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Promega rnasin inhibitor
Rnasin Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rnasin inhibitor - by Bioz Stars, 2020-04
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Clone Assay:

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: Paragraph title: Single B cell sorting, RT–PCR, sequencing, cloning, and expression of mAbs ... The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA).

Centrifugation:

Article Title: ZNF598 Is a Quality Control Sensor of Collided Ribosomes
Article Snippet: 4 × 1 mL of non-nucleased RRL supplemented with 500 nM eRF1AAQ was allowed to translate at 32°C for 25 min. Ribosomes from each reaction were pelleted through a 2 mL cushion of 15% (w/v) sucrose in PS buffer (50 mM HEPES, 100 mM KOAc, 2 mM Mg(OAc)2 , 0.5 mM TCEP, pH 7.4) by centrifugation at 424,480 x g for 1 hr at 4°C in a TLA-100.3 rotor (Beckman). .. The pellets were resuspended by soaking and repeated pipetting in 800 μL PS buffer supplemented with 1 U/μl RNasin inhibitor and carefully loaded onto two 14 mL sucrose gradients (10%–50%) in PS buffer supplemented with 20 U/ml RNasin inhibitor (Promega).

Article Title: Maf1p, a Negative Effector of RNA Polymerase III in Saccharomyces cerevisiae
Article Snippet: Insoluble material was removed by low-speed centrifugation. .. Transcription mixtures (40 μl) contained transcription buffer with 0.2 mM ATP, CTP, and GTP; 0.01 mM UTP; 10 μCi of [α-32 P]UTP (400 Ci/mmol; Amersham); 8 U of RNasin inhibitor (Promega), 40 ng of DNA per template; and protein crude extract.

Amplification:

Article Title: Effect of Vasoactive Intestinal Peptide (VIP) on NKG2D Signal Pathway and Its Contribution to Immune Escape of MKN45 Cells
Article Snippet: A typical 25-μ L reaction contained 10 μ L total RNA, 200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase, 500 ng oligo(dT)15 , 1 × 5 μ L reverse transcription buffer, 10 mmol/L deoxy-ribonucleoside triphosphate (dNTP), RNasin inhibitor (Promega, Madison, WI, USA), and ddH2 O. .. The cDNA was used as a template for PCR amplification.

Article Title: A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation
Article Snippet: Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega). .. PCR amplification was analysed using 2nd derivative maximum algorithm (LightCycler 480 Sw 1.5, Roche) and the expression of the gene of interest was normalised to the housekeeping gene Rn18s .

Article Title: Chitinase-like proteins promote IL-17-mediated neutrophilia in a trade-off between nematode killing and host damage
Article Snippet: Reverse transcription was performed using 0.25-1.00 μg of total RNA using 50 U Tetro reverse transcriptase (Bioline), 40 mM dNTPs (Promega), and 0.5 μg Oligo dT15 (Roche) and RNasin inhibitor (Promega). .. PCR amplification was analyzed using 2nd derivative maximum algorithm (LightCycler 480 Sw 1.5, Roche) and the expression of the gene of interest was normalized to a housekeeping gene Rpl13a .

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA). .. Briefly, RT was carried out by adding 1 µL of random hexamers (150 ng/µL; Promega), 2 µL dNTPs (each at 10 mM), and 0.5 µL SuperScript III (Invitrogen) to each well, followed by incubation at 42°C for 1 h. IgG heavy and light chain variable regions were independently amplified with nested PCR.

Article Title: Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion
Article Snippet: Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega). .. PCR amplification was analyzed using 2nd derivative maximum algorithm (LightCycler 480 Sw 1.5, Roche) and the expression of the gene of interest was normalized to the housekeeping gene Rn18s .

Article Title: Nemo-like Kinase Drives Foxp3 Stability and Is Critical for Maintenance of Immune Tolerance by Regulatory T Cells
Article Snippet: Reverse transcription was performed using 1 mg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 mg Oligo dT15 and RNasin inhibitor (Promega). .. PCR amplification was analyzed using 2nd derivative maximum algorithm (LightCycler 480 Sw 1.5, Roche) and Nlk expression was normalized to the housekeeping gene Rn18s .

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Total RNA (5 μg) was reverse transcribed using 8 μL of Molony murine leukemia virus RT (M-MLV RT) 5 × buffer, 3 μL of 10 mM dNTPs, 0.45 μL of 40 U/μL RNasein inhibitor, 0.3 μL of 200 U/μL M-MLV RT (Promega, Madison, WI, USA), and 1.5 μL of 50 μM oligo dT (Bioneer, Daejeon, Korea) in a 40 μL volume. .. Single-stranded cDNA was then amplified by PCR using 4 μL of 5 × green Go Taq flexi buffer, 0.4 μL of 10 mM dNTPs, 0.1 μL of 5 U/μL Taq polymerase, 1.2 μL of 25 mM MgCl2 (Promega), and 0.4 μL of 20 μM of specific sense and anti-sense primers of tyrosinase, TRP-1, TRP-2, MITF-M, or β-actin.

Article Title: Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles
Article Snippet: After incubation, PVDF membranes were removed and unbound RNA was amplified by RT-PCR using Invitrogen™ SuperScript® 3 One-Step RT-PCR system (Life Technologies) under the following conditions: first-strand synthesis step at 55°C for 30 minutes, and 15 cycles of denaturing step at 94°C for 1 minute, hybridization step at 55°C for 45 seconds, and polymerization step at 72°C for 2 minutes. .. Positive selection was performed by incubating the preselected RNA pool with PVDF-immobilized HPV-16 L1 VLPs (60 ng VLP protein/0.5 cm2 PVDF) and 20 U RNasin® inhibitor (Promega) in 100 μL 1× D-PBS for 1 hour at room temperature.

Synthesized:

Article Title: Impact and mechanism of non-steroidal anti-inflammatory drugs combined with chemotherapeutic drugs on human lung cancer-nude mouse transplanted tumors
Article Snippet: Reverse transcriptase, RNasin® inhibitor, deoxynucleotides, Taq DNA polymerization enzyme and agarose were purchased from Promega Corp. (Madison, WI, USA). .. Random primers were synthesized by Sangon Biotech Co., Ltd (Shanghai.

Cytometry:

Article Title: Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition
Article Snippet: .. Single synovial B cells expressing CD19 and IgG, while lacking CD3 and CD14, were sorted by flow cytometry using a Cytomation MoFlo (Dako) into 96-well PCR plates containing 5 µl/well of 0.5× PBS containing 10 mM DTT and 8 U RNAsin inhibitor (Promega) as previously described ( ; ). .. cDNA synthesis and PCR amplification Single cell cDNA was synthesized in a total volume of 15 µl in the original 96-well PCR plate using the SuperScript III RT (Gibco Invitrogen).

Quantitative RT-PCR:

Article Title: Impact and mechanism of non-steroidal anti-inflammatory drugs combined with chemotherapeutic drugs on human lung cancer-nude mouse transplanted tumors
Article Snippet: Paragraph title: Detection of CD44v6, MMP-2 and survivin mRNA expression by fluorescence reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay ... Reverse transcriptase, RNasin® inhibitor, deoxynucleotides, Taq DNA polymerization enzyme and agarose were purchased from Promega Corp. (Madison, WI, USA).

Real-time Polymerase Chain Reaction:

Article Title: Patient-derived multicellular tumor spheroids towards optimized treatment for patients with hepatocellular carcinoma
Article Snippet: Paragraph title: Real-time PCR ... The reaction mixture contained RT buffer (Bio Basic, Amherst, NY, USA), dNTP solution (Bio Basic), RNasin® inhibitor (Promega), oligo (dT)15 primer (Bioneer, Daejeon, Korea), total RNA, and M-MLV reverse transcriptase (Invitrogen).

Article Title: A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation
Article Snippet: Paragraph title: RNA Extraction and Quantitative Real-Time PCR ... Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega).

Article Title: Chitinase-like proteins promote IL-17-mediated neutrophilia in a trade-off between nematode killing and host damage
Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR ... Reverse transcription was performed using 0.25-1.00 μg of total RNA using 50 U Tetro reverse transcriptase (Bioline), 40 mM dNTPs (Promega), and 0.5 μg Oligo dT15 (Roche) and RNasin inhibitor (Promega).

Article Title: Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion
Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR ... Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega).

Article Title: Nemo-like Kinase Drives Foxp3 Stability and Is Critical for Maintenance of Immune Tolerance by Regulatory T Cells
Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR ... Reverse transcription was performed using 1 mg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 mg Oligo dT15 and RNasin inhibitor (Promega).

Article Title: RNA Secondary Structure Motifs of the Influenza A Virus as Targets for siRNA-Mediated RNA Interference
Article Snippet: Paragraph title: Virus Quantitative Real-Time PCR Analysis ... Then, 10 mM DTT, 2.5 mM 2′-deoxynucleoside 5′-triphosphates (dNTPs), 1× first-strand buffer, 10 U of RNasin inhibitor (Promega), and 50 U of SuperScript III reverse transcriptase were added to the final volume of 10 μL and incubated for 50 min at 55°C.

Incubation:

Article Title: Effect of Vasoactive Intestinal Peptide (VIP) on NKG2D Signal Pathway and Its Contribution to Immune Escape of MKN45 Cells
Article Snippet: After NK cells were incubated by the VIP and its antagonist for 48 h as mentioned above, the total RNA of the NK cells was extracted according to the procedure of the Trizol reagent (TianGen Biotechnology Co., Beijing, China). .. A typical 25-μ L reaction contained 10 μ L total RNA, 200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase, 500 ng oligo(dT)15 , 1 × 5 μ L reverse transcription buffer, 10 mmol/L deoxy-ribonucleoside triphosphate (dNTP), RNasin inhibitor (Promega, Madison, WI, USA), and ddH2 O.

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA). .. Briefly, RT was carried out by adding 1 µL of random hexamers (150 ng/µL; Promega), 2 µL dNTPs (each at 10 mM), and 0.5 µL SuperScript III (Invitrogen) to each well, followed by incubation at 42°C for 1 h. IgG heavy and light chain variable regions were independently amplified with nested PCR.

