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GE Healthcare rnaguard
Rnaguard, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 28 article reviews
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rnaguard - by Bioz Stars, 2019-10
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Amplification:

Article Title: N1303K (c.3909C > G) Mutation and Splicing: Implication of Its c.[744-33GATT(6); 869+11C > T] Complex Allele in CFTR Exon 7 Aberrant Splicing
Article Snippet: Total mRNA was extracted from cell lysates using the RNeasy Mini Kit (Qiagen, Germany) and dissolved in 30 μ L of sterile water. cDNA synthesis was carried out at 37°C for 1 h after adjustment of the mixture to contain 5 μ L of 5x buffer (Gibco-BRL, France; 250 mmol/L of Tris-HCl pH 8.3, 375 mmol/L of KCl, 15 mmol/L of MgCl2 ), 10 mmol/L of dithiothreitol (Gibco-BRL, France), 1 mmol/L of dNTPs (Roche Diagnostics, France), 2.4 μ g of random hexamer primers, 10 μ L of RNA, 40 U RNAguard (Amersham Biosciences, Orsay, France), and 400 U Moloney murine leukemia virus (MMLV) reverse transcriptase. .. PCRs were performed using a 9700 GeneAmp Thermo Cycler (Perkin Elmer) with the following cycling conditions: initial denaturation (94°C, 2 min), followed by 30 cycles (94°C, 30 sec; 58°C, 30 sec; 72°C, 30 sec), and a final extension step (72°C, 5 min).

Article Title: Identifying single-cell molecular programs by stochastic profiling
Article Snippet: Paragraph title: Small-cell quantitative mRNA amplification ... Digested samples were spun into PCR tubes and quenched with 1 μl of digestion stop buffer (1.5 U ml−1 Prime RNAse inhibitor [Eppendorf], 1.5 U ml−1 RNAguard [Amersham], and 5 mM freshly prepared PMSF).

Article Title: Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery
Article Snippet: RNA was treated with TURBO DNase (Ambion, Austin, TX, USA) to remove any remaining contaminating DNA and then reverse-transcribed in the presence of RNAguard (GE Healthcare, Chalfont St Giles, UK) by using random hexamers and revert-aid reverse transcriptase (Fermentas International, York, UK). .. MacVector™ (version 7.2) and Ensembl Genome Browser ( www.emsembl.org ) were used to design RT–PCR primer pairs.

Article Title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein
Article Snippet: Reverse transcriptions were performed by addition of 2 µl of this solution in 20 µl of 1 × MMLV buffer (Promega) containing 5 pmol of O-5825 primer, 1 mM of each dNTP, 1 µl of RNAguard (GE Healthcare) and 200 U of MMLV reverse transcriptase (Promega). .. In order to detect spliced RNAs, PCR amplifications were performed with the O-6246/O-6519 primer pair.

Article Title: PPARγ ligands inhibit telomerase activity and hTERT expression through modulation of the Myc/Mad/Max network in colon cancer cells
Article Snippet: Total RNA was isolated using the RNA fast Kit (Molecular System, Genenco, Milano, Italy). cDNA synthesis was performed with 4 μg of total RNA in a reaction volume of 40 μl containing 1.25 μg of random primers, l mM of dATP, dGTP, dCTP and dTTP (Invitrogen, Milano, Italy), 66 units of RNAguard (Amersham Biosciences, Cologno Monzese, Milano, Italy), 8 μl of 5× first strand buffer, 10 mM DTT, 300 units of MMLV reverse transcriptase (Invitrogen). .. Total RNA was isolated using the RNA fast Kit (Molecular System, Genenco, Milano, Italy). cDNA synthesis was performed with 4 μg of total RNA in a reaction volume of 40 μl containing 1.25 μg of random primers, l mM of dATP, dGTP, dCTP and dTTP (Invitrogen, Milano, Italy), 66 units of RNAguard (Amersham Biosciences, Cologno Monzese, Milano, Italy), 8 μl of 5× first strand buffer, 10 mM DTT, 300 units of MMLV reverse transcriptase (Invitrogen).

