Structured Review

Promega rnase inhibitor
RRM1 but not RRM2 is co-purified with endogenous <t>RNA</t> from E. coli lysates. (A) Schematic representation of a TDP-43 domain structure. (B – D) RRM1 (solid curve) and RRM2 (broken curve) were overexpressed in E. coli BL21(DE3) and purified with Ni 2+ -affinity chromatography. ( B ) Crude lysates were loaded on the Ni 2+ -affinity resins, which were washed with a TN-low buffer. The bound proteins were eluted from the resins and examined spectroscopically. ( C , D ) Resins incubated with crude lysates were washed with a TN-high buffer, and the bound proteins were eluted. Fractions obtained in ( C ) the wash and ( D ) the elute steps were examined spectroscopically. (E) The fraction washed out by a TN-high buffer from the resins incubated with RRM1 crude lysates was treated with either DNase or <t>RNase</t> and analyzed with urea-PAGE.
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Images

1) Product Images from "A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43"

Article Title: A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43

Journal: Scientific Reports

doi: 10.1038/srep20576

RRM1 but not RRM2 is co-purified with endogenous RNA from E. coli lysates. (A) Schematic representation of a TDP-43 domain structure. (B – D) RRM1 (solid curve) and RRM2 (broken curve) were overexpressed in E. coli BL21(DE3) and purified with Ni 2+ -affinity chromatography. ( B ) Crude lysates were loaded on the Ni 2+ -affinity resins, which were washed with a TN-low buffer. The bound proteins were eluted from the resins and examined spectroscopically. ( C , D ) Resins incubated with crude lysates were washed with a TN-high buffer, and the bound proteins were eluted. Fractions obtained in ( C ) the wash and ( D ) the elute steps were examined spectroscopically. (E) The fraction washed out by a TN-high buffer from the resins incubated with RRM1 crude lysates was treated with either DNase or RNase and analyzed with urea-PAGE.
Figure Legend Snippet: RRM1 but not RRM2 is co-purified with endogenous RNA from E. coli lysates. (A) Schematic representation of a TDP-43 domain structure. (B – D) RRM1 (solid curve) and RRM2 (broken curve) were overexpressed in E. coli BL21(DE3) and purified with Ni 2+ -affinity chromatography. ( B ) Crude lysates were loaded on the Ni 2+ -affinity resins, which were washed with a TN-low buffer. The bound proteins were eluted from the resins and examined spectroscopically. ( C , D ) Resins incubated with crude lysates were washed with a TN-high buffer, and the bound proteins were eluted. Fractions obtained in ( C ) the wash and ( D ) the elute steps were examined spectroscopically. (E) The fraction washed out by a TN-high buffer from the resins incubated with RRM1 crude lysates was treated with either DNase or RNase and analyzed with urea-PAGE.

Techniques Used: Purification, Affinity Chromatography, Incubation, Polyacrylamide Gel Electrophoresis

2) Product Images from "hnRNPM induces translation switch under hypoxia to promote colon cancer development"

Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2019.02.059

Hypoxia enhances interaction between hnRNPM and FGF9 mRNA and promotes translation. (a) Gel image of FGF9 RT-PCR from hnRNPM-immunoprecipitation assay. RNase treatment was performed to indicate that PCR products were amplified from RNA origin. (b) The quantitative measurement of FGF9 TaqMan assays from hnRNPM-immunoprecipitation assay. The relative FGF9 mRNA level was shown as the amount measured in hypoxia normalized to the amount in normoxia. (c) Representative Western blot image shows levels of hnRNPM pulldown by full length FGF9 RNA 5’UTR (FL) or just IRES sequences from cells cultured under normoxia or hypoxia conditions. (d) Immunofluorescent images showed cellular distribution of hnRNPM (red) in HEK293 cells cultured in normoxia or hypoxia conditions. The arrowheads indicate cytosolic hnRNPM. Nuclei were marked with DAPI (blue). (e) Number of cytosolic hnRNPM positive cells in normoxia or hypoxia (A total of 100 cells per independent experiment were counted). (f) Representative Western blot image shows the expression of hnRNPM in the cytosolic and nuclear fractions of indicated cells treated with normoxia or hypoxia for 9 h ( n = 3 independent experiments). The levels of α-tubulin and lamin A/C are used as markers for cytosolic and nuclear fractions, respectively. (g) The 18S and 28S rRNAs were resolved on a 1% formaldehyde/agarose gel and visualized by ethidium bromide staining (Upper panel). Immunoblotting analysis of gradient fractions was performed using antibodies against hnRNPM, PTBP1, eIF3, eIF4A1, and ribosomal protein RPS6 (Lower panel). (h) The quantitative measurement of mRNAs from hnRNPM-knockdown cytoplasmic extracts. Bar graphs represent the proportion of actively translated mRNA of FGF9 (left panel) and β-actin (right panel) from 3 independent experiments. ⁎ P
Figure Legend Snippet: Hypoxia enhances interaction between hnRNPM and FGF9 mRNA and promotes translation. (a) Gel image of FGF9 RT-PCR from hnRNPM-immunoprecipitation assay. RNase treatment was performed to indicate that PCR products were amplified from RNA origin. (b) The quantitative measurement of FGF9 TaqMan assays from hnRNPM-immunoprecipitation assay. The relative FGF9 mRNA level was shown as the amount measured in hypoxia normalized to the amount in normoxia. (c) Representative Western blot image shows levels of hnRNPM pulldown by full length FGF9 RNA 5’UTR (FL) or just IRES sequences from cells cultured under normoxia or hypoxia conditions. (d) Immunofluorescent images showed cellular distribution of hnRNPM (red) in HEK293 cells cultured in normoxia or hypoxia conditions. The arrowheads indicate cytosolic hnRNPM. Nuclei were marked with DAPI (blue). (e) Number of cytosolic hnRNPM positive cells in normoxia or hypoxia (A total of 100 cells per independent experiment were counted). (f) Representative Western blot image shows the expression of hnRNPM in the cytosolic and nuclear fractions of indicated cells treated with normoxia or hypoxia for 9 h ( n = 3 independent experiments). The levels of α-tubulin and lamin A/C are used as markers for cytosolic and nuclear fractions, respectively. (g) The 18S and 28S rRNAs were resolved on a 1% formaldehyde/agarose gel and visualized by ethidium bromide staining (Upper panel). Immunoblotting analysis of gradient fractions was performed using antibodies against hnRNPM, PTBP1, eIF3, eIF4A1, and ribosomal protein RPS6 (Lower panel). (h) The quantitative measurement of mRNAs from hnRNPM-knockdown cytoplasmic extracts. Bar graphs represent the proportion of actively translated mRNA of FGF9 (left panel) and β-actin (right panel) from 3 independent experiments. ⁎ P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Western Blot, Cell Culture, Expressing, Agarose Gel Electrophoresis, Staining