Article Title: RNA Secondary Structure Motifs of the Influenza A Virus as Targets for siRNA-Mediated RNA Interference
Article Snippet: .. Then, 10 mM DTT, 2.5 mM 2′-deoxynucleoside 5′-triphosphates (dNTPs), 1× first-strand buffer, 10 U of RNasin inhibitor (Promega), and 50 U of SuperScript III reverse transcriptase were added to the final volume of 10 μL and incubated for 50 min at 55°C. .. The reaction was inactivated by heating at 70°C for 15 min. One microliter of the obtained cDNA was subjected to real-time PCR with gene-specific primers ( ) and 5× HOT FIREPol Probe qPCR Mix Plus (Solis BioDyne) according to the manufacturer’s protocol.

Article Title: Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles
Article Snippet: After incubation, PVDF membranes were removed and unbound RNA was amplified by RT-PCR using Invitrogen™ SuperScript® 3 One-Step RT-PCR system (Life Technologies) under the following conditions: first-strand synthesis step at 55°C for 30 minutes, and 15 cycles of denaturing step at 94°C for 1 minute, hybridization step at 55°C for 45 seconds, and polymerization step at 72°C for 2 minutes. .. Positive selection was performed by incubating the preselected RNA pool with PVDF-immobilized HPV-16 L1 VLPs (60 ng VLP protein/0.5 cm2 PVDF) and 20 U RNasin® inhibitor (Promega) in 100 μL 1× D-PBS for 1 hour at room temperature.

Article Title: Frequent Reassortments May Explain the Genetic Heterogeneity of Rotaviruses: Analysis of Finnish Rotavirus Strains
Article Snippet: The RT was run separately for 1 h at 37°C, in a 20-μl reaction mixture containing 50 mM Tris-HCl (pH 8.8), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2 , 0.5 mM deoxynucleoside triphosphates (Pharmacia, Uppsala, Sweden), 0.5 μM primer, 1 U of RNAsin inhibitor (Promega, Madison, Wis.)/μl, 10 U of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, Md.)/μl, and rotavirus RNA that had been pretreated by heating for 5 min in presence of 37.5% dimethyl sulfoxide and chilling on ice for 5 min. For PCR, 5 μl of the RT reaction mixture was added to a 100-μl reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 0.001% gelatin, 0.1 mM deoxynucleoside triphosphates, 0.2 μM each primer, and 2.5 U of AmpliTaq DNA polymerase (Perkin Elmer, Branchburg, N.J.)/100 μl. .. The PCR program was as follows: 33 cycles of 94°C for 1.0 min, 40 to 42°C for 2.0 min, and 72°C for 2.0 min, followed by a final incubation for 7 min at 72°C.

Expressing:

Article Title: A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation
Article Snippet: Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega). .. PCR amplification was analysed using 2nd derivative maximum algorithm (LightCycler 480 Sw 1.5, Roche) and the expression of the gene of interest was normalised to the housekeeping gene Rn18s .

Article Title: Chitinase-like proteins promote IL-17-mediated neutrophilia in a trade-off between nematode killing and host damage
Article Snippet: Reverse transcription was performed using 0.25-1.00 μg of total RNA using 50 U Tetro reverse transcriptase (Bioline), 40 mM dNTPs (Promega), and 0.5 μg Oligo dT15 (Roche) and RNasin inhibitor (Promega). .. PCR amplification was analyzed using 2nd derivative maximum algorithm (LightCycler 480 Sw 1.5, Roche) and the expression of the gene of interest was normalized to a housekeeping gene Rpl13a .

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: Paragraph title: Single B cell sorting, RT–PCR, sequencing, cloning, and expression of mAbs ... The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA).

Article Title: Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition
Article Snippet: .. Single synovial B cells expressing CD19 and IgG, while lacking CD3 and CD14, were sorted by flow cytometry using a Cytomation MoFlo (Dako) into 96-well PCR plates containing 5 µl/well of 0.5× PBS containing 10 mM DTT and 8 U RNAsin inhibitor (Promega) as previously described ( ; ). .. cDNA synthesis and PCR amplification Single cell cDNA was synthesized in a total volume of 15 µl in the original 96-well PCR plate using the SuperScript III RT (Gibco Invitrogen).

Article Title: Impact and mechanism of non-steroidal anti-inflammatory drugs combined with chemotherapeutic drugs on human lung cancer-nude mouse transplanted tumors
Article Snippet: Paragraph title: Detection of CD44v6, MMP-2 and survivin mRNA expression by fluorescence reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay ... Reverse transcriptase, RNasin® inhibitor, deoxynucleotides, Taq DNA polymerization enzyme and agarose were purchased from Promega Corp. (Madison, WI, USA).

Article Title: Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion
Article Snippet: Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega). .. Expression of genes of interest was measured by real-time PCR with the Lightcycler 480 II system (Roche) using Taqman Master kit and specific primers (specified in the KEY RESOURCES TABLE), as previously described ( ).

Article Title: Nemo-like Kinase Drives Foxp3 Stability and Is Critical for Maintenance of Immune Tolerance by Regulatory T Cells
Article Snippet: Reverse transcription was performed using 1 mg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 mg Oligo dT15 and RNasin inhibitor (Promega). .. Expression of genes of interest was measured by real-time PCR with the Lightcycler 480 II system (Roche) using Taqman Master kit and specific primers for Nlk , as previously described.

RNA Binding Assay:

Article Title: Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles
Article Snippet: Briefly, three initial preselection cycles were performed to remove unspecific RNA binding by incubating the RNA pool with activated 0.5 cm2 polyvinylidene fluoride (PVDF) membranes (EMD Millipore Corporation, Billerica, MA) and 20 U RNasin® ribonuclease inhibitor (Promega) in 100 μL 1× D-PBS for 1 hour at room temperature. .. Positive selection was performed by incubating the preselected RNA pool with PVDF-immobilized HPV-16 L1 VLPs (60 ng VLP protein/0.5 cm2 PVDF) and 20 U RNasin® inhibitor (Promega) in 100 μL 1× D-PBS for 1 hour at room temperature.

Hybridization:

Article Title: Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles
Article Snippet: After incubation, PVDF membranes were removed and unbound RNA was amplified by RT-PCR using Invitrogen™ SuperScript® 3 One-Step RT-PCR system (Life Technologies) under the following conditions: first-strand synthesis step at 55°C for 30 minutes, and 15 cycles of denaturing step at 94°C for 1 minute, hybridization step at 55°C for 45 seconds, and polymerization step at 72°C for 2 minutes. .. Positive selection was performed by incubating the preselected RNA pool with PVDF-immobilized HPV-16 L1 VLPs (60 ng VLP protein/0.5 cm2 PVDF) and 20 U RNasin® inhibitor (Promega) in 100 μL 1× D-PBS for 1 hour at room temperature.

Countercurrent Chromatography:

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Total RNA (5 μg) was reverse transcribed using 8 μL of Molony murine leukemia virus RT (M-MLV RT) 5 × buffer, 3 μL of 10 mM dNTPs, 0.45 μL of 40 U/μL RNasein inhibitor, 0.3 μL of 200 U/μL M-MLV RT (Promega, Madison, WI, USA), and 1.5 μL of 50 μM oligo dT (Bioneer, Daejeon, Korea) in a 40 μL volume. .. The primer sequences used for PCR were as follows: tyrosinase forward 5′-CAT TTT TGA TTT GAG TGT CT-3′, reverse 5′-TGT GGT AGT CGT CTT TGT CC-3′; TRP-1 forward 5′-GCT GCA GGA GCC TTC TTT CTC-3′, reverse 5′-AAG ACG CTG CAC TGC TGG TCT-3′; TRP-2 forward 5′-GGA TGA CCG TGA GCA ATG GCC-3′, reverse 5′-CGG TTG TGA CCA ATG GGT GCC-3′; MITF-M forward 5′-TAC AGA AAG TAG AGG GAG GAG GAC TAA G-3′, reverse 5′-CAC AGT TGG AGT TAA GAG TGA GCA TAG CC-3′; β-actin forward 5′-ACC GTG AAA AGA TGA CCC AG-3′, reverse 5′-TAC GGA TGT CAA CGT CAC AC-3′.

Flow Cytometry:

Article Title: Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition
Article Snippet: .. Single synovial B cells expressing CD19 and IgG, while lacking CD3 and CD14, were sorted by flow cytometry using a Cytomation MoFlo (Dako) into 96-well PCR plates containing 5 µl/well of 0.5× PBS containing 10 mM DTT and 8 U RNAsin inhibitor (Promega) as previously described ( ; ). .. cDNA synthesis and PCR amplification Single cell cDNA was synthesized in a total volume of 15 µl in the original 96-well PCR plate using the SuperScript III RT (Gibco Invitrogen).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Effect of Vasoactive Intestinal Peptide (VIP) on NKG2D Signal Pathway and Its Contribution to Immune Escape of MKN45 Cells
Article Snippet: Paragraph title: 2.4.1. RNA Isolation and Reverse Transcription Polymerase Chain Reaction (RT-PCR) ... A typical 25-μ L reaction contained 10 μ L total RNA, 200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase, 500 ng oligo(dT)15 , 1 × 5 μ L reverse transcription buffer, 10 mmol/L deoxy-ribonucleoside triphosphate (dNTP), RNasin inhibitor (Promega, Madison, WI, USA), and ddH2 O.

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: Paragraph title: Single B cell sorting, RT–PCR, sequencing, cloning, and expression of mAbs ... The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA).

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Paragraph title: Reverse transcription-polymerase chain reaction (RT-PCR) ... Total RNA (5 μg) was reverse transcribed using 8 μL of Molony murine leukemia virus RT (M-MLV RT) 5 × buffer, 3 μL of 10 mM dNTPs, 0.45 μL of 40 U/μL RNasein inhibitor, 0.3 μL of 200 U/μL M-MLV RT (Promega, Madison, WI, USA), and 1.5 μL of 50 μM oligo dT (Bioneer, Daejeon, Korea) in a 40 μL volume.