Mouse Assay:

Article Title: Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice
Article Snippet: For reverse transcription 2 μg total RNA was incubated for 1 h at 37°C in buffer containing 50mM Tris-HCl pH 8.3, 75 mM KCl, 3mM MgCl2 , 10mM DTT and 1mM dNTPs, supplemented with 400 U M-MLV-reverse transcriptase (Thermo Fisher Scientific, Waltham, MA), 80 U RNAguard (Amersham Biosciences, Little Chalfont, UK) and 1 μg oligodT primer. .. For reverse transcription 2 μg total RNA was incubated for 1 h at 37°C in buffer containing 50mM Tris-HCl pH 8.3, 75 mM KCl, 3mM MgCl2 , 10mM DTT and 1mM dNTPs, supplemented with 400 U M-MLV-reverse transcriptase (Thermo Fisher Scientific, Waltham, MA), 80 U RNAguard (Amersham Biosciences, Little Chalfont, UK) and 1 μg oligodT primer.

Quantitative RT-PCR:

Article Title: Molecular analysis of human cancer cells infected by an oncolytic HSV-1 reveals multiple upregulated cellular genes and a role for SOCS1 in virus replication
Article Snippet: A total of 4 µl of 5 × first-strand synthesis buffer (Invitrogen), 2 µl of 0.1 M dithiothreotol, 0.5 µl of 0.1M dNTP, 0.5 µl of RNAguard (Amersham Biosciences, Piscataway, NJ) and 1 µl of M-MLV RT (Promega, San Luis Obispo, CA) were added and incubated at 37 °C for 1 h. The reaction was stopped by 10 min incubation at 70 °C. .. A total of 4 µl of 5 × first-strand synthesis buffer (Invitrogen), 2 µl of 0.1 M dithiothreotol, 0.5 µl of 0.1M dNTP, 0.5 µl of RNAguard (Amersham Biosciences, Piscataway, NJ) and 1 µl of M-MLV RT (Promega, San Luis Obispo, CA) were added and incubated at 37 °C for 1 h. The reaction was stopped by 10 min incubation at 70 °C.

Real-time Polymerase Chain Reaction:

Article Title: Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice
Article Snippet: Paragraph title: RNA extraction, cDNA preparation and QPCR analysis ... For reverse transcription 2 μg total RNA was incubated for 1 h at 37°C in buffer containing 50mM Tris-HCl pH 8.3, 75 mM KCl, 3mM MgCl2 , 10mM DTT and 1mM dNTPs, supplemented with 400 U M-MLV-reverse transcriptase (Thermo Fisher Scientific, Waltham, MA), 80 U RNAguard (Amersham Biosciences, Little Chalfont, UK) and 1 μg oligodT primer.

Article Title: New functionally-enhanced soy proteins as food ingredients with anti-viral activity
Article Snippet: The mixture was centrifuged at 6000 rpm for 5 min and supernatant was used for real time-PCR quantification of viral RNA [ ]. .. The RNA was converted to full-length cDNA in the following reaction: 2.5 μl of DMPC (di-methyl pyrocarbonate) water, 5 μl of 5× First Strand buffer (Invitrogen), 0.5 μl of 10 mMdNTP mix (Amersham Biosciences), 2 μl of 50 mM UNi12 primer, 32 U of RNAguard (Amersham Biosciences), 200 U of MMLV reverse transcriptase (Invitrogen) and 5 μl RNA solution in total volume of 25 μl.

Article Title: Molecular analysis of human cancer cells infected by an oncolytic HSV-1 reveals multiple upregulated cellular genes and a role for SOCS1 in virus replication
Article Snippet: Paragraph title: Real-time PCR ... A total of 4 µl of 5 × first-strand synthesis buffer (Invitrogen), 2 µl of 0.1 M dithiothreotol, 0.5 µl of 0.1M dNTP, 0.5 µl of RNAguard (Amersham Biosciences, Piscataway, NJ) and 1 µl of M-MLV RT (Promega, San Luis Obispo, CA) were added and incubated at 37 °C for 1 h. The reaction was stopped by 10 min incubation at 70 °C.