3) Product Images from "Expression of Vascular Endothelial Growth Factor A During Ligand-Induced Down-Regulation of Luteinizing Hormone Receptor in the Ovary ☆"

Article Title: Expression of Vascular Endothelial Growth Factor A During Ligand-Induced Down-Regulation of Luteinizing Hormone Receptor in the Ovary ☆

Journal: Molecular and cellular endocrinology

doi: 10.1016/j.mce.2010.06.015

3β-HSD staining of purified luteal cells. Isolated ovaries were minced and incubated with collagenase, RNase free DNase-I, and RNase inhibitor. Dispersed cells were purified based on size as described in Materials and Methods. An aliquot of the cells was incubated for 1h at 37 C with staining solution containing 100 μg/ml dehydroepiandrosterone and 100 μg/ml pregnenolone as substrates (A). Cells incubated with staining solution without substrates are shown in (B).
Figure Legend Snippet: 3β-HSD staining of purified luteal cells. Isolated ovaries were minced and incubated with collagenase, RNase free DNase-I, and RNase inhibitor. Dispersed cells were purified based on size as described in Materials and Methods. An aliquot of the cells was incubated for 1h at 37 C with staining solution containing 100 μg/ml dehydroepiandrosterone and 100 μg/ml pregnenolone as substrates (A). Cells incubated with staining solution without substrates are shown in (B).

Techniques Used: Staining, Purification, Isolation, Incubation

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

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks1132

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

Techniques Used: Activity Assay, Western Blot

Related Articles

Clone Assay:

Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus
Article Snippet: Paragraph title: nifH cloning and RT–PCR ... RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above.

Centrifugation:

Article Title: Activating frataxin expression by repeat-targeted nucleic acids
Article Snippet: Pelleted nuclei were washed 3 × with ice-cold hypotonic lysis buffer followed by 5 min incubation on ice, pipetting and vortexing, then centrifugation at 4 °C at 100g for 2 min. .. Nuclei were resuspended in ice-cold nuclear lysis buffer (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 3 mM MgCl2 , 0.3% NP-40 and 10% glycerol) supplemented with 1% Protease Inhibitor Cocktail (Roche), 5 μl ml−1 RNaseIn (Promega) at a final of 0.5 ml per 75 mg of original wet cell pellet weight.

Article Title: Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing *
Article Snippet: HEK 293 cells were lysed with immunoprecipitation buffer containing either 0.05% RNasin (Promega) or 0.2 μg/μl RNase A (Roche Applied Science), incubated for 10 min on ice, and centrifuged for 10 min. .. The supernatant was loaded on top of a 10–40% sucrose gradient, and centrifugation was performed for 16 h at 23,000 rpm and 4 °C in an ultracentrifuge (Sorvall WX ultra 100, Thermo Scientific).

Amplification:

Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
Article Snippet: .. Total RNA was isolated with the Qiagen RNeasy mini kit (Qiagen, Inc.) and subjected to amplification-grade DNase I (Gibco BRL) treatment in the presence of RNasin (Promega Corporation). cDNA was generated by using oligonucleotide 728 (Fig. ) and treated with RNase H (Gibco BRL), prior to being used as a template for subsequent PCR. .. RT-PCR products were obtained from intergenic regions between lbpB :: lbpA and lbpA :: orf3 (see Fig. , lanes 2 and 4, respectively).

Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus
Article Snippet: .. RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above. .. A second round amplification was done using nifH1 and nifH2 primers and Taq polymerase mix from Qiagen (Valencia, CA, USA) as described above.