Article Title: Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles
Article Snippet: After incubation, PVDF membranes were removed and unbound RNA was amplified by RT-PCR using Invitrogen™ SuperScript® 3 One-Step RT-PCR system (Life Technologies) under the following conditions: first-strand synthesis step at 55°C for 30 minutes, and 15 cycles of denaturing step at 94°C for 1 minute, hybridization step at 55°C for 45 seconds, and polymerization step at 72°C for 2 minutes. .. Positive selection was performed by incubating the preselected RNA pool with PVDF-immobilized HPV-16 L1 VLPs (60 ng VLP protein/0.5 cm2 PVDF) and 20 U RNasin® inhibitor (Promega) in 100 μL 1× D-PBS for 1 hour at room temperature.

Article Title: Frequent Reassortments May Explain the Genetic Heterogeneity of Rotaviruses: Analysis of Finnish Rotavirus Strains
Article Snippet: Paragraph title: RNA extraction and RT-PCR. ... The RT was run separately for 1 h at 37°C, in a 20-μl reaction mixture containing 50 mM Tris-HCl (pH 8.8), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2 , 0.5 mM deoxynucleoside triphosphates (Pharmacia, Uppsala, Sweden), 0.5 μM primer, 1 U of RNAsin inhibitor (Promega, Madison, Wis.)/μl, 10 U of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, Md.)/μl, and rotavirus RNA that had been pretreated by heating for 5 min in presence of 37.5% dimethyl sulfoxide and chilling on ice for 5 min. For PCR, 5 μl of the RT reaction mixture was added to a 100-μl reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 0.001% gelatin, 0.1 mM deoxynucleoside triphosphates, 0.2 μM each primer, and 2.5 U of AmpliTaq DNA polymerase (Perkin Elmer, Branchburg, N.J.)/100 μl.

Sequencing:

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: Paragraph title: Single B cell sorting, RT–PCR, sequencing, cloning, and expression of mAbs ... The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA).

Article Title: RNA Secondary Structure Motifs of the Influenza A Virus as Targets for siRNA-Mediated RNA Interference
Article Snippet: Reverse transcription was performed with gene-specific primers complementary to the vRNA7 sequence , ( ) and SuperScript III reverse transcriptase (Invitrogen). .. Then, 10 mM DTT, 2.5 mM 2′-deoxynucleoside 5′-triphosphates (dNTPs), 1× first-strand buffer, 10 U of RNasin inhibitor (Promega), and 50 U of SuperScript III reverse transcriptase were added to the final volume of 10 μL and incubated for 50 min at 55°C.

Cellular Antioxidant Activity Assay:

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Total RNA (5 μg) was reverse transcribed using 8 μL of Molony murine leukemia virus RT (M-MLV RT) 5 × buffer, 3 μL of 10 mM dNTPs, 0.45 μL of 40 U/μL RNasein inhibitor, 0.3 μL of 200 U/μL M-MLV RT (Promega, Madison, WI, USA), and 1.5 μL of 50 μM oligo dT (Bioneer, Daejeon, Korea) in a 40 μL volume. .. The primer sequences used for PCR were as follows: tyrosinase forward 5′-CAT TTT TGA TTT GAG TGT CT-3′, reverse 5′-TGT GGT AGT CGT CTT TGT CC-3′; TRP-1 forward 5′-GCT GCA GGA GCC TTC TTT CTC-3′, reverse 5′-AAG ACG CTG CAC TGC TGG TCT-3′; TRP-2 forward 5′-GGA TGA CCG TGA GCA ATG GCC-3′, reverse 5′-CGG TTG TGA CCA ATG GGT GCC-3′; MITF-M forward 5′-TAC AGA AAG TAG AGG GAG GAG GAC TAA G-3′, reverse 5′-CAC AGT TGG AGT TAA GAG TGA GCA TAG CC-3′; β-actin forward 5′-ACC GTG AAA AGA TGA CCC AG-3′, reverse 5′-TAC GGA TGT CAA CGT CAC AC-3′.

Fluorescence:

Article Title: Impact and mechanism of non-steroidal anti-inflammatory drugs combined with chemotherapeutic drugs on human lung cancer-nude mouse transplanted tumors
Article Snippet: Paragraph title: Detection of CD44v6, MMP-2 and survivin mRNA expression by fluorescence reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay ... Reverse transcriptase, RNasin® inhibitor, deoxynucleotides, Taq DNA polymerization enzyme and agarose were purchased from Promega Corp. (Madison, WI, USA).

Isolation:

Article Title: Effect of Vasoactive Intestinal Peptide (VIP) on NKG2D Signal Pathway and Its Contribution to Immune Escape of MKN45 Cells
Article Snippet: Paragraph title: 2.4.1. RNA Isolation and Reverse Transcription Polymerase Chain Reaction (RT-PCR) ... A typical 25-μ L reaction contained 10 μ L total RNA, 200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase, 500 ng oligo(dT)15 , 1 × 5 μ L reverse transcription buffer, 10 mmol/L deoxy-ribonucleoside triphosphate (dNTP), RNasin inhibitor (Promega, Madison, WI, USA), and ddH2 O.

Article Title: Patient-derived multicellular tumor spheroids towards optimized treatment for patients with hepatocellular carcinoma
Article Snippet: Real-time PCR Total RNA was isolated from cells using TRIzol® (Invitrogen) according to the manufacturer’s instructions. .. The reaction mixture contained RT buffer (Bio Basic, Amherst, NY, USA), dNTP solution (Bio Basic), RNasin® inhibitor (Promega), oligo (dT)15 primer (Bioneer, Daejeon, Korea), total RNA, and M-MLV reverse transcriptase (Invitrogen).

Article Title: A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation
Article Snippet: RNA Extraction and Quantitative Real-Time PCR Tissue or cells in culture were homogenised in TRIzol with a TissueLyser (Qiagen) and RNA was isolated following manufacturer’s instructions. .. Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega).

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: Single B cell sorting, RT–PCR, sequencing, cloning, and expression of mAbs Freshly isolated PBMCs were stained with a cocktail of the following antibodies: anti-human CD20-FITC (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA); IgG-APC-H7/CD3-Pacific Blue/CD27-PerCP-Cy5.5 (BD Biosciences, San Jose, CA, USA); and anti-HIS-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). .. The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA).

Article Title: Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion
Article Snippet: Tissue was homogenized in TRIzol with a TissueLyser (QIAGEN) and RNA was isolated following manufacturer’s instructions. .. Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega).

Article Title: Nemo-like Kinase Drives Foxp3 Stability and Is Critical for Maintenance of Immune Tolerance by Regulatory T Cells
Article Snippet: RNA extraction and quantitative real-time PCR TREG cells were homogenized in TRIzol and RNA was isolated following manufacturer’s instructions. .. Reverse transcription was performed using 1 mg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 mg Oligo dT15 and RNasin inhibitor (Promega).

Article Title: RNA Secondary Structure Motifs of the Influenza A Virus as Targets for siRNA-Mediated RNA Interference
Article Snippet: Enzyme inactivation was performed by adding 1 μL of 25 mM EDTA to reach a final concentration of 2.3 mM and heating at 75°C for 10 min. Agarose gel electrophoresis was conducted to check the quality of isolated RNA. .. Then, 10 mM DTT, 2.5 mM 2′-deoxynucleoside 5′-triphosphates (dNTPs), 1× first-strand buffer, 10 U of RNasin inhibitor (Promega), and 50 U of SuperScript III reverse transcriptase were added to the final volume of 10 μL and incubated for 50 min at 55°C.

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated using Trizol-Reagent (Invitrogen, New York, NY, USA) according to the manufacturer’s instructions. .. Total RNA (5 μg) was reverse transcribed using 8 μL of Molony murine leukemia virus RT (M-MLV RT) 5 × buffer, 3 μL of 10 mM dNTPs, 0.45 μL of 40 U/μL RNasein inhibitor, 0.3 μL of 200 U/μL M-MLV RT (Promega, Madison, WI, USA), and 1.5 μL of 50 μM oligo dT (Bioneer, Daejeon, Korea) in a 40 μL volume.

RNA Extraction:

Article Title: A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation
Article Snippet: Paragraph title: RNA Extraction and Quantitative Real-Time PCR ... Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega).

Article Title: Chitinase-like proteins promote IL-17-mediated neutrophilia in a trade-off between nematode killing and host damage
Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR ... Reverse transcription was performed using 0.25-1.00 μg of total RNA using 50 U Tetro reverse transcriptase (Bioline), 40 mM dNTPs (Promega), and 0.5 μg Oligo dT15 (Roche) and RNasin inhibitor (Promega).

Article Title: Impact and mechanism of non-steroidal anti-inflammatory drugs combined with chemotherapeutic drugs on human lung cancer-nude mouse transplanted tumors
Article Snippet: TRIzol® reagent (RNA extraction solution) was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). .. Reverse transcriptase, RNasin® inhibitor, deoxynucleotides, Taq DNA polymerization enzyme and agarose were purchased from Promega Corp. (Madison, WI, USA).

Article Title: Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion
Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR ... Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega).

Article Title: Nemo-like Kinase Drives Foxp3 Stability and Is Critical for Maintenance of Immune Tolerance by Regulatory T Cells
Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR ... Reverse transcription was performed using 1 mg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 mg Oligo dT15 and RNasin inhibitor (Promega).

Article Title: Frequent Reassortments May Explain the Genetic Heterogeneity of Rotaviruses: Analysis of Finnish Rotavirus Strains
Article Snippet: Paragraph title: RNA extraction and RT-PCR. ... The RT was run separately for 1 h at 37°C, in a 20-μl reaction mixture containing 50 mM Tris-HCl (pH 8.8), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2 , 0.5 mM deoxynucleoside triphosphates (Pharmacia, Uppsala, Sweden), 0.5 μM primer, 1 U of RNAsin inhibitor (Promega, Madison, Wis.)/μl, 10 U of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, Md.)/μl, and rotavirus RNA that had been pretreated by heating for 5 min in presence of 37.5% dimethyl sulfoxide and chilling on ice for 5 min. For PCR, 5 μl of the RT reaction mixture was added to a 100-μl reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 0.001% gelatin, 0.1 mM deoxynucleoside triphosphates, 0.2 μM each primer, and 2.5 U of AmpliTaq DNA polymerase (Perkin Elmer, Branchburg, N.J.)/100 μl.