Random Hexamer Labeling:

Article Title: N1303K (c.3909C > G) Mutation and Splicing: Implication of Its c.[744-33GATT(6); 869+11C > T] Complex Allele in CFTR Exon 7 Aberrant Splicing
Article Snippet: At least three independent transfections for each cell line were performed for RNA extraction experiments. .. Total mRNA was extracted from cell lysates using the RNeasy Mini Kit (Qiagen, Germany) and dissolved in 30 μ L of sterile water. cDNA synthesis was carried out at 37°C for 1 h after adjustment of the mixture to contain 5 μ L of 5x buffer (Gibco-BRL, France; 250 mmol/L of Tris-HCl pH 8.3, 375 mmol/L of KCl, 15 mmol/L of MgCl2 ), 10 mmol/L of dithiothreitol (Gibco-BRL, France), 1 mmol/L of dNTPs (Roche Diagnostics, France), 2.4 μ g of random hexamer primers, 10 μ L of RNA, 40 U RNAguard (Amersham Biosciences, Orsay, France), and 400 U Moloney murine leukemia virus (MMLV) reverse transcriptase. .. The reaction medium was made up to 25 μ L with sterile water and the reaction was stopped by incubation at 100°C for 2 min.

Activity Assay:

Article Title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein
Article Snippet: To study the influence of protein 9G8 on NRS activity, splicing reactions were performed with a 1:1 mixture of HeLa cell cytoplasmic extract and HeLa cell nuclear extract and 180 or 270 ng of purified recombinant human 9G8 protein (produced in baculovirus) or 500 ng of commercial purified BSA used as a control. .. Reverse transcriptions were performed by addition of 2 µl of this solution in 20 µl of 1 × MMLV buffer (Promega) containing 5 pmol of O-5825 primer, 1 mM of each dNTP, 1 µl of RNAguard (GE Healthcare) and 200 U of MMLV reverse transcriptase (Promega).

Expressing:

Article Title: Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition
Article Snippet: Immunoprecipitations were performed using the FLAG Tagged Protein Immunoprecipitation Kit (FLAGIPT-1 Sigma), essentially following manufacturer’s recommendations. .. Typically, 1.5 mg of proteinase-K-treated mitochondria isolated from HEK293T cells expressing mtPABP1 or mtLUC were re-suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with Complete EDTA free Protease Inhibitor Cocktail (Roche) and RNAguard (GE Healthcare). .. Lysis was performed for 20 min rotating at 4°C, followed by centrifugation at 12 000g for 10 min to remove insoluble material.

Hybridization:

Article Title: Maternal Argonaute 2 Is Essential for Early Mouse Development at the Maternal-Zygotic Transition
Article Snippet: Paragraph title: cDNA Synthesis and Subtractive Hybridization (Representational Difference Analysis) ... Briefly, the embryo was lysed at 65°C for 1 min in lysis buffer (0.5% NP40, 1.25 mM each of dATP, dCTP, dGTP, and dTTP, 1 μl of 50 OD/ml oligo d(T)24 , 0.4 U primeRNase inhibitor [Roche] and 0.38 U RNAguard [Amersham Biotechnology, Piscataway, NJ [ in 1× MMLV (Moloney murine leukemia virus) buffer (Roche).

Article Title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis
Article Snippet: Paragraph title: In situ hybridization using insl3 on zebrafish testis ... For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis).

Immunoprecipitation:

Article Title: Yeast targets for mRNA methylation
Article Snippet: Paragraph title: Immunoprecipitation of m6 A containing messages ... Following the washes 3.8 μg Ab and 3 μl RNAguard (Amersham Pharmacia) was added to the oligo(dT) bound mRNA, in a total volume of 500 μl PBS.

Article Title: Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition
Article Snippet: Immunoprecipitations were performed using the FLAG Tagged Protein Immunoprecipitation Kit (FLAGIPT-1 Sigma), essentially following manufacturer’s recommendations. .. Typically, 1.5 mg of proteinase-K-treated mitochondria isolated from HEK293T cells expressing mtPABP1 or mtLUC were re-suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with Complete EDTA free Protease Inhibitor Cocktail (Roche) and RNAguard (GE Healthcare).

Protease Inhibitor:

Article Title: Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition
Article Snippet: Immunoprecipitations were performed using the FLAG Tagged Protein Immunoprecipitation Kit (FLAGIPT-1 Sigma), essentially following manufacturer’s recommendations. .. Typically, 1.5 mg of proteinase-K-treated mitochondria isolated from HEK293T cells expressing mtPABP1 or mtLUC were re-suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with Complete EDTA free Protease Inhibitor Cocktail (Roche) and RNAguard (GE Healthcare). .. Lysis was performed for 20 min rotating at 4°C, followed by centrifugation at 12 000g for 10 min to remove insoluble material.