DNA Synthesis:

Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus
Article Snippet: .. RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above. .. A second round amplification was done using nifH1 and nifH2 primers and Taq polymerase mix from Qiagen (Valencia, CA, USA) as described above.

Autoradiography:

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: Briefly, a duplex unwinding reaction was performed in a 16-μl volume with 30 mM Tris-HCl (pH 7.5), 3 mM MgCl2 , 10 mM DTT, 5 mM ATP, 1 μl of RNasin (Promega), 1 nM VP6 protein, and 1 nM 32 P-labeled RNA duplex. .. The reaction mixture was incubated at 37°C for 30 min, and the reaction was terminated by the addition of 4 μl of 5× SDS-PAGE sample buffer and analyzed by 12% (wt/vol) nondenaturing PAGE in Tris-glycine buffer followed by autoradiography as described previously ( ).

Construct:

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: The oligos were constructed by using standard methods ( ) and are the following: 5′-AGAGAGAGAGGUUGAGAGAGAGAGAGUUUGAGAGAGAGAG-3′ (40-mer, template strand) and 5′-CAAACUCUCUCUCUCUCAAAAAAAAA-3′ (26-mer, release strand). .. Unlabeled nucleotides were removed by using Sephadex G-50 spin columns (Pharmacia) and stored at −20°C in sterile H2 0 containing 0.25 U of RNasin (Promega) per μl.

Incubation:

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: Briefly, a duplex unwinding reaction was performed in a 16-μl volume with 30 mM Tris-HCl (pH 7.5), 3 mM MgCl2 , 10 mM DTT, 5 mM ATP, 1 μl of RNasin (Promega), 1 nM VP6 protein, and 1 nM 32 P-labeled RNA duplex. .. The reaction mixture was incubated at 37°C for 30 min, and the reaction was terminated by the addition of 4 μl of 5× SDS-PAGE sample buffer and analyzed by 12% (wt/vol) nondenaturing PAGE in Tris-glycine buffer followed by autoradiography as described previously ( ).

Article Title: Activating frataxin expression by repeat-targeted nucleic acids
Article Snippet: Pelleted nuclei were washed 3 × with ice-cold hypotonic lysis buffer followed by 5 min incubation on ice, pipetting and vortexing, then centrifugation at 4 °C at 100g for 2 min. .. Nuclei were resuspended in ice-cold nuclear lysis buffer (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 3 mM MgCl2 , 0.3% NP-40 and 10% glycerol) supplemented with 1% Protease Inhibitor Cocktail (Roche), 5 μl ml−1 RNaseIn (Promega) at a final of 0.5 ml per 75 mg of original wet cell pellet weight.

Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development
Article Snippet: The RIP was conducted under the use of RNase inhibitor to protect RNA from RNase degradation (Recombinant RNasin® Ribonuclease Inhibitor, Promega Cat. No. N2515). .. In brief, cells were lysed and incubated with hnRNPM antibody (sc-20,002; 5 μg) or control IgG (SC-2025; 5 μg) conjugated with magnetic beads (50 μl) for 4 h. The protein-RNA complexes were immunoprecipitated and magnetically separated.

Article Title: Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing *
Article Snippet: .. HEK 293 cells were lysed with immunoprecipitation buffer containing either 0.05% RNasin (Promega) or 0.2 μg/μl RNase A (Roche Applied Science), incubated for 10 min on ice, and centrifuged for 10 min. .. The supernatant was loaded on top of a 10–40% sucrose gradient, and centrifugation was performed for 16 h at 23,000 rpm and 4 °C in an ultracentrifuge (Sorvall WX ultra 100, Thermo Scientific).

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis
Article Snippet: .. For UV-cross-linking experiments, equal moles of wild-type and mutant GST-GRSF-1 were incubated with 106 dpm of radiolabeled NP 5′ UTR RNA in buffer containing 5 mM HEPES (pH 7.6), 25 mM KCl, 2 mM MgCl2 , 5% glycerol, 100 mM NaCl, 2 mM dithiothreitol, and 20 U of RNasin (Promega) at 30°C for 15 min. .. Samples were then exposed to UV light (1.0 J) in a Stratalinker (Stratagene), followed by incubation with 10 mg of RNase A per ml and 2 U of RNase T1 at 37°C for 30 min.

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

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: .. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled ssRNA (26-mer) oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl2 , 10 mM DTT, and 1 μl of RNasin [Promega]). .. The binding reaction mixture was incubated at 37°C for 15 min, and then the reaction was terminated by adding 5× RNA loading dye containing 0.5% Nonidet P-40 and subsequently analyzed on a 4% polyacrylamide-0.5× Tris-borate-EDTA gel.

Activity Assay:

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

Expressing:

Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
Article Snippet: Neither TbpA expression (solid box) nor TbpB expression (solid circle) was altered by any of the kan insertions. .. Total RNA was isolated with the Qiagen RNeasy mini kit (Qiagen, Inc.) and subjected to amplification-grade DNase I (Gibco BRL) treatment in the presence of RNasin (Promega Corporation). cDNA was generated by using oligonucleotide 728 (Fig. ) and treated with RNase H (Gibco BRL), prior to being used as a template for subsequent PCR.