Purification:

Article Title: ZNF598 Is a Quality Control Sensor of Collided Ribosomes
Article Snippet: Paragraph title: Purification of Stalled Poly-ribosomes for Cryo-EM ... The pellets were resuspended by soaking and repeated pipetting in 800 μL PS buffer supplemented with 1 U/μl RNasin inhibitor and carefully loaded onto two 14 mL sucrose gradients (10%–50%) in PS buffer supplemented with 20 U/ml RNasin inhibitor (Promega).

Polymerase Chain Reaction:

Article Title: Effect of Vasoactive Intestinal Peptide (VIP) on NKG2D Signal Pathway and Its Contribution to Immune Escape of MKN45 Cells
Article Snippet: A typical 25-μ L reaction contained 10 μ L total RNA, 200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase, 500 ng oligo(dT)15 , 1 × 5 μ L reverse transcription buffer, 10 mmol/L deoxy-ribonucleoside triphosphate (dNTP), RNasin inhibitor (Promega, Madison, WI, USA), and ddH2 O. .. The cDNA was used as a template for PCR amplification.

Article Title: Patient-derived multicellular tumor spheroids towards optimized treatment for patients with hepatocellular carcinoma
Article Snippet: The reaction mixture contained RT buffer (Bio Basic, Amherst, NY, USA), dNTP solution (Bio Basic), RNasin® inhibitor (Promega), oligo (dT)15 primer (Bioneer, Daejeon, Korea), total RNA, and M-MLV reverse transcriptase (Invitrogen). .. PCR reactions were performed in 96-well plates in a mixture composed of cDNA, SYBR Green master mix (Applied Biosystems, Waltham, MA, USA), primers, and DEPC using a StepOnePlus real-time PCR system (Applied Biosystems).

Article Title: A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation
Article Snippet: Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega). .. PCR amplification was analysed using 2nd derivative maximum algorithm (LightCycler 480 Sw 1.5, Roche) and the expression of the gene of interest was normalised to the housekeeping gene Rn18s .

Article Title: Chitinase-like proteins promote IL-17-mediated neutrophilia in a trade-off between nematode killing and host damage
Article Snippet: Reverse transcription was performed using 0.25-1.00 μg of total RNA using 50 U Tetro reverse transcriptase (Bioline), 40 mM dNTPs (Promega), and 0.5 μg Oligo dT15 (Roche) and RNasin inhibitor (Promega). .. PCR amplification was analyzed using 2nd derivative maximum algorithm (LightCycler 480 Sw 1.5, Roche) and the expression of the gene of interest was normalized to a housekeeping gene Rpl13a .

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: Individual B cells were sorted into 96-well PCR plates containing 20 µL of lysis buffer per cell. .. The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA).

Article Title: Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition
Article Snippet: .. Single synovial B cells expressing CD19 and IgG, while lacking CD3 and CD14, were sorted by flow cytometry using a Cytomation MoFlo (Dako) into 96-well PCR plates containing 5 µl/well of 0.5× PBS containing 10 mM DTT and 8 U RNAsin inhibitor (Promega) as previously described ( ; ). .. cDNA synthesis and PCR amplification Single cell cDNA was synthesized in a total volume of 15 µl in the original 96-well PCR plate using the SuperScript III RT (Gibco Invitrogen).

Article Title: Impact and mechanism of non-steroidal anti-inflammatory drugs combined with chemotherapeutic drugs on human lung cancer-nude mouse transplanted tumors
Article Snippet: Paragraph title: Detection of CD44v6, MMP-2 and survivin mRNA expression by fluorescence reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay ... Reverse transcriptase, RNasin® inhibitor, deoxynucleotides, Taq DNA polymerization enzyme and agarose were purchased from Promega Corp. (Madison, WI, USA).

Article Title: Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion
Article Snippet: Reverse transcription was performed using 1 μg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 μg Oligo dT15 and RNasin inhibitor (Promega). .. PCR amplification was analyzed using 2nd derivative maximum algorithm (LightCycler 480 Sw 1.5, Roche) and the expression of the gene of interest was normalized to the housekeeping gene Rn18s .

Article Title: Nemo-like Kinase Drives Foxp3 Stability and Is Critical for Maintenance of Immune Tolerance by Regulatory T Cells
Article Snippet: Reverse transcription was performed using 1 mg of total RNA using 200 U of M-MLV reverse transcriptase, 10 mM dNTPs, and 0.5 mg Oligo dT15 and RNasin inhibitor (Promega). .. PCR amplification was analyzed using 2nd derivative maximum algorithm (LightCycler 480 Sw 1.5, Roche) and Nlk expression was normalized to the housekeeping gene Rn18s .

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Total RNA (5 μg) was reverse transcribed using 8 μL of Molony murine leukemia virus RT (M-MLV RT) 5 × buffer, 3 μL of 10 mM dNTPs, 0.45 μL of 40 U/μL RNasein inhibitor, 0.3 μL of 200 U/μL M-MLV RT (Promega, Madison, WI, USA), and 1.5 μL of 50 μM oligo dT (Bioneer, Daejeon, Korea) in a 40 μL volume. .. Single-stranded cDNA was then amplified by PCR using 4 μL of 5 × green Go Taq flexi buffer, 0.4 μL of 10 mM dNTPs, 0.1 μL of 5 U/μL Taq polymerase, 1.2 μL of 25 mM MgCl2 (Promega), and 0.4 μL of 20 μM of specific sense and anti-sense primers of tyrosinase, TRP-1, TRP-2, MITF-M, or β-actin.

Article Title: Frequent Reassortments May Explain the Genetic Heterogeneity of Rotaviruses: Analysis of Finnish Rotavirus Strains
Article Snippet: .. The RT was run separately for 1 h at 37°C, in a 20-μl reaction mixture containing 50 mM Tris-HCl (pH 8.8), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2 , 0.5 mM deoxynucleoside triphosphates (Pharmacia, Uppsala, Sweden), 0.5 μM primer, 1 U of RNAsin inhibitor (Promega, Madison, Wis.)/μl, 10 U of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, Md.)/μl, and rotavirus RNA that had been pretreated by heating for 5 min in presence of 37.5% dimethyl sulfoxide and chilling on ice for 5 min. For PCR, 5 μl of the RT reaction mixture was added to a 100-μl reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 0.001% gelatin, 0.1 mM deoxynucleoside triphosphates, 0.2 μM each primer, and 2.5 U of AmpliTaq DNA polymerase (Perkin Elmer, Branchburg, N.J.)/100 μl. .. The PCR program was as follows: 33 cycles of 94°C for 1.0 min, 40 to 42°C for 2.0 min, and 72°C for 2.0 min, followed by a final incubation for 7 min at 72°C.

FACS:

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: Paragraph title: Single B cell sorting, RT–PCR, sequencing, cloning, and expression of mAbs ... The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA).

Article Title: Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition
Article Snippet: Paragraph title: Single B cell sorting ... Single synovial B cells expressing CD19 and IgG, while lacking CD3 and CD14, were sorted by flow cytometry using a Cytomation MoFlo (Dako) into 96-well PCR plates containing 5 µl/well of 0.5× PBS containing 10 mM DTT and 8 U RNAsin inhibitor (Promega) as previously described ( ; ).

Lysis:

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: .. The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA). .. PCR plates containing the sorted cells were frozen on dry ice then stored at – 80°C or used for reverse transcription (RT).

Nested PCR:

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA). .. Briefly, RT was carried out by adding 1 µL of random hexamers (150 ng/µL; Promega), 2 µL dNTPs (each at 10 mM), and 0.5 µL SuperScript III (Invitrogen) to each well, followed by incubation at 42°C for 1 h. IgG heavy and light chain variable regions were independently amplified with nested PCR.

SYBR Green Assay:

Article Title: Patient-derived multicellular tumor spheroids towards optimized treatment for patients with hepatocellular carcinoma
Article Snippet: The reaction mixture contained RT buffer (Bio Basic, Amherst, NY, USA), dNTP solution (Bio Basic), RNasin® inhibitor (Promega), oligo (dT)15 primer (Bioneer, Daejeon, Korea), total RNA, and M-MLV reverse transcriptase (Invitrogen). .. PCR reactions were performed in 96-well plates in a mixture composed of cDNA, SYBR Green master mix (Applied Biosystems, Waltham, MA, USA), primers, and DEPC using a StepOnePlus real-time PCR system (Applied Biosystems).

Article Title: Chitinase-like proteins promote IL-17-mediated neutrophilia in a trade-off between nematode killing and host damage
Article Snippet: Reverse transcription was performed using 0.25-1.00 μg of total RNA using 50 U Tetro reverse transcriptase (Bioline), 40 mM dNTPs (Promega), and 0.5 μg Oligo dT15 (Roche) and RNasin inhibitor (Promega). .. Transcript abundance of genes of interest were measured by real-time PCR with the Lightcycler 480 II system (Roche) using SYBR Green I Master kit and specific primer pairs ( ) as previously described .

Article Title: Impact and mechanism of non-steroidal anti-inflammatory drugs combined with chemotherapeutic drugs on human lung cancer-nude mouse transplanted tumors
Article Snippet: Reverse transcriptase, RNasin® inhibitor, deoxynucleotides, Taq DNA polymerization enzyme and agarose were purchased from Promega Corp. (Madison, WI, USA). .. RealMasterMix (SYBR Green) kit and DNAse was purchased from Tiangen Biotech Co., Ltd. (Beijing, China).

Negative Control:

Article Title: RNA Secondary Structure Motifs of the Influenza A Virus as Targets for siRNA-Mediated RNA Interference
Article Snippet: Then, 10 mM DTT, 2.5 mM 2′-deoxynucleoside 5′-triphosphates (dNTPs), 1× first-strand buffer, 10 U of RNasin inhibitor (Promega), and 50 U of SuperScript III reverse transcriptase were added to the final volume of 10 μL and incubated for 50 min at 55°C. .. The viral RNA copy number for each siRNA-treated sample was calculated as a percentage of viral copy number detected in the negative control, which was established as 100% of the viral RNA copies.