Northern Blot:

Article Title: Yeast targets for mRNA methylation
Article Snippet: After ethanol precipitation, the mRNA samples from the Ab bound fraction and from the Ab depleted fraction were subjected to northern blot analysis. .. Following the washes 3.8 μg Ab and 3 μl RNAguard (Amersham Pharmacia) was added to the oligo(dT) bound mRNA, in a total volume of 500 μl PBS.

Infection:

Article Title: Molecular analysis of human cancer cells infected by an oncolytic HSV-1 reveals multiple upregulated cellular genes and a role for SOCS1 in virus replication
Article Snippet: Malignant peripheral nerve sheath tumor cells were infected with G207 at a multiplicity of infection = 5 and total RNA collected at 6 h using Trizol. .. A total of 4 µl of 5 × first-strand synthesis buffer (Invitrogen), 2 µl of 0.1 M dithiothreotol, 0.5 µl of 0.1M dNTP, 0.5 µl of RNAguard (Amersham Biosciences, Piscataway, NJ) and 1 µl of M-MLV RT (Promega, San Luis Obispo, CA) were added and incubated at 37 °C for 1 h. The reaction was stopped by 10 min incubation at 70 °C.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Maternal Argonaute 2 Is Essential for Early Mouse Development at the Maternal-Zygotic Transition
Article Snippet: Briefly, the embryo was lysed at 65°C for 1 min in lysis buffer (0.5% NP40, 1.25 mM each of dATP, dCTP, dGTP, and dTTP, 1 μl of 50 OD/ml oligo d(T)24 , 0.4 U primeRNase inhibitor [Roche] and 0.38 U RNAguard [Amersham Biotechnology, Piscataway, NJ [ in 1× MMLV (Moloney murine leukemia virus) buffer (Roche). .. The reaction was allowed to cool down at RT for 1 min and kept on ice before adding AMV-and MMLV-reverse transcriptases according to the manufacturer's instructions (Roche). cDNA libraries for subtractive hybridization were further treated with the tailing reaction using terminal deoxynucleotidyltransferase according the manufacturer's instructions (10533-065, Invitrogen) and subsequently amplified using the OL-1 primer to allow the insertion of restriction enzyme site.

Article Title: N1303K (c.3909C > G) Mutation and Splicing: Implication of Its c.[744-33GATT(6); 869+11C > T] Complex Allele in CFTR Exon 7 Aberrant Splicing
Article Snippet: Paragraph title: 2.6. RT-PCR Analyses ... Total mRNA was extracted from cell lysates using the RNeasy Mini Kit (Qiagen, Germany) and dissolved in 30 μ L of sterile water. cDNA synthesis was carried out at 37°C for 1 h after adjustment of the mixture to contain 5 μ L of 5x buffer (Gibco-BRL, France; 250 mmol/L of Tris-HCl pH 8.3, 375 mmol/L of KCl, 15 mmol/L of MgCl2 ), 10 mmol/L of dithiothreitol (Gibco-BRL, France), 1 mmol/L of dNTPs (Roche Diagnostics, France), 2.4 μ g of random hexamer primers, 10 μ L of RNA, 40 U RNAguard (Amersham Biosciences, Orsay, France), and 400 U Moloney murine leukemia virus (MMLV) reverse transcriptase.

Article Title: Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins
Article Snippet: Paragraph title: RT-PCR and sequencing. ... The reaction mix (20 μl), which contained, in addition to pre-supplied buffer, 35 ng of primer, 10–100 ng of viral RNA, 1 mM dNTPs, 10 mM DTT, 25 U of RNAguard (Amersham, Little Chalfont, United Kingdom), and 200 U of reverse transcriptase, was incubated at 42 °C for 60 min and then at 94 °C for 2 min.

Article Title: PPARγ ligands inhibit telomerase activity and hTERT expression through modulation of the Myc/Mad/Max network in colon cancer cells
Article Snippet: Paragraph title: RNA isolation and semi-quantitative RT-PCR analysis ... Total RNA was isolated using the RNA fast Kit (Molecular System, Genenco, Milano, Italy). cDNA synthesis was performed with 4 μg of total RNA in a reaction volume of 40 μl containing 1.25 μg of random primers, l mM of dATP, dGTP, dCTP and dTTP (Invitrogen, Milano, Italy), 66 units of RNAguard (Amersham Biosciences, Cologno Monzese, Milano, Italy), 8 μl of 5× first strand buffer, 10 mM DTT, 300 units of MMLV reverse transcriptase (Invitrogen).