Modification:

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis
Article Snippet: A binding reaction was a modification of that described by Meerovitch et al. ( ). .. For UV-cross-linking experiments, equal moles of wild-type and mutant GST-GRSF-1 were incubated with 106 dpm of radiolabeled NP 5′ UTR RNA in buffer containing 5 mM HEPES (pH 7.6), 25 mM KCl, 2 mM MgCl2 , 5% glycerol, 100 mM NaCl, 2 mM dithiothreitol, and 20 U of RNasin (Promega) at 30°C for 15 min.

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

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

RNA Binding Assay:

Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development
Article Snippet: 2.7 RNA immunoprecipitation (RIP) and RIP sequencing RNA immunoprecipitation (RIP) assay was performed using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore). .. The RIP was conducted under the use of RNase inhibitor to protect RNA from RNase degradation (Recombinant RNasin® Ribonuclease Inhibitor, Promega Cat. No. N2515).

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis
Article Snippet: Paragraph title: RNA binding analysis. ... For UV-cross-linking experiments, equal moles of wild-type and mutant GST-GRSF-1 were incubated with 106 dpm of radiolabeled NP 5′ UTR RNA in buffer containing 5 mM HEPES (pH 7.6), 25 mM KCl, 2 mM MgCl2 , 5% glycerol, 100 mM NaCl, 2 mM dithiothreitol, and 20 U of RNasin (Promega) at 30°C for 15 min.

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: Paragraph title: RNA binding assays. ... One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled ssRNA (26-mer) oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl2 , 10 mM DTT, and 1 μl of RNasin [Promega]).

Transformation Assay:

Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus
Article Snippet: Amplified products from the second round of PCR were ligated into vector pCR2.1 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and ligation products were transformed into chemically competent One Shot Top10 Escherichia coli (Invitrogen) and transformants were selected on LB agar with ampicillin (50 μg ml−1 ) and X-gal (40 μg ml−1 ). .. RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above.

Sequencing:

Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development
Article Snippet: Paragraph title: RNA immunoprecipitation (RIP) and RIP sequencing ... The RIP was conducted under the use of RNase inhibitor to protect RNA from RNase degradation (Recombinant RNasin® Ribonuclease Inhibitor, Promega Cat. No. N2515).

Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus
Article Snippet: Transformants harboring plasmids with inserts were collected and DNA sequence was determined by the Institute of Marine and Environmental Technology BioAnalytical Services Laboratory using an ABI 3130 XL Genetic Analyzer (Life Technologies, Grand Island, NY, USA). .. RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above.

Mobility Shift:

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: Binding of small, labeled RNA oligonucleotides to the BTV VP6 and the mutant proteins was compared by gel mobility shift assay. .. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled ssRNA (26-mer) oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl2 , 10 mM DTT, and 1 μl of RNasin [Promega]).

Protease Inhibitor:

Article Title: Activating frataxin expression by repeat-targeted nucleic acids
Article Snippet: .. Nuclei were resuspended in ice-cold nuclear lysis buffer (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 3 mM MgCl2 , 0.3% NP-40 and 10% glycerol) supplemented with 1% Protease Inhibitor Cocktail (Roche), 5 μl ml−1 RNaseIn (Promega) at a final of 0.5 ml per 75 mg of original wet cell pellet weight. .. After high-speed centrifugation at 4 °C to remove insoluble cell debris, the soluble fraction was kept as nuclear extract.

Generated:

Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
Article Snippet: .. Total RNA was isolated with the Qiagen RNeasy mini kit (Qiagen, Inc.) and subjected to amplification-grade DNase I (Gibco BRL) treatment in the presence of RNasin (Promega Corporation). cDNA was generated by using oligonucleotide 728 (Fig. ) and treated with RNase H (Gibco BRL), prior to being used as a template for subsequent PCR. .. RT-PCR products were obtained from intergenic regions between lbpB :: lbpA and lbpA :: orf3 (see Fig. , lanes 2 and 4, respectively).

Reverse Transcription Polymerase Chain Reaction:

Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development
Article Snippet: The isolated RNAs were reverse-transcribed to cDNA for RT-PCR or high-throughput sequencing (RIP-Seq). .. The RIP was conducted under the use of RNase inhibitor to protect RNA from RNase degradation (Recombinant RNasin® Ribonuclease Inhibitor, Promega Cat. No. N2515).

Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
Article Snippet: The RT-PCR experiments were performed with SuperScript II RNase H− reverse transcriptase (Gibco BRL) according to the manufacturer’s instructions. .. Total RNA was isolated with the Qiagen RNeasy mini kit (Qiagen, Inc.) and subjected to amplification-grade DNase I (Gibco BRL) treatment in the presence of RNasin (Promega Corporation). cDNA was generated by using oligonucleotide 728 (Fig. ) and treated with RNase H (Gibco BRL), prior to being used as a template for subsequent PCR.

Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus
Article Snippet: .. RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above. .. A second round amplification was done using nifH1 and nifH2 primers and Taq polymerase mix from Qiagen (Valencia, CA, USA) as described above.