Selection:

Article Title: Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles
Article Snippet: .. Positive selection was performed by incubating the preselected RNA pool with PVDF-immobilized HPV-16 L1 VLPs (60 ng VLP protein/0.5 cm2 PVDF) and 20 U RNasin® inhibitor (Promega) in 100 μL 1× D-PBS for 1 hour at room temperature. .. After incubation, unbound RNA was removed by three 1× D-PBS washes and membranes were placed in 50 μL 1× D-PBS at room temperature.

Agarose Gel Electrophoresis:

Article Title: RNA Secondary Structure Motifs of the Influenza A Virus as Targets for siRNA-Mediated RNA Interference
Article Snippet: Enzyme inactivation was performed by adding 1 μL of 25 mM EDTA to reach a final concentration of 2.3 mM and heating at 75°C for 10 min. Agarose gel electrophoresis was conducted to check the quality of isolated RNA. .. Then, 10 mM DTT, 2.5 mM 2′-deoxynucleoside 5′-triphosphates (dNTPs), 1× first-strand buffer, 10 U of RNasin inhibitor (Promega), and 50 U of SuperScript III reverse transcriptase were added to the final volume of 10 μL and incubated for 50 min at 55°C.

In Vitro:

Article Title: Maf1p, a Negative Effector of RNA Polymerase III in Saccharomyces cerevisiae
Article Snippet: Paragraph title: In vitro transcription. ... Transcription mixtures (40 μl) contained transcription buffer with 0.2 mM ATP, CTP, and GTP; 0.01 mM UTP; 10 μCi of [α-32 P]UTP (400 Ci/mmol; Amersham); 8 U of RNasin inhibitor (Promega), 40 ng of DNA per template; and protein crude extract.

Electrophoresis:

Article Title: Effect of Vasoactive Intestinal Peptide (VIP) on NKG2D Signal Pathway and Its Contribution to Immune Escape of MKN45 Cells
Article Snippet: A typical 25-μ L reaction contained 10 μ L total RNA, 200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase, 500 ng oligo(dT)15 , 1 × 5 μ L reverse transcription buffer, 10 mmol/L deoxy-ribonucleoside triphosphate (dNTP), RNasin inhibitor (Promega, Madison, WI, USA), and ddH2 O. .. Electrophoresis and mRNA Semiquantity .

Spectrophotometry:

Article Title: Impact and mechanism of non-steroidal anti-inflammatory drugs combined with chemotherapeutic drugs on human lung cancer-nude mouse transplanted tumors
Article Snippet: Reverse transcriptase, RNasin® inhibitor, deoxynucleotides, Taq DNA polymerization enzyme and agarose were purchased from Promega Corp. (Madison, WI, USA). .. The tumor tissue RNA was extracted using 2 µl DNAse per sample, and a UV spectrophotometer (UV-2550 2450; Shimadzu, Corp., Kyoto, Japan) was used to detect the optical density (OD) 260/OD280 ratio.

Concentration Assay:

Article Title: RNA Secondary Structure Motifs of the Influenza A Virus as Targets for siRNA-Mediated RNA Interference
Article Snippet: Enzyme inactivation was performed by adding 1 μL of 25 mM EDTA to reach a final concentration of 2.3 mM and heating at 75°C for 10 min. Agarose gel electrophoresis was conducted to check the quality of isolated RNA. .. Then, 10 mM DTT, 2.5 mM 2′-deoxynucleoside 5′-triphosphates (dNTPs), 1× first-strand buffer, 10 U of RNasin inhibitor (Promega), and 50 U of SuperScript III reverse transcriptase were added to the final volume of 10 μL and incubated for 50 min at 55°C.

CTG Assay:

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Total RNA (5 μg) was reverse transcribed using 8 μL of Molony murine leukemia virus RT (M-MLV RT) 5 × buffer, 3 μL of 10 mM dNTPs, 0.45 μL of 40 U/μL RNasein inhibitor, 0.3 μL of 200 U/μL M-MLV RT (Promega, Madison, WI, USA), and 1.5 μL of 50 μM oligo dT (Bioneer, Daejeon, Korea) in a 40 μL volume. .. The primer sequences used for PCR were as follows: tyrosinase forward 5′-CAT TTT TGA TTT GAG TGT CT-3′, reverse 5′-TGT GGT AGT CGT CTT TGT CC-3′; TRP-1 forward 5′-GCT GCA GGA GCC TTC TTT CTC-3′, reverse 5′-AAG ACG CTG CAC TGC TGG TCT-3′; TRP-2 forward 5′-GGA TGA CCG TGA GCA ATG GCC-3′, reverse 5′-CGG TTG TGA CCA ATG GGT GCC-3′; MITF-M forward 5′-TAC AGA AAG TAG AGG GAG GAG GAC TAA G-3′, reverse 5′-CAC AGT TGG AGT TAA GAG TGA GCA TAG CC-3′; β-actin forward 5′-ACC GTG AAA AGA TGA CCC AG-3′, reverse 5′-TAC GGA TGT CAA CGT CAC AC-3′.

Staining:

Article Title: Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response
Article Snippet: Single B cell sorting, RT–PCR, sequencing, cloning, and expression of mAbs Freshly isolated PBMCs were stained with a cocktail of the following antibodies: anti-human CD20-FITC (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA); IgG-APC-H7/CD3-Pacific Blue/CD27-PerCP-Cy5.5 (BD Biosciences, San Jose, CA, USA); and anti-HIS-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). .. The lysis buffer contained 0.25 µL RNasin® inhibitor (Promega, Madison, WI, USA), 5 µL 5 × first-strand buffer, 1.25 µL 0.1 M DTT, and 0.0625 µL IGEPAL (Sigma-Aldrich, St. Louis, MO, USA).

Article Title: Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition
Article Snippet: Single B cell sorting Cryopreserved synovial mononuclear cells were thawed, and the cell suspensions were stained with anti–human CD19 conjugated to PE (Miltenyi Biotec), anti–human IgG conjugated to APC (Miltenyi Biotec), and anti–human CD3 (BD) and anti–human CD14 (BD) both conjugated to FITC. .. Single synovial B cells expressing CD19 and IgG, while lacking CD3 and CD14, were sorted by flow cytometry using a Cytomation MoFlo (Dako) into 96-well PCR plates containing 5 µl/well of 0.5× PBS containing 10 mM DTT and 8 U RNAsin inhibitor (Promega) as previously described ( ; ).

