_018890 and NM_198829 panels), CDC42 (oligo 4 and 7 out of the NM_001039802, NM_001791 and NM_044472 panels), STARD13 (oligo 4 out of the NM_001243466 panel], RHOA (oligo 1 and 6 out of the NM_001664 panel), RHOC (oligo 5 and 6 out of the NM_001042678, NM_00104269 and NM_175744 panels), ROCK1 (oligo 9 and 10 out of the NM_005406 panel) and ROCK2 (oligo 5 and 6 out of the NM_004850 panel) were brought from Qiagen (Qiagen, Hilden, Germany).

    Techniques: Transfection, Luciferase, Tube Formation Assay, Quantitation Assay

    RHOG/CDC42/RAC1 leads to tube formation through ERK and downstream from PI3K. ( A ) Representative images of a tube formation assay (72 h after plating) of ECV cells transfected with the vector alone, CDC42-DA, or RAC1-DA, and treated with DMSO or with 10 μM U0126 (24 h before imaging). The scale bar is 100 μm. ( B , C ) ECV cells were transfected either with luciferase, RHOG, CDC42, or RAC1 siRNA, or transfected with the vector alone, CDC42-DA, or RAC1-DA, and treated with 10 μM U0126 (24 h) or with the carrier (DMSO). ( B ) Western blot of the lysates from the different conditions mentioned above against p-ERK and ERK as controls. ( C ) Quantitation of (B) normalized to ERK and expressed as a fold difference from the luciferase control. Data are the mean ± SEM of three independent experiments. * p

    Journal: Cells

    Article Title: RHOG Activates RAC1 through CDC42 Leading to Tube Formation in Vascular Endothelial Cells

    doi: 10.3390/cells8020171

    Figure Lengend Snippet: RHOG/CDC42/RAC1 leads to tube formation through ERK and downstream from PI3K. ( A ) Representative images of a tube formation assay (72 h after plating) of ECV cells transfected with the vector alone, CDC42-DA, or RAC1-DA, and treated with DMSO or with 10 μM U0126 (24 h before imaging). The scale bar is 100 μm. ( B , C ) ECV cells were transfected either with luciferase, RHOG, CDC42, or RAC1 siRNA, or transfected with the vector alone, CDC42-DA, or RAC1-DA, and treated with 10 μM U0126 (24 h) or with the carrier (DMSO). ( B ) Western blot of the lysates from the different conditions mentioned above against p-ERK and ERK as controls. ( C ) Quantitation of (B) normalized to ERK and expressed as a fold difference from the luciferase control. Data are the mean ± SEM of three independent experiments. * p

    Article Snippet: Human FlexiTube siRNA for each of RHOG [oligo 1, 2 and 3 out of the NM_001665 panel), RAC1 (oligo 5 and 6 out of the NM_006908, NM_018890 and NM_198829 panels), CDC42 (oligo 4 and 7 out of the NM_001039802, NM_001791 and NM_044472 panels), STARD13 (oligo 4 out of the NM_001243466 panel], RHOA (oligo 1 and 6 out of the NM_001664 panel), RHOC (oligo 5 and 6 out of the NM_001042678, NM_00104269 and NM_175744 panels), ROCK1 (oligo 9 and 10 out of the NM_005406 panel) and ROCK2 (oligo 5 and 6 out of the NM_004850 panel) were brought from Qiagen (Qiagen, Hilden, Germany).

    Techniques: Tube Formation Assay, Transfection, Plasmid Preparation, Imaging, Luciferase, Western Blot, Quantitation Assay

    Suggested model based on the data presented in this study: RHOG positively regulates tube formation assay in ECV cells through a CDC42-RAC1-ERK. PI3K appears to be an upstream regulator of RHO GTPases leading to tube formation. RHOA and C seem to have different roles in tube formation. While RHOA leads to positive regulation of the process through ROCK1/2 as downstream effectors, RHOC does not seem to be necessary for tube formation. Finally, RHOA, unlike the RHOG-CDC42-RAC1 pathway, does not seem to exert its effect on tube formation through the MAPK pathway.

    Journal: Cells

    Article Title: RHOG Activates RAC1 through CDC42 Leading to Tube Formation in Vascular Endothelial Cells

    doi: 10.3390/cells8020171

    Figure Lengend Snippet: Suggested model based on the data presented in this study: RHOG positively regulates tube formation assay in ECV cells through a CDC42-RAC1-ERK. PI3K appears to be an upstream regulator of RHO GTPases leading to tube formation. RHOA and C seem to have different roles in tube formation. While RHOA leads to positive regulation of the process through ROCK1/2 as downstream effectors, RHOC does not seem to be necessary for tube formation. Finally, RHOA, unlike the RHOG-CDC42-RAC1 pathway, does not seem to exert its effect on tube formation through the MAPK pathway.

    Article Snippet: Human FlexiTube siRNA for each of RHOG [oligo 1, 2 and 3 out of the NM_001665 panel), RAC1 (oligo 5 and 6 out of the NM_006908, NM_018890 and NM_198829 panels), CDC42 (oligo 4 and 7 out of the NM_001039802, NM_001791 and NM_044472 panels), STARD13 (oligo 4 out of the NM_001243466 panel], RHOA (oligo 1 and 6 out of the NM_001664 panel), RHOC (oligo 5 and 6 out of the NM_001042678, NM_00104269 and NM_175744 panels), ROCK1 (oligo 9 and 10 out of the NM_005406 panel) and ROCK2 (oligo 5 and 6 out of the NM_004850 panel) were brought from Qiagen (Qiagen, Hilden, Germany).

    Techniques: Tube Formation Assay