Binding Assay:

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis
Article Snippet: A binding reaction was a modification of that described by Meerovitch et al. ( ). .. For UV-cross-linking experiments, equal moles of wild-type and mutant GST-GRSF-1 were incubated with 106 dpm of radiolabeled NP 5′ UTR RNA in buffer containing 5 mM HEPES (pH 7.6), 25 mM KCl, 2 mM MgCl2 , 5% glycerol, 100 mM NaCl, 2 mM dithiothreitol, and 20 U of RNasin (Promega) at 30°C for 15 min.

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: Binding of small, labeled RNA oligonucleotides to the BTV VP6 and the mutant proteins was compared by gel mobility shift assay. .. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled ssRNA (26-mer) oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl2 , 10 mM DTT, and 1 μl of RNasin [Promega]).

Fluorescence:

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

Magnetic Beads:

Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development
Article Snippet: The RIP was conducted under the use of RNase inhibitor to protect RNA from RNase degradation (Recombinant RNasin® Ribonuclease Inhibitor, Promega Cat. No. N2515). .. In brief, cells were lysed and incubated with hnRNPM antibody (sc-20,002; 5 μg) or control IgG (SC-2025; 5 μg) conjugated with magnetic beads (50 μl) for 4 h. The protein-RNA complexes were immunoprecipitated and magnetically separated.

Mutagenesis:

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis
Article Snippet: .. For UV-cross-linking experiments, equal moles of wild-type and mutant GST-GRSF-1 were incubated with 106 dpm of radiolabeled NP 5′ UTR RNA in buffer containing 5 mM HEPES (pH 7.6), 25 mM KCl, 2 mM MgCl2 , 5% glycerol, 100 mM NaCl, 2 mM dithiothreitol, and 20 U of RNasin (Promega) at 30°C for 15 min. .. Samples were then exposed to UV light (1.0 J) in a Stratalinker (Stratagene), followed by incubation with 10 mg of RNase A per ml and 2 U of RNase T1 at 37°C for 30 min.

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: .. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled ssRNA (26-mer) oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl2 , 10 mM DTT, and 1 μl of RNasin [Promega]). .. The binding reaction mixture was incubated at 37°C for 15 min, and then the reaction was terminated by adding 5× RNA loading dye containing 0.5% Nonidet P-40 and subsequently analyzed on a 4% polyacrylamide-0.5× Tris-borate-EDTA gel.

Isolation:

Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development
Article Snippet: The isolated RNAs were reverse-transcribed to cDNA for RT-PCR or high-throughput sequencing (RIP-Seq). .. The RIP was conducted under the use of RNase inhibitor to protect RNA from RNase degradation (Recombinant RNasin® Ribonuclease Inhibitor, Promega Cat. No. N2515).

Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
Article Snippet: .. Total RNA was isolated with the Qiagen RNeasy mini kit (Qiagen, Inc.) and subjected to amplification-grade DNase I (Gibco BRL) treatment in the presence of RNasin (Promega Corporation). cDNA was generated by using oligonucleotide 728 (Fig. ) and treated with RNase H (Gibco BRL), prior to being used as a template for subsequent PCR. .. RT-PCR products were obtained from intergenic regions between lbpB :: lbpA and lbpA :: orf3 (see Fig. , lanes 2 and 4, respectively).

Labeling:

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: Paragraph title: Labeling of RNA oligonucleotide. ... Unlabeled nucleotides were removed by using Sephadex G-50 spin columns (Pharmacia) and stored at −20°C in sterile H2 0 containing 0.25 U of RNasin (Promega) per μl.

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: Binding of small, labeled RNA oligonucleotides to the BTV VP6 and the mutant proteins was compared by gel mobility shift assay. .. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled ssRNA (26-mer) oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl2 , 10 mM DTT, and 1 μl of RNasin [Promega]).

Concentration Assay:

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

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

Polymerase Chain Reaction:

Article Title: Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants
Article Snippet: .. Total RNA was isolated with the Qiagen RNeasy mini kit (Qiagen, Inc.) and subjected to amplification-grade DNase I (Gibco BRL) treatment in the presence of RNasin (Promega Corporation). cDNA was generated by using oligonucleotide 728 (Fig. ) and treated with RNase H (Gibco BRL), prior to being used as a template for subsequent PCR. .. RT-PCR products were obtained from intergenic regions between lbpB :: lbpA and lbpA :: orf3 (see Fig. , lanes 2 and 4, respectively).

Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus
Article Snippet: Amplified products from the second round of PCR were ligated into vector pCR2.1 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and ligation products were transformed into chemically competent One Shot Top10 Escherichia coli (Invitrogen) and transformants were selected on LB agar with ampicillin (50 μg ml−1 ) and X-gal (40 μg ml−1 ). .. RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above.

Polyacrylamide Gel Electrophoresis:

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: Briefly, a duplex unwinding reaction was performed in a 16-μl volume with 30 mM Tris-HCl (pH 7.5), 3 mM MgCl2 , 10 mM DTT, 5 mM ATP, 1 μl of RNasin (Promega), 1 nM VP6 protein, and 1 nM 32 P-labeled RNA duplex. .. The reaction mixture was incubated at 37°C for 30 min, and the reaction was terminated by the addition of 4 μl of 5× SDS-PAGE sample buffer and analyzed by 12% (wt/vol) nondenaturing PAGE in Tris-glycine buffer followed by autoradiography as described previously ( ).