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  • 85
    Promega rac1 luciferase activity
    Molecular alterations attenuated by Smad7 ( a ) Immunostaining of NF-κB subunit p50, TGF-β1 and pSmad2 in irradiated tongue sections of WT mice adjacent to an ulcer and sections from the damaged area of K5.Smad7 mice, as well as in human samples from nonirradiated oral mucosa and radiation-induced mucositis. Dotted lines delineate epithelial-stromal boundary. Scale bar, 25 μm. ( b ) Quantification (mean ± s.d.) of nuclear NF-κB subunit p50 and pSmad2 in a . n = 3 or 4 per group. ( c ) Quantitative RT-PCR (mean ± s.d.) of TGF-β1 (normalized to keratin 5; n = 6 per group for day 0, n = 4 for day 7 and day 9, and n = 7 for day 10). ( d ) Quantification (mean ± s.d.) of human oral keratinocyte migration (see images in Supplementary Fig. 2 ). Scrambled, scrambled siRNA; siSmad7-1 and siSmad7-2, two different siRNAs specific to Smad7. n = 3 per group. ( e ) Western blot analysis of <t>Rac1</t> 72 h after Smad7 knockdown. The knockdown efficiency of siSmad7-1 and siSmad7-2 can be estimated from the blot. M: molecular markers; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( f ) Western blot analysis of total and activated (GTP-bound) Rac1 (GTP-Rac1) protein. ( g ) Effect of Rac1 knockdown on Smad7-mediated keratinocyte migration (see knockdown efficiency in Supplementary Fig. 3a and images in Supplementary Fig. 3d ). n = 3 per group. Data are presented as mean ± s.d. siRac-1 and siRac1-2 are two siRNAs specific for Rac1. * P
    Rac1 Luciferase Activity, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rac1 luciferase activity/product/Promega
    Average 85 stars, based on 1 article reviews
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    rac1 luciferase activity - by Bioz Stars, 2020-04
    85/100 stars
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    91
    Promega rnase h buffer
    <t>RNase</t> H activation by LNA/DNA and 2′- O -methyl gapmers. (Top) Results of RNase H assay. Full-length VR1 mRNA was incubated with a 5-fold excess of oligonucleotides in the presence of RNase H for 7.5 min at 37°C. Substrate (S) and cleavage products (P1 + P2) were separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, control; lane 2, DNA 1; lane 3, LNA 9; lane 4, LNA 12; lane 5, LNA 13; lane 6, LNA 14; lane 7, LNA 15; lane 8, OMe 1; Lane 9, OMe 2; lane 10, OMe 3. (Bottom) Quantitative evaluation of the gel. All values are averages and standard deviations of at least three independent experiments and were normalized.
    Rnase H Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase h buffer/product/Promega
    Average 91 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    rnase h buffer - by Bioz Stars, 2020-04
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    89
    Promega luciferase reporter constructs psicheck2 rac1 3 utr wt
    Rac1 and ICMT are direct targets of miR-100 (A) miR-100 and its putative binding sequences in the <t>3′-UTR</t> of Rac1 and ICMT. Mutations were generated in the complementary site that binds to the seed region of miR-100. (B) miR-100 overexpression suppressed the activity of renilla luciferase that carried the wild-type but not mutant 3′-UTR of Rac1 and ICMT. QGY-7703 cells were co-transfected with the indicated RNA duplex and <t>psiCHECK2</t> luciferase reporter plasmid containing wild-type or mutant 3′-UTR (indicated as WT or MUT on the X axis) of putative target genes. The values for the luciferase activity assays were from three independent experiments that were performed in duplicate. (C) Reintroduction of miR-100 reduced the endogenous level of Rac1 and ICMT proteins in HCC cell lines. Left and middle panels, QGY-7703 and SMMC-7721 cells without treatment (lane 1), treated with Lipofectamine RNAiMax (lane 2), or transfected with the indicated RNA duplex (lanes 3-4). Right panel, Hepa1-6 stable subclones. (D) Inhibition of miR-100 increased the protein levels of Rac1 and ICMT. Forty-eight hours after transfection with anti-miR-C or anti-miR-100, SMMC-7721 cells were analyzed by immunoblotting. For (C and D), the results were reproducible in three independent experiments. β-actin, internal control. (E and F) Mouse orthotopic xenografts of Hepa-miR-100 cells showed much lower Rac1 and ICMT expression than those of Hepa-Ctrl cells. (G) The level of miR-100 was inversely correlated with Rac1 expression in human HCC tissues. Rac1 expression was quantified based on immunohistochemical staining and miR-100 levels were detected by qPCR. Brown signal was considered as positive staining. Scale bar, 50 μm. * P
    Luciferase Reporter Constructs Psicheck2 Rac1 3 Utr Wt, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase reporter constructs psicheck2 rac1 3 utr wt/product/Promega
    Average 89 stars, based on 2 article reviews
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    99
    Promega tnf α
    Fig. 5. Inhibition of synthesis of the luciferase reporter gene by P56 in vivo . ( A ) Interaction of P48/Int-6 with P56 but not MP56. pCMV-P56 (lanes 1 and 3) or pCMV-MP56 (lanes 2 and 4) was co-transfected with pCMV-P48Fl into cells. At 48 h post-transfection, cells were harvested and whole-cell extracts were prepared. A 50 µg aliquot of total cell protein was subjected to gel electrophoresis followed by western blotting with P56 antibody (lanes 1 and 2). A 1 mg aliquot of cell protein was subjected to immunoprecipitation with anti-Flag-conjugated Sepharose beads followed by western blot analysis with P56 antibody (lanes 3 and 4). ( B ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (bar 4), pCMV-MP56 (bar 5), pCMV-DRBP76 (bar 3) or the empty expression vector (bars 1 and 2). After 48 h, cells were treated with <t>TNF-α</t> (bars 2–5) for 4 h. Cell extracts were made and luciferase activity was measured. The averages of results from three experiments are shown. ( C ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (+) or vector (–). At 48 h post-transfection, cells were treated with TNF-α for 4 h. Cells were harvested and total RNA was isolated for RNase protection assay. A 40 µg aliquot of total RNA was hybridized with 32 P-labeled Luc (370 bases) and γ-actin (140 bases) antisense RNA probes shown on the left as undigested probes. Following RNase digestion, the protected RNA probes were resolved in a 6% polyacrylamide, 8 M urea gel. Luciferase mRNA levels, shown on the right as protected probes, were quantified by phosphorimager and, after normalizing against the γ-actin mRNA levels, they were comparable in the two samples. ( D ) Cells were co-transfected with E-selectin-Luc and vector, pCMV-P56 or pCMV-MP56, as indicated. The experimental protocol was the same as in (B). ( E ) The same three cell extracts from (D) were western blotted with P56 antibody.
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    Molecular alterations attenuated by Smad7 ( a ) Immunostaining of NF-κB subunit p50, TGF-β1 and pSmad2 in irradiated tongue sections of WT mice adjacent to an ulcer and sections from the damaged area of K5.Smad7 mice, as well as in human samples from nonirradiated oral mucosa and radiation-induced mucositis. Dotted lines delineate epithelial-stromal boundary. Scale bar, 25 μm. ( b ) Quantification (mean ± s.d.) of nuclear NF-κB subunit p50 and pSmad2 in a . n = 3 or 4 per group. ( c ) Quantitative RT-PCR (mean ± s.d.) of TGF-β1 (normalized to keratin 5; n = 6 per group for day 0, n = 4 for day 7 and day 9, and n = 7 for day 10). ( d ) Quantification (mean ± s.d.) of human oral keratinocyte migration (see images in Supplementary Fig. 2 ). Scrambled, scrambled siRNA; siSmad7-1 and siSmad7-2, two different siRNAs specific to Smad7. n = 3 per group. ( e ) Western blot analysis of Rac1 72 h after Smad7 knockdown. The knockdown efficiency of siSmad7-1 and siSmad7-2 can be estimated from the blot. M: molecular markers; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( f ) Western blot analysis of total and activated (GTP-bound) Rac1 (GTP-Rac1) protein. ( g ) Effect of Rac1 knockdown on Smad7-mediated keratinocyte migration (see knockdown efficiency in Supplementary Fig. 3a and images in Supplementary Fig. 3d ). n = 3 per group. Data are presented as mean ± s.d. siRac-1 and siRac1-2 are two siRNAs specific for Rac1. * P

    Journal: Nature medicine

    Article Title: Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis

    doi: 10.1038/nm.3118

    Figure Lengend Snippet: Molecular alterations attenuated by Smad7 ( a ) Immunostaining of NF-κB subunit p50, TGF-β1 and pSmad2 in irradiated tongue sections of WT mice adjacent to an ulcer and sections from the damaged area of K5.Smad7 mice, as well as in human samples from nonirradiated oral mucosa and radiation-induced mucositis. Dotted lines delineate epithelial-stromal boundary. Scale bar, 25 μm. ( b ) Quantification (mean ± s.d.) of nuclear NF-κB subunit p50 and pSmad2 in a . n = 3 or 4 per group. ( c ) Quantitative RT-PCR (mean ± s.d.) of TGF-β1 (normalized to keratin 5; n = 6 per group for day 0, n = 4 for day 7 and day 9, and n = 7 for day 10). ( d ) Quantification (mean ± s.d.) of human oral keratinocyte migration (see images in Supplementary Fig. 2 ). Scrambled, scrambled siRNA; siSmad7-1 and siSmad7-2, two different siRNAs specific to Smad7. n = 3 per group. ( e ) Western blot analysis of Rac1 72 h after Smad7 knockdown. The knockdown efficiency of siSmad7-1 and siSmad7-2 can be estimated from the blot. M: molecular markers; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( f ) Western blot analysis of total and activated (GTP-bound) Rac1 (GTP-Rac1) protein. ( g ) Effect of Rac1 knockdown on Smad7-mediated keratinocyte migration (see knockdown efficiency in Supplementary Fig. 3a and images in Supplementary Fig. 3d ). n = 3 per group. Data are presented as mean ± s.d. siRac-1 and siRac1-2 are two siRNAs specific for Rac1. * P

    Article Snippet: We measured Rac1-luciferase activity with the Glomax machine (Promega) and expressed by the ratio of firefly activity to Renilla activity.

    Techniques: Immunostaining, Irradiation, Mouse Assay, Quantitative RT-PCR, Migration, Western Blot

    Smad7 leads to higher Rac1 expression by repressing individual Smad proteins and CtBP1 binding to the SBE of the Rac1 promoter (a ) Rac1 mRNA levels (mean ± s.d.) in keratinocytes from WT and Smad7 transgenic mice. n = 4 per group. ( b ) Western blot analysis of GTP-bound Rac1 (GTP-Rac1) and total Rac1 in WT and Smad7 transgenic keratinocytes. Smad7 protein levels were determined by re-probing the tubulin western blot with an antibody to Smad7 (see an additional western blot and quantification in Supplementary Fig. 4a, b ). (c) The amount of Rac1 protein after knocking down Smad2, Smad3 or Smad4 individually in human keratinocytes (See Supplementary Fig. 4c–e for Smad knockdown efficiencies). siSamd2-4, siRNAs specific for Smad2-4. ( d ) ChIP assay for Smad2, Smad3, Smad4, and Smad7 binding to the SBE -1.5 kb site of the Rac1 promoter in keratinocytes from WT and Smad7 transgenic mice. ( e ) Rac1 luciferase reporter assay in mouse keratinocytes. n = 6 per group. siSmad7, siRNA specific for Smad7; Rac1-SBE, the SBE-1.5 kb site of the Rac1 promoter. Data are the mean ± s.d. ( f ) Activities (mean ± s.d.) of Rac1- luc reporters containing SBE (Rac1-SBE) or mutant SBE (Mut Rac1-SBE) in keratinocytes from WT and Smad7 transgenic mice. n = 6 per group. ( g ) Images of ChIP assays of CtBP1 binding to the SBE-1.5 kb site of the Rac1 promoter in keratinocytes from WT or K5.Smad7 mice. ( h ) ChIP-quantitative PCR (mean ± s.d.) of CtBP1 binding to the SBE in g in keratinocytes from WT and Smad7 transgenic mice. n = 4 per group. * P

    Journal: Nature medicine

    Article Title: Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis

    doi: 10.1038/nm.3118

    Figure Lengend Snippet: Smad7 leads to higher Rac1 expression by repressing individual Smad proteins and CtBP1 binding to the SBE of the Rac1 promoter (a ) Rac1 mRNA levels (mean ± s.d.) in keratinocytes from WT and Smad7 transgenic mice. n = 4 per group. ( b ) Western blot analysis of GTP-bound Rac1 (GTP-Rac1) and total Rac1 in WT and Smad7 transgenic keratinocytes. Smad7 protein levels were determined by re-probing the tubulin western blot with an antibody to Smad7 (see an additional western blot and quantification in Supplementary Fig. 4a, b ). (c) The amount of Rac1 protein after knocking down Smad2, Smad3 or Smad4 individually in human keratinocytes (See Supplementary Fig. 4c–e for Smad knockdown efficiencies). siSamd2-4, siRNAs specific for Smad2-4. ( d ) ChIP assay for Smad2, Smad3, Smad4, and Smad7 binding to the SBE -1.5 kb site of the Rac1 promoter in keratinocytes from WT and Smad7 transgenic mice. ( e ) Rac1 luciferase reporter assay in mouse keratinocytes. n = 6 per group. siSmad7, siRNA specific for Smad7; Rac1-SBE, the SBE-1.5 kb site of the Rac1 promoter. Data are the mean ± s.d. ( f ) Activities (mean ± s.d.) of Rac1- luc reporters containing SBE (Rac1-SBE) or mutant SBE (Mut Rac1-SBE) in keratinocytes from WT and Smad7 transgenic mice. n = 6 per group. ( g ) Images of ChIP assays of CtBP1 binding to the SBE-1.5 kb site of the Rac1 promoter in keratinocytes from WT or K5.Smad7 mice. ( h ) ChIP-quantitative PCR (mean ± s.d.) of CtBP1 binding to the SBE in g in keratinocytes from WT and Smad7 transgenic mice. n = 4 per group. * P

    Article Snippet: We measured Rac1-luciferase activity with the Glomax machine (Promega) and expressed by the ratio of firefly activity to Renilla activity.