Indirect Immunoperoxidase Assay:

Article Title: Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery
Article Snippet: 2.3 Total RNA was extracted from IPA or PASMC using the Qiagen RNeasy mini kit and TissueLyser (Qiagen, Crawley, UK). .. RNA was treated with TURBO DNase (Ambion, Austin, TX, USA) to remove any remaining contaminating DNA and then reverse-transcribed in the presence of RNAguard (GE Healthcare, Chalfont St Giles, UK) by using random hexamers and revert-aid reverse transcriptase (Fermentas International, York, UK).

Generated:

Article Title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis
Article Snippet: A zebrafish-specific insl3 -specific PCR product was generated with primer 2126 (5'- GGGCGGGTG TTATTAACCCTCACTAAA GGGAGTGAAGATGTGCGAGTGAAGC-3'; containing the T3 RNA polymerase promoter sequence [underlined]) and primer 2127 (5'-CCGGGGGGTG TAATACGACTCACTATA GGGGTACTGAATCAGTT CATTCATGGTGCA-3'; containing the T7 RNA polymerase promoter sequence [underlined]). .. For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis).

Polymerase Chain Reaction:

Article Title: N1303K (c.3909C > G) Mutation and Splicing: Implication of Its c.[744-33GATT(6); 869+11C > T] Complex Allele in CFTR Exon 7 Aberrant Splicing
Article Snippet: Total mRNA was extracted from cell lysates using the RNeasy Mini Kit (Qiagen, Germany) and dissolved in 30 μ L of sterile water. cDNA synthesis was carried out at 37°C for 1 h after adjustment of the mixture to contain 5 μ L of 5x buffer (Gibco-BRL, France; 250 mmol/L of Tris-HCl pH 8.3, 375 mmol/L of KCl, 15 mmol/L of MgCl2 ), 10 mmol/L of dithiothreitol (Gibco-BRL, France), 1 mmol/L of dNTPs (Roche Diagnostics, France), 2.4 μ g of random hexamer primers, 10 μ L of RNA, 40 U RNAguard (Amersham Biosciences, Orsay, France), and 400 U Moloney murine leukemia virus (MMLV) reverse transcriptase. .. Total mRNA was extracted from cell lysates using the RNeasy Mini Kit (Qiagen, Germany) and dissolved in 30 μ L of sterile water. cDNA synthesis was carried out at 37°C for 1 h after adjustment of the mixture to contain 5 μ L of 5x buffer (Gibco-BRL, France; 250 mmol/L of Tris-HCl pH 8.3, 375 mmol/L of KCl, 15 mmol/L of MgCl2 ), 10 mmol/L of dithiothreitol (Gibco-BRL, France), 1 mmol/L of dNTPs (Roche Diagnostics, France), 2.4 μ g of random hexamer primers, 10 μ L of RNA, 40 U RNAguard (Amersham Biosciences, Orsay, France), and 400 U Moloney murine leukemia virus (MMLV) reverse transcriptase.

Article Title: Identifying single-cell molecular programs by stochastic profiling
Article Snippet: Samples were eluted from the microdissection caps by adding 4 μl digestion buffer (1.25× MMLV RT buffer [Invitrogen], 100 μM dNTPs [Roche], 0.08 OD ml−1 oligo(dT)24 , and 250 μg ml−1 proteinase K [Sigma]) and incubating at 42°C for 1 hr. .. Digested samples were spun into PCR tubes and quenched with 1 μl of digestion stop buffer (1.5 U ml−1 Prime RNAse inhibitor [Eppendorf], 1.5 U ml−1 RNAguard [Amersham], and 5 mM freshly prepared PMSF). .. The quenched samples were then processed by using poly(A) PCR that was heavily modified to allow quantitative amplification of high- and low-abundance transcripts.

Article Title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis
Article Snippet: The ~ 450 bp PCR product was gel purified, and served as a template for digoxigenin-labeled cRNA probe synthesis. .. For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis). .. To visualize cellular expression sites of insl3 mRNA in zebrafish testis, whole mount in situ hybridization was performed on zebrafish testicular tissue, fixed in 4% paraformaldehyde in PBS (pH 7.4), as described by Westerfield (2000) http://zfin.org/zf_info/zfbook/chapt9/9.82.html with some modifications to the protocol.