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis
Article Snippet: For UV-cross-linking experiments, equal moles of wild-type and mutant GST-GRSF-1 were incubated with 106 dpm of radiolabeled NP 5′ UTR RNA in buffer containing 5 mM HEPES (pH 7.6), 25 mM KCl, 2 mM MgCl2 , 5% glycerol, 100 mM NaCl, 2 mM dithiothreitol, and 20 U of RNasin (Promega) at 30°C for 15 min. .. Samples were suspended in 1× Laemeli buffer and separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE).

SDS Page:

Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6
Article Snippet: Briefly, a duplex unwinding reaction was performed in a 16-μl volume with 30 mM Tris-HCl (pH 7.5), 3 mM MgCl2 , 10 mM DTT, 5 mM ATP, 1 μl of RNasin (Promega), 1 nM VP6 protein, and 1 nM 32 P-labeled RNA duplex. .. The reaction mixture was incubated at 37°C for 30 min, and the reaction was terminated by the addition of 4 μl of 5× SDS-PAGE sample buffer and analyzed by 12% (wt/vol) nondenaturing PAGE in Tris-glycine buffer followed by autoradiography as described previously ( ).

Article Title: Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis
Article Snippet: For UV-cross-linking experiments, equal moles of wild-type and mutant GST-GRSF-1 were incubated with 106 dpm of radiolabeled NP 5′ UTR RNA in buffer containing 5 mM HEPES (pH 7.6), 25 mM KCl, 2 mM MgCl2 , 5% glycerol, 100 mM NaCl, 2 mM dithiothreitol, and 20 U of RNasin (Promega) at 30°C for 15 min. .. Samples were suspended in 1× Laemeli buffer and separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE).

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

Plasmid Preparation:

Article Title: Expression of Vascular Endothelial Growth Factor A During Ligand-Induced Down-Regulation of Luteinizing Hormone Receptor in the Ovary ☆
Article Snippet: .. RNase free DNase-I, RNase inhibitor, and pGEM T-Easy vector system were purchased from Promega (Madison, WI). .. O.C.T. compound was purchased from Sakura Finetek (Torrance, CA).

Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus
Article Snippet: Amplified products from the second round of PCR were ligated into vector pCR2.1 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and ligation products were transformed into chemically competent One Shot Top10 Escherichia coli (Invitrogen) and transformants were selected on LB agar with ampicillin (50 μg ml−1 ) and X-gal (40 μg ml−1 ). .. RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above.

Negative Control:

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

Recombinant:

Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development
Article Snippet: .. The RIP was conducted under the use of RNase inhibitor to protect RNA from RNase degradation (Recombinant RNasin® Ribonuclease Inhibitor, Promega Cat. No. N2515). .. In brief, cells were lysed and incubated with hnRNPM antibody (sc-20,002; 5 μg) or control IgG (SC-2025; 5 μg) conjugated with magnetic beads (50 μl) for 4 h. The protein-RNA complexes were immunoprecipitated and magnetically separated.

In Vitro:

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

Ligation:

Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus
Article Snippet: Amplified products from the second round of PCR were ligated into vector pCR2.1 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and ligation products were transformed into chemically competent One Shot Top10 Escherichia coli (Invitrogen) and transformants were selected on LB agar with ampicillin (50 μg ml−1 ) and X-gal (40 μg ml−1 ). .. RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above.

Immunoprecipitation:

Article Title: Activating frataxin expression by repeat-targeted nucleic acids
Article Snippet: Paragraph title: RNA immunoprecipitation ... Nuclei were resuspended in ice-cold nuclear lysis buffer (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 3 mM MgCl2 , 0.3% NP-40 and 10% glycerol) supplemented with 1% Protease Inhibitor Cocktail (Roche), 5 μl ml−1 RNaseIn (Promega) at a final of 0.5 ml per 75 mg of original wet cell pellet weight.

Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development
Article Snippet: Paragraph title: RNA immunoprecipitation (RIP) and RIP sequencing ... The RIP was conducted under the use of RNase inhibitor to protect RNA from RNase degradation (Recombinant RNasin® Ribonuclease Inhibitor, Promega Cat. No. N2515).

Article Title: Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing *
Article Snippet: .. HEK 293 cells were lysed with immunoprecipitation buffer containing either 0.05% RNasin (Promega) or 0.2 μg/μl RNase A (Roche Applied Science), incubated for 10 min on ice, and centrifuged for 10 min. .. The supernatant was loaded on top of a 10–40% sucrose gradient, and centrifugation was performed for 16 h at 23,000 rpm and 4 °C in an ultracentrifuge (Sorvall WX ultra 100, Thermo Scientific).

High Throughput Screening Assay:

Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development
Article Snippet: The isolated RNAs were reverse-transcribed to cDNA for RT-PCR or high-throughput sequencing (RIP-Seq). .. The RIP was conducted under the use of RNase inhibitor to protect RNA from RNase degradation (Recombinant RNasin® Ribonuclease Inhibitor, Promega Cat. No. N2515).

Lysis:

Article Title: Activating frataxin expression by repeat-targeted nucleic acids
Article Snippet: .. Nuclei were resuspended in ice-cold nuclear lysis buffer (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 3 mM MgCl2 , 0.3% NP-40 and 10% glycerol) supplemented with 1% Protease Inhibitor Cocktail (Roche), 5 μl ml−1 RNaseIn (Promega) at a final of 0.5 ml per 75 mg of original wet cell pellet weight. .. After high-speed centrifugation at 4 °C to remove insoluble cell debris, the soluble fraction was kept as nuclear extract.

Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
Article Snippet: .. Cell lysis buffer conditions Total RNAs or sorted single cells were diluted or lysed in 1 μL of cell lysis buffer containing 1 U RNasein plus (Promega), 10% RealTime ready Cell Lysis Buffer (Roche), 0.3% NP40 (Thermo Fisher), and RNase-free water (TaKaRa). .. The lysate solution was immediately centrifuged and mixed using a ThermoMixer C (Eppendorf) at 2000 rpm for 1 min at 4 °C.

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

Article Title: Anti-prion activity of an RNA aptamer and its structural basis
Article Snippet: For the conditions with an RNase inhibitor, 800 U of RNasin (Promega) was added to the medium 5 min before the addition of the R12 solution. .. After 72 h of treatment, cells were lysed in 150 μl of 1× Triton X-100-deoxycholate lysis buffer [150 mM of NaCl, 0.5% of Triton X-100, 0.5% of sodium deoxycholate, 50 mM of Tris–HCl (pH 7.5)], and the supernatant was collected.

Gradient Centrifugation:

Article Title: Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing *
Article Snippet: Paragraph title: Sucrose Gradient Centrifugation ... HEK 293 cells were lysed with immunoprecipitation buffer containing either 0.05% RNasin (Promega) or 0.2 μg/μl RNase A (Roche Applied Science), incubated for 10 min on ice, and centrifuged for 10 min.

Fluorescence In Situ Hybridization:

Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus
Article Snippet: RNA for RT–PCR was extracted from GI tract sections of wood-only fed fish using the PureLink RNA kit from Invitrogen following the manufacturer's instructions. .. RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above.

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    Promega rnase inhibitor
    Coligo topology is necessary but not sufficient to template the synthesis of stable released sRNA transcripts in human <t>WCE.</t> ( A ) Circularization stabilizes oligonucleotides in human WCE. Circular (C) or linear (L) templates (Input) were recovered (Post) from HEK293T WCE IVT, digested with <t>RNase</t> cocktail to reduce cellular RNA, and stained after DPAGE. Linear forms were degraded during IVT; coligos were stable. Coligo 19aRL sequence is shown in Supplementary Figure S2 . ( B ) DPAGE separation of HEK293T WCE IVT of the three coligos and linear precursors from the reactions shown in panel A. ( C ) Transcripts are released from the coligo template during IVT. RNase H (RH) was added to (+) or withheld from (−) the indicated coligo IVT reactions at the end of a typical 90-min incubation period. Following additional incubation, the RNA products were separated by DPAGE. Lanes 1 and 2, validation of exhaustive RNase H activity on a 32 P-RNA:DNA hybrid. Reaction in lane 2 was supplemented with total HEK293T cellular RNA to normalize non-specific competing RNAs among all RNase H reactions. The result shows that the coligo 19aTAR ’s transcripts do not remain hybridized to the coligo template, while ∼20% of coligo 122 ’s transcripts do remain bound to the coligo template.
    Rnase Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Coligo topology is necessary but not sufficient to template the synthesis of stable released sRNA transcripts in human WCE. ( A ) Circularization stabilizes oligonucleotides in human WCE. Circular (C) or linear (L) templates (Input) were recovered (Post) from HEK293T WCE IVT, digested with RNase cocktail to reduce cellular RNA, and stained after DPAGE. Linear forms were degraded during IVT; coligos were stable. Coligo 19aRL sequence is shown in Supplementary Figure S2 . ( B ) DPAGE separation of HEK293T WCE IVT of the three coligos and linear precursors from the reactions shown in panel A. ( C ) Transcripts are released from the coligo template during IVT. RNase H (RH) was added to (+) or withheld from (−) the indicated coligo IVT reactions at the end of a typical 90-min incubation period. Following additional incubation, the RNA products were separated by DPAGE. Lanes 1 and 2, validation of exhaustive RNase H activity on a 32 P-RNA:DNA hybrid. Reaction in lane 2 was supplemented with total HEK293T cellular RNA to normalize non-specific competing RNAs among all RNase H reactions. The result shows that the coligo 19aTAR ’s transcripts do not remain hybridized to the coligo template, while ∼20% of coligo 122 ’s transcripts do remain bound to the coligo template.

    Journal: Nucleic Acids Research

    Article Title: Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells

    doi: 10.1093/nar/gks1334

    Figure Lengend Snippet: Coligo topology is necessary but not sufficient to template the synthesis of stable released sRNA transcripts in human WCE. ( A ) Circularization stabilizes oligonucleotides in human WCE. Circular (C) or linear (L) templates (Input) were recovered (Post) from HEK293T WCE IVT, digested with RNase cocktail to reduce cellular RNA, and stained after DPAGE. Linear forms were degraded during IVT; coligos were stable. Coligo 19aRL sequence is shown in Supplementary Figure S2 . ( B ) DPAGE separation of HEK293T WCE IVT of the three coligos and linear precursors from the reactions shown in panel A. ( C ) Transcripts are released from the coligo template during IVT. RNase H (RH) was added to (+) or withheld from (−) the indicated coligo IVT reactions at the end of a typical 90-min incubation period. Following additional incubation, the RNA products were separated by DPAGE. Lanes 1 and 2, validation of exhaustive RNase H activity on a 32 P-RNA:DNA hybrid. Reaction in lane 2 was supplemented with total HEK293T cellular RNA to normalize non-specific competing RNAs among all RNase H reactions. The result shows that the coligo 19aTAR ’s transcripts do not remain hybridized to the coligo template, while ∼20% of coligo 122 ’s transcripts do remain bound to the coligo template.