    Techniques: Expressing, Binding Assay, Transgenic Assay, Mouse Assay, Western Blot, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Mutagenesis, Real-time Polymerase Chain Reaction

    CtBP1-associated Rac1 repression contributes to the inhibition of keratinocyte migration ( a ) Western blot analysis of Rac1 protein after knockdown of CtBP1 in human oral keratinocytes. siCtBP-1 and siCtBP1-2 are two different siRNAs specific for CtBP1. ( b ) SBE-containing Rac1 -luc reporter activity (mean ± s.d.). n = 6 per group. ( c ) Effect of CtBP1 knockdown on human oral keratinocyte migration (mean ± s.d.). n = 3 per group. ( d ) Immunostaining of CtBP1 in irradiated sections adjacent to the ulcer (WT) or from the damaged area (K5.Smad7). Dotted lines denote the basement membrane. Scale bar, 50 μm. ( e ) Immunostaining of CtBP1 in nonirradiated oral mucosa and radiation-induced oral mucositis in human specimens. Dotted lines denote the basement membrane. Scale bar, 50 μm. ( f ) Quantification of nuclear CtBP1-positive cells (mean ± s.d.) in d and e. n = 3 or 4 per group. ( g ) Quantitative RT-PCR (mean ± s.d.) for CtBP1 (normalized to keratin 5). n = 6 per group for day 0, n = 4 for day 7 and day 9, and n = 7 for day 10. * P

    Journal: Nature medicine

    Article Title: Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis

    doi: 10.1038/nm.3118

    Figure Lengend Snippet: CtBP1-associated Rac1 repression contributes to the inhibition of keratinocyte migration ( a ) Western blot analysis of Rac1 protein after knockdown of CtBP1 in human oral keratinocytes. siCtBP-1 and siCtBP1-2 are two different siRNAs specific for CtBP1. ( b ) SBE-containing Rac1 -luc reporter activity (mean ± s.d.). n = 6 per group. ( c ) Effect of CtBP1 knockdown on human oral keratinocyte migration (mean ± s.d.). n = 3 per group. ( d ) Immunostaining of CtBP1 in irradiated sections adjacent to the ulcer (WT) or from the damaged area (K5.Smad7). Dotted lines denote the basement membrane. Scale bar, 50 μm. ( e ) Immunostaining of CtBP1 in nonirradiated oral mucosa and radiation-induced oral mucositis in human specimens. Dotted lines denote the basement membrane. Scale bar, 50 μm. ( f ) Quantification of nuclear CtBP1-positive cells (mean ± s.d.) in d and e. n = 3 or 4 per group. ( g ) Quantitative RT-PCR (mean ± s.d.) for CtBP1 (normalized to keratin 5). n = 6 per group for day 0, n = 4 for day 7 and day 9, and n = 7 for day 10. * P

    Article Snippet: We measured Rac1-luciferase activity with the Glomax machine (Promega) and expressed by the ratio of firefly activity to Renilla activity.

    Techniques: Inhibition, Migration, Western Blot, Activity Assay, Immunostaining, Irradiation, Quantitative RT-PCR

    Tat-Smad7 treatment on oral mucositis ( a ) Dot blot graph (mean ± s.e.m.) of ulcer sizes measured on day 10 after initiation of 8 Gy x 3 radiation. Glycerol is 50% glycerol/PBS. ( b ) H E staining of an open ulcer in palifermin-treated but not Tat-Smad7-treated mucosa (top) and a comparison of epithelial thickness between palifermin-treated and Tat-Smad7-treated mucosa (bottom). Dotted lines delineate the basement membrane, and the vertical lines highlight the ulcer boundary. Scale bar, 50 μm. ( c ) Tat-Smad7 treatment in 20 Gy-induced oral mucositis after ulcers healed. V5 immunostaining visualizes Tat-Smad7 in oral epithelia (sections are away from the damaged regions); K14 immunostaining was used as counterstain. Green in muscle cells represents autofluorescence. Dotted lines delineate the basement membrane. Scale bar, 25 μm. ( d ) Rac1 western blot analysis of Tat-Smad7-treated mouse tongues on day 10 after initiation of 8 Gy x 3 radiation. M, molecular markers. ( e ) Rac1 western blot analysis of Tat-Smad7-treated normal human oral keratinocytes 48 h after treatment. Control, PBS. ( f ) Effect of Tat-Smad7 treatment on oral human keratinocyte migration (NOK-SI, see images in Supplementary Fig. 7a ). n = 4 per group. Data are the mean ± s.d. ( g ) Survival curves (mean ± s.d.) of NOK-SI keratinocytes and SCC lines (Cal27 and MSK921) with or without Tat-Smad7 treatment. n = 4 per group for each radiation dose. * P

    Journal: Nature medicine

    Article Title: Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis

    doi: 10.1038/nm.3118

    Figure Lengend Snippet: Tat-Smad7 treatment on oral mucositis ( a ) Dot blot graph (mean ± s.e.m.) of ulcer sizes measured on day 10 after initiation of 8 Gy x 3 radiation. Glycerol is 50% glycerol/PBS. ( b ) H E staining of an open ulcer in palifermin-treated but not Tat-Smad7-treated mucosa (top) and a comparison of epithelial thickness between palifermin-treated and Tat-Smad7-treated mucosa (bottom). Dotted lines delineate the basement membrane, and the vertical lines highlight the ulcer boundary. Scale bar, 50 μm. ( c ) Tat-Smad7 treatment in 20 Gy-induced oral mucositis after ulcers healed. V5 immunostaining visualizes Tat-Smad7 in oral epithelia (sections are away from the damaged regions); K14 immunostaining was used as counterstain. Green in muscle cells represents autofluorescence. Dotted lines delineate the basement membrane. Scale bar, 25 μm. ( d ) Rac1 western blot analysis of Tat-Smad7-treated mouse tongues on day 10 after initiation of 8 Gy x 3 radiation. M, molecular markers. ( e ) Rac1 western blot analysis of Tat-Smad7-treated normal human oral keratinocytes 48 h after treatment. Control, PBS. ( f ) Effect of Tat-Smad7 treatment on oral human keratinocyte migration (NOK-SI, see images in Supplementary Fig. 7a ). n = 4 per group. Data are the mean ± s.d. ( g ) Survival curves (mean ± s.d.) of NOK-SI keratinocytes and SCC lines (Cal27 and MSK921) with or without Tat-Smad7 treatment. n = 4 per group for each radiation dose. * P

    Article Snippet: We measured Rac1-luciferase activity with the Glomax machine (Promega) and expressed by the ratio of firefly activity to Renilla activity.

    Techniques: Dot Blot, Staining, Immunostaining, Western Blot, Migration

    RNase H activation by LNA/DNA and 2′- O -methyl gapmers. (Top) Results of RNase H assay. Full-length VR1 mRNA was incubated with a 5-fold excess of oligonucleotides in the presence of RNase H for 7.5 min at 37°C. Substrate (S) and cleavage products (P1 + P2) were separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, control; lane 2, DNA 1; lane 3, LNA 9; lane 4, LNA 12; lane 5, LNA 13; lane 6, LNA 14; lane 7, LNA 15; lane 8, OMe 1; Lane 9, OMe 2; lane 10, OMe 3. (Bottom) Quantitative evaluation of the gel. All values are averages and standard deviations of at least three independent experiments and were normalized.

    Journal: Nucleic Acids Research

    Article Title: Design of antisense oligonucleotides stabilized by locked nucleic acids

    doi:

    Figure Lengend Snippet: RNase H activation by LNA/DNA and 2′- O -methyl gapmers. (Top) Results of RNase H assay. Full-length VR1 mRNA was incubated with a 5-fold excess of oligonucleotides in the presence of RNase H for 7.5 min at 37°C. Substrate (S) and cleavage products (P1 + P2) were separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, control; lane 2, DNA 1; lane 3, LNA 9; lane 4, LNA 12; lane 5, LNA 13; lane 6, LNA 14; lane 7, LNA 15; lane 8, OMe 1; Lane 9, OMe 2; lane 10, OMe 3. (Bottom) Quantitative evaluation of the gel. All values are averages and standard deviations of at least three independent experiments and were normalized.

    Article Snippet: The standard RNase H assay was performed as described previously ( ): 100 nM VR1 mRNA were incubated with a 5-fold excess of an antisense oligonucleotide in a total volume of 10 µl in RNase H buffer (40 mM Tris–HCl pH 7.2, 4 mM MgCl2 , 1 mM DTT, 150 mM NaCl and 1.25 U/µl RNasin; Promega, Madison, WI) for 7.5 min at 37°C in the presence of 0.4 U E.coli RNase H (Promega).

    Techniques: Activation Assay, Rnase H Assay, Incubation, Agarose Gel Electrophoresis, Staining

    RNase H assays with a short RNA target. Lane 1, one base ladder produced by alkaline hydrolysis; lane 2, cleavage products after incubation with RNase T1; lane 3, 18mer RNA; lanes 4 and 5, products after incubation of target RNA with all DNA antisense oligonucleotide (DNA 1) in the presence of RNase H after 10 and 30 min, respectively; lanes 6 and 7, products after incubation of target RNA with end-block LNA/DNA/LNA oligonucleotide (LNA 17) in the presence of RNase H after 10 and 30 min; lanes 8 and 9, products after incubation of target RNA with LNA/DNA mixmer (LNA 9) in the presence of RNase H after 10 and 30 min; lanes 10 and 11, products after incubation of target RNA with phosphorothioate in the presence of RNase H after 10 and 30 min. For further details see text.