Article Title: Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice
Article Snippet: For reverse transcription 2 μg total RNA was incubated for 1 h at 37°C in buffer containing 50mM Tris-HCl pH 8.3, 75 mM KCl, 3mM MgCl2 , 10mM DTT and 1mM dNTPs, supplemented with 400 U M-MLV-reverse transcriptase (Thermo Fisher Scientific, Waltham, MA), 80 U RNAguard (Amersham Biosciences, Little Chalfont, UK) and 1 μg oligodT primer. .. The procedure for QPCR analysis was described earlier [ ].

Article Title: New functionally-enhanced soy proteins as food ingredients with anti-viral activity
Article Snippet: The RNA was converted to full-length cDNA in the following reaction: 2.5 μl of DMPC (di-methyl pyrocarbonate) water, 5 μl of 5× First Strand buffer (Invitrogen), 0.5 μl of 10 mMdNTP mix (Amersham Biosciences), 2 μl of 50 mM UNi12 primer, 32 U of RNAguard (Amersham Biosciences), 200 U of MMLV reverse transcriptase (Invitrogen) and 5 μl RNA solution in total volume of 25 μl. .. Reactions were incubated at 42 °C for 60 min followed by inactivation of the enzyme at 95 °C for 5 min. PCR amplification with HA gene specific primers (Forward primer: 5′-AGCAAAAGCAGGGGA-3′, Reverse primer: 5′-AGTAGAAACAAGGGTGTT-3′) was performed on the PikoReal 96 Real Time PCR System (Thermo Scientific, USA) to amplify the product containing the fragment HA gene.

Article Title: Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins
Article Snippet: The reaction mix (20 μl), which contained, in addition to pre-supplied buffer, 35 ng of primer, 10–100 ng of viral RNA, 1 mM dNTPs, 10 mM DTT, 25 U of RNAguard (Amersham, Little Chalfont, United Kingdom), and 200 U of reverse transcriptase, was incubated at 42 °C for 60 min and then at 94 °C for 2 min. .. The reaction mix (100 μl), which contained, in addition to pre-supplied buffer, 70 ng of primer pair, 1 μl of RT reaction product, 200 μM dNTPs, 2 mM MgCl2 , and 2.5 U of DNA polymerase, was incubated at 94 °C for 1 min, then 94 °C for 20 s, 50 °C for 20 s, 68 °C for 3 min, for a total of 35 cycles and a final 10-min extension at 68 °C.

Article Title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein
Article Snippet: Reverse transcriptions were performed by addition of 2 µl of this solution in 20 µl of 1 × MMLV buffer (Promega) containing 5 pmol of O-5825 primer, 1 mM of each dNTP, 1 µl of RNAguard (GE Healthcare) and 200 U of MMLV reverse transcriptase (Promega). .. In order to detect spliced RNAs, PCR amplifications were performed with the O-6246/O-6519 primer pair.

Article Title: PPARγ ligands inhibit telomerase activity and hTERT expression through modulation of the Myc/Mad/Max network in colon cancer cells
Article Snippet: RNA analyses were performed by a semi-quantitative PCR method as previously described [ ]. .. Total RNA was isolated using the RNA fast Kit (Molecular System, Genenco, Milano, Italy). cDNA synthesis was performed with 4 μg of total RNA in a reaction volume of 40 μl containing 1.25 μg of random primers, l mM of dATP, dGTP, dCTP and dTTP (Invitrogen, Milano, Italy), 66 units of RNAguard (Amersham Biosciences, Cologno Monzese, Milano, Italy), 8 μl of 5× first strand buffer, 10 mM DTT, 300 units of MMLV reverse transcriptase (Invitrogen).

Binding Assay:

Article Title: Direct measurement of the mechanical work during translocation by the ribosome
Article Snippet: Since free tRNAs, which are not productive in translation, tend to increase the noise in the translation traces (probably by binding to the single-stranded mRNA), we developed a procedure to purify the ternary complexes using the 6xHis-tag present in EF-Tu. .. Next, 50 µl purified ternary complexes (containing 0.2 U total aa-tRNA) were diluted with 390 µl (total 440 µl) buffer TL containing 1 mM GTP, 1 mM ATP, 40 U RNAguard (GE Healthcare, Piscataway, NJ) and 1 µM EF-G (final concentrations).