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

    Techniques: Staining, Sequencing, Incubation, Activity Assay

    Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative RNase protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total RNA from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).

    Journal: Infection and Immunity

    Article Title: Calprotectin Expression by Gingival Epithelial Cells

    doi: 10.1128/IAI.69.5.3248-3254.2001

    Figure Lengend Snippet: Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative RNase protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total RNA from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).

    Article Snippet: The radiolabeled probes were synthesized under the following reaction conditions: 500 μM concentrations each of rCTP, rGTP, and rATP, and 1 μM rUTP; 3 μM [α-32 P]UTP (800 Ci/mmol, 10 mCi/ml) (DuPont NEN Research Products, Boston, Mass.); 0.5 μl of PCR template; 1 U of T7 or T3 RNA polymerase (Stratagene); 2 μl of transcription buffer (Stratagene); 40 U of RNase inhibitor (Promega, Madison, Wis.); and distilled H2 O to a total volume of 10 μl.

    Techniques: Expressing, Rnase Protection Assay, Binding Assay, Quantitation Assay, Standard Deviation

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

    Journal: mBio

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

    doi: 10.1128/mBio.01355-17

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

    Article Snippet: Reaction mixtures were identical to those in the gel shift assay except that RNase inhibitor was omitted, the concentration of labeled RNA was increased to 10 nM, and 200 μg/ml acetylated bovine serum albumin (BSA) was added.

    Techniques: Labeling, Concentration Assay, Sequencing

    FUS forms a complex with itself, PABP and RNA. ( A ) Co-immunoprecipitation of HA-FUS, Myc-FUS and PABP from HEK293T cells. Immunoprecipitation with a Myc (mouse anti-Myc, CST) antibody pulled down PABP (mouse anti-PABP, Sigma) along with anti-HA-FUS (rabbit HA, CST). RNasin was added to block all RNase activity. ( B ) The interaction between HA-FUS and Myc-FUS was not altered by the addition of RNase, while the co-immunoprecipitation of PABP was completely abolished. ( C ) Immunoprecipitation of FUS (mouse anti-FUS, Santa Cruz) from untransfected cells shows that endogenous FUS (rabbit anti-FUS, Novus Biologicals) also interacts with PABP (mouse anti-PABP, Sigma) and treatment with RNase shows that this interaction is also dependent on RNA. ( D ) Mock immunoprecipitation experiments with either untransfected cells or a single transfection of either HA-FUS WT or Myc-FUS WT with no antibody showed that neither endogenous nor tagged FUS binds to the beads. ( E ) Immunoprecipitation of single transfections with an antibody to the wrong tag showed that a Myc antibody does not pull down HA-FUS and a HA antibody does not precipitate Myc-FUS. FT indicates flow-through of proteins that did not bind to the beads.

    Journal: Human Molecular Genetics

    Article Title: ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules

    doi: 10.1093/hmg/ddt117

    Figure Lengend Snippet: FUS forms a complex with itself, PABP and RNA. ( A ) Co-immunoprecipitation of HA-FUS, Myc-FUS and PABP from HEK293T cells. Immunoprecipitation with a Myc (mouse anti-Myc, CST) antibody pulled down PABP (mouse anti-PABP, Sigma) along with anti-HA-FUS (rabbit HA, CST). RNasin was added to block all RNase activity. ( B ) The interaction between HA-FUS and Myc-FUS was not altered by the addition of RNase, while the co-immunoprecipitation of PABP was completely abolished. ( C ) Immunoprecipitation of FUS (mouse anti-FUS, Santa Cruz) from untransfected cells shows that endogenous FUS (rabbit anti-FUS, Novus Biologicals) also interacts with PABP (mouse anti-PABP, Sigma) and treatment with RNase shows that this interaction is also dependent on RNA. ( D ) Mock immunoprecipitation experiments with either untransfected cells or a single transfection of either HA-FUS WT or Myc-FUS WT with no antibody showed that neither endogenous nor tagged FUS binds to the beads. ( E ) Immunoprecipitation of single transfections with an antibody to the wrong tag showed that a Myc antibody does not pull down HA-FUS and a HA antibody does not precipitate Myc-FUS. FT indicates flow-through of proteins that did not bind to the beads.

    Article Snippet: 5 µl of an RNase A/T1 mix (Fermentas, Yorkshire, UK) or RNasin Plus RNase inhibitor (Promega, Southampton, UK) was added to the tubes and incubated at 37°C for 5 min, then placed back on ice.

    Techniques: Immunoprecipitation, Blocking Assay, Activity Assay, Transfection, Flow Cytometry