    Journal: Nucleic Acids Research

    Article Title: Design of antisense oligonucleotides stabilized by locked nucleic acids

    doi:

    Figure Lengend Snippet: RNase H assays with a short RNA target. Lane 1, one base ladder produced by alkaline hydrolysis; lane 2, cleavage products after incubation with RNase T1; lane 3, 18mer RNA; lanes 4 and 5, products after incubation of target RNA with all DNA antisense oligonucleotide (DNA 1) in the presence of RNase H after 10 and 30 min, respectively; lanes 6 and 7, products after incubation of target RNA with end-block LNA/DNA/LNA oligonucleotide (LNA 17) in the presence of RNase H after 10 and 30 min; lanes 8 and 9, products after incubation of target RNA with LNA/DNA mixmer (LNA 9) in the presence of RNase H after 10 and 30 min; lanes 10 and 11, products after incubation of target RNA with phosphorothioate in the presence of RNase H after 10 and 30 min. For further details see text.

    Article Snippet: The standard RNase H assay was performed as described previously ( ): 100 nM VR1 mRNA were incubated with a 5-fold excess of an antisense oligonucleotide in a total volume of 10 µl in RNase H buffer (40 mM Tris–HCl pH 7.2, 4 mM MgCl2 , 1 mM DTT, 150 mM NaCl and 1.25 U/µl RNasin; Promega, Madison, WI) for 7.5 min at 37°C in the presence of 0.4 U E.coli RNase H (Promega).

    Techniques: Produced, Incubation, Blocking Assay

    Rac1 and ICMT are direct targets of miR-100 (A) miR-100 and its putative binding sequences in the 3′-UTR of Rac1 and ICMT. Mutations were generated in the complementary site that binds to the seed region of miR-100. (B) miR-100 overexpression suppressed the activity of renilla luciferase that carried the wild-type but not mutant 3′-UTR of Rac1 and ICMT. QGY-7703 cells were co-transfected with the indicated RNA duplex and psiCHECK2 luciferase reporter plasmid containing wild-type or mutant 3′-UTR (indicated as WT or MUT on the X axis) of putative target genes. The values for the luciferase activity assays were from three independent experiments that were performed in duplicate. (C) Reintroduction of miR-100 reduced the endogenous level of Rac1 and ICMT proteins in HCC cell lines. Left and middle panels, QGY-7703 and SMMC-7721 cells without treatment (lane 1), treated with Lipofectamine RNAiMax (lane 2), or transfected with the indicated RNA duplex (lanes 3-4). Right panel, Hepa1-6 stable subclones. (D) Inhibition of miR-100 increased the protein levels of Rac1 and ICMT. Forty-eight hours after transfection with anti-miR-C or anti-miR-100, SMMC-7721 cells were analyzed by immunoblotting. For (C and D), the results were reproducible in three independent experiments. β-actin, internal control. (E and F) Mouse orthotopic xenografts of Hepa-miR-100 cells showed much lower Rac1 and ICMT expression than those of Hepa-Ctrl cells. (G) The level of miR-100 was inversely correlated with Rac1 expression in human HCC tissues. Rac1 expression was quantified based on immunohistochemical staining and miR-100 levels were detected by qPCR. Brown signal was considered as positive staining. Scale bar, 50 μm. * P

    Journal: Oncotarget

    Article Title: Downregulation of microRNA-100 enhances the ICMT-Rac1 signaling and promotes metastasis of hepatocellular carcinoma cells

    doi:

    Figure Lengend Snippet: Rac1 and ICMT are direct targets of miR-100 (A) miR-100 and its putative binding sequences in the 3′-UTR of Rac1 and ICMT. Mutations were generated in the complementary site that binds to the seed region of miR-100. (B) miR-100 overexpression suppressed the activity of renilla luciferase that carried the wild-type but not mutant 3′-UTR of Rac1 and ICMT. QGY-7703 cells were co-transfected with the indicated RNA duplex and psiCHECK2 luciferase reporter plasmid containing wild-type or mutant 3′-UTR (indicated as WT or MUT on the X axis) of putative target genes. The values for the luciferase activity assays were from three independent experiments that were performed in duplicate. (C) Reintroduction of miR-100 reduced the endogenous level of Rac1 and ICMT proteins in HCC cell lines. Left and middle panels, QGY-7703 and SMMC-7721 cells without treatment (lane 1), treated with Lipofectamine RNAiMax (lane 2), or transfected with the indicated RNA duplex (lanes 3-4). Right panel, Hepa1-6 stable subclones. (D) Inhibition of miR-100 increased the protein levels of Rac1 and ICMT. Forty-eight hours after transfection with anti-miR-C or anti-miR-100, SMMC-7721 cells were analyzed by immunoblotting. For (C and D), the results were reproducible in three independent experiments. β-actin, internal control. (E and F) Mouse orthotopic xenografts of Hepa-miR-100 cells showed much lower Rac1 and ICMT expression than those of Hepa-Ctrl cells. (G) The level of miR-100 was inversely correlated with Rac1 expression in human HCC tissues. Rac1 expression was quantified based on immunohistochemical staining and miR-100 levels were detected by qPCR. Brown signal was considered as positive staining. Scale bar, 50 μm. * P

    Article Snippet: To create luciferase reporter constructs psiCHECK2-Rac1-3′UTR-WT and psiCHECK2-ICMT-3′UTR-WT, a wild-type 3′-UTR segments of human Rac1 (461 bp) or ICMT (271 bp) mRNA that contained putative binding sites for miR-100 were inserted downstream the renilla luciferase coding region in psiCHECK2 (Promega, WI, USA).

    Techniques: Binding Assay, Generated, Over Expression, Activity Assay, Luciferase, Mutagenesis, Transfection, Plasmid Preparation, Inhibition, Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

    Fig. 5. Inhibition of synthesis of the luciferase reporter gene by P56 in vivo . ( A ) Interaction of P48/Int-6 with P56 but not MP56. pCMV-P56 (lanes 1 and 3) or pCMV-MP56 (lanes 2 and 4) was co-transfected with pCMV-P48Fl into cells. At 48 h post-transfection, cells were harvested and whole-cell extracts were prepared. A 50 µg aliquot of total cell protein was subjected to gel electrophoresis followed by western blotting with P56 antibody (lanes 1 and 2). A 1 mg aliquot of cell protein was subjected to immunoprecipitation with anti-Flag-conjugated Sepharose beads followed by western blot analysis with P56 antibody (lanes 3 and 4). ( B ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (bar 4), pCMV-MP56 (bar 5), pCMV-DRBP76 (bar 3) or the empty expression vector (bars 1 and 2). After 48 h, cells were treated with TNF-α (bars 2–5) for 4 h. Cell extracts were made and luciferase activity was measured. The averages of results from three experiments are shown. ( C ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (+) or vector (–). At 48 h post-transfection, cells were treated with TNF-α for 4 h. Cells were harvested and total RNA was isolated for RNase protection assay. A 40 µg aliquot of total RNA was hybridized with 32 P-labeled Luc (370 bases) and γ-actin (140 bases) antisense RNA probes shown on the left as undigested probes. Following RNase digestion, the protected RNA probes were resolved in a 6% polyacrylamide, 8 M urea gel. Luciferase mRNA levels, shown on the right as protected probes, were quantified by phosphorimager and, after normalizing against the γ-actin mRNA levels, they were comparable in the two samples. ( D ) Cells were co-transfected with E-selectin-Luc and vector, pCMV-P56 or pCMV-MP56, as indicated. The experimental protocol was the same as in (B). ( E ) The same three cell extracts from (D) were western blotted with P56 antibody.

    Journal: The EMBO Journal

    Article Title: A new pathway of translational regulation mediated by eukaryotic initiation factor 3

    doi: 10.1093/emboj/19.24.6891

    Figure Lengend Snippet: Fig. 5. Inhibition of synthesis of the luciferase reporter gene by P56 in vivo . ( A ) Interaction of P48/Int-6 with P56 but not MP56. pCMV-P56 (lanes 1 and 3) or pCMV-MP56 (lanes 2 and 4) was co-transfected with pCMV-P48Fl into cells. At 48 h post-transfection, cells were harvested and whole-cell extracts were prepared. A 50 µg aliquot of total cell protein was subjected to gel electrophoresis followed by western blotting with P56 antibody (lanes 1 and 2). A 1 mg aliquot of cell protein was subjected to immunoprecipitation with anti-Flag-conjugated Sepharose beads followed by western blot analysis with P56 antibody (lanes 3 and 4). ( B ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (bar 4), pCMV-MP56 (bar 5), pCMV-DRBP76 (bar 3) or the empty expression vector (bars 1 and 2). After 48 h, cells were treated with TNF-α (bars 2–5) for 4 h. Cell extracts were made and luciferase activity was measured. The averages of results from three experiments are shown. ( C ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (+) or vector (–). At 48 h post-transfection, cells were treated with TNF-α for 4 h. Cells were harvested and total RNA was isolated for RNase protection assay. A 40 µg aliquot of total RNA was hybridized with 32 P-labeled Luc (370 bases) and γ-actin (140 bases) antisense RNA probes shown on the left as undigested probes. Following RNase digestion, the protected RNA probes were resolved in a 6% polyacrylamide, 8 M urea gel. Luciferase mRNA levels, shown on the right as protected probes, were quantified by phosphorimager and, after normalizing against the γ-actin mRNA levels, they were comparable in the two samples. ( D ) Cells were co-transfected with E-selectin-Luc and vector, pCMV-P56 or pCMV-MP56, as indicated. The experimental protocol was the same as in (B). ( E ) The same three cell extracts from (D) were western blotted with P56 antibody.

    Article Snippet: After 48 h, cells were induced with 20 ng/ml TNF-α for 4 h. Cell extracts were prepared in 1× reporter lysis buffer (Promega) and luciferase activity was measured using the luciferase reporter gene assay kit (Promega).

    Techniques: Inhibition, Luciferase, In Vivo, Transfection, Nucleic Acid Electrophoresis, Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation, Activity Assay, Isolation, Rnase Protection Assay, Labeling