Fluorescence:

Article Title: Molecular analysis of human cancer cells infected by an oncolytic HSV-1 reveals multiple upregulated cellular genes and a role for SOCS1 in virus replication
Article Snippet: A total of 4 µl of 5 × first-strand synthesis buffer (Invitrogen), 2 µl of 0.1 M dithiothreotol, 0.5 µl of 0.1M dNTP, 0.5 µl of RNAguard (Amersham Biosciences, Piscataway, NJ) and 1 µl of M-MLV RT (Promega, San Luis Obispo, CA) were added and incubated at 37 °C for 1 h. The reaction was stopped by 10 min incubation at 70 °C. .. A total of 4 µl of 5 × first-strand synthesis buffer (Invitrogen), 2 µl of 0.1 M dithiothreotol, 0.5 µl of 0.1M dNTP, 0.5 µl of RNAguard (Amersham Biosciences, Piscataway, NJ) and 1 µl of M-MLV RT (Promega, San Luis Obispo, CA) were added and incubated at 37 °C for 1 h. The reaction was stopped by 10 min incubation at 70 °C.

Magnetic Beads:

Article Title: Yeast targets for mRNA methylation
Article Snippet: For the immunoprecipitation (IP) experiments on yeast mRNA, 1.2 μg poly(A)RNA was bound to oligo(dT) magnetic beads, using the PolyTtrack System 1000 (Promega) and washed 3× with PBS buffer. .. Following the washes 3.8 μg Ab and 3 μl RNAguard (Amersham Pharmacia) was added to the oligo(dT) bound mRNA, in a total volume of 500 μl PBS.

Mutagenesis:

Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
Article Snippet: The samples were frozen on dry ice and combined with an equal volume of denaturing loading buffer (10 M urea, 1X TBE). .. The 32 P-labeled PHGPx SECIS or U40C mutant RNAs (10 fmol) were incubated in buffer containing 0.7X PBS, 11 mM DTT, 250 ng/μL yeast tRNA and RNAguard (Amersham). .. The indicated amounts of L30 were added last and the reactions incubated at 37°C for 30 min.

Article Title: Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins
Article Snippet: The entire replicase gene-coding region (ORF1a and ORF1b) was sequenced for eight ts mutant and revertant pairs. .. The reaction mix (20 μl), which contained, in addition to pre-supplied buffer, 35 ng of primer, 10–100 ng of viral RNA, 1 mM dNTPs, 10 mM DTT, 25 U of RNAguard (Amersham, Little Chalfont, United Kingdom), and 200 U of reverse transcriptase, was incubated at 42 °C for 60 min and then at 94 °C for 2 min.

Article Title: Characterization of the Mechanisms Controlling Greatwall Activity
Article Snippet: Extracts were prepared 50 min after ionophore addition by the same procedure as that described for CSF extracts. mRNAs encoding different wild-type (Wt) and mutant forms of Gwl were transcribed in vitro with SP6 RNA polymerase. .. For mRNA translation, CSF egg extracts were supplemented with dithiothreitol (DTT; 1 mM), RNAguard (0.4 U/μl extract; Amersham Biosciences), tRNA (0.1 μg/μl), and the corresponding mRNA (0.05 μg/μl extract) and incubated for 2 h at 20°C ( ).

Isolation:

Article Title: Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition
Article Snippet: Immunoprecipitations were performed using the FLAG Tagged Protein Immunoprecipitation Kit (FLAGIPT-1 Sigma), essentially following manufacturer’s recommendations. .. Typically, 1.5 mg of proteinase-K-treated mitochondria isolated from HEK293T cells expressing mtPABP1 or mtLUC were re-suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with Complete EDTA free Protease Inhibitor Cocktail (Roche) and RNAguard (GE Healthcare). .. Lysis was performed for 20 min rotating at 4°C, followed by centrifugation at 12 000g for 10 min to remove insoluble material.

Article Title: Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice
Article Snippet: RNA was isolated from snap frozen mouse prostates using the Qiagen Easy RNA isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer’s guidelines, including an on column DNAseI digestion. .. For reverse transcription 2 μg total RNA was incubated for 1 h at 37°C in buffer containing 50mM Tris-HCl pH 8.3, 75 mM KCl, 3mM MgCl2 , 10mM DTT and 1mM dNTPs, supplemented with 400 U M-MLV-reverse transcriptase (Thermo Fisher Scientific, Waltham, MA), 80 U RNAguard (Amersham Biosciences, Little Chalfont, UK) and 1 μg oligodT primer.

Article Title: Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery
Article Snippet: Paragraph title: RNA isolation and reverse transcriptase–polymerase chain reaction ... RNA was treated with TURBO DNase (Ambion, Austin, TX, USA) to remove any remaining contaminating DNA and then reverse-transcribed in the presence of RNAguard (GE Healthcare, Chalfont St Giles, UK) by using random hexamers and revert-aid reverse transcriptase (Fermentas International, York, UK).

Article Title: PPARγ ligands inhibit telomerase activity and hTERT expression through modulation of the Myc/Mad/Max network in colon cancer cells
Article Snippet: RNA analyses were performed by a semi-quantitative PCR method as previously described [ ]. .. Total RNA was isolated using the RNA fast Kit (Molecular System, Genenco, Milano, Italy). cDNA synthesis was performed with 4 μg of total RNA in a reaction volume of 40 μl containing 1.25 μg of random primers, l mM of dATP, dGTP, dCTP and dTTP (Invitrogen, Milano, Italy), 66 units of RNAguard (Amersham Biosciences, Cologno Monzese, Milano, Italy), 8 μl of 5× first strand buffer, 10 mM DTT, 300 units of MMLV reverse transcriptase (Invitrogen). .. PCR reactions were performed in a GeneAmp PCR System 9600 (Perkin Elmer, Monza, Milano, Italy), with 1 μl of cDNA reaction mixture in a volume of 50 μl containing 200 μM of dATP, dTTP, dGTP and dCTP, 1 μM of 5′ and 3′ primer and 1.25 units of TAQ DNA polymerase (Finnzymes, Milano, Italy).

Size-exclusion Chromatography:

Article Title: N1303K (c.3909C > G) Mutation and Splicing: Implication of Its c.[744-33GATT(6); 869+11C > T] Complex Allele in CFTR Exon 7 Aberrant Splicing
Article Snippet: Total mRNA was extracted from cell lysates using the RNeasy Mini Kit (Qiagen, Germany) and dissolved in 30 μ L of sterile water. cDNA synthesis was carried out at 37°C for 1 h after adjustment of the mixture to contain 5 μ L of 5x buffer (Gibco-BRL, France; 250 mmol/L of Tris-HCl pH 8.3, 375 mmol/L of KCl, 15 mmol/L of MgCl2 ), 10 mmol/L of dithiothreitol (Gibco-BRL, France), 1 mmol/L of dNTPs (Roche Diagnostics, France), 2.4 μ g of random hexamer primers, 10 μ L of RNA, 40 U RNAguard (Amersham Biosciences, Orsay, France), and 400 U Moloney murine leukemia virus (MMLV) reverse transcriptase. .. The PCR reaction (25 μ L) consisted of 5 μ L of cDNA, 2.5 μ L of 10x buffer, 2 mmol/L of MgCl2 , 250 μ mol/L of each dNTP, 10 pM of specific primers for the cDNA , and 3 U Taq polymerase.

Article Title: PPARγ ligands inhibit telomerase activity and hTERT expression through modulation of the Myc/Mad/Max network in colon cancer cells
Article Snippet: Total RNA was isolated using the RNA fast Kit (Molecular System, Genenco, Milano, Italy). cDNA synthesis was performed with 4 μg of total RNA in a reaction volume of 40 μl containing 1.25 μg of random primers, l mM of dATP, dGTP, dCTP and dTTP (Invitrogen, Milano, Italy), 66 units of RNAguard (Amersham Biosciences, Cologno Monzese, Milano, Italy), 8 μl of 5× first strand buffer, 10 mM DTT, 300 units of MMLV reverse transcriptase (Invitrogen). .. PCR reactions were performed in a GeneAmp PCR System 9600 (Perkin Elmer, Monza, Milano, Italy), with 1 μl of cDNA reaction mixture in a volume of 50 μl containing 200 μM of dATP, dTTP, dGTP and dCTP, 1 μM of 5′ and 3′ primer and 1.25 units of TAQ DNA polymerase (Finnzymes, Milano, Italy).

Labeling:

Article Title: Yeast targets for mRNA methylation
Article Snippet: The probes were DNA fragments identical to the in vitro transcribed sequences ( Supplementary Data 2 ) and were radio labeled with [α-32 P] dCTP using Rediprime Kit (Amersham). .. Following the washes 3.8 μg Ab and 3 μl RNAguard (Amersham Pharmacia) was added to the oligo(dT) bound mRNA, in a total volume of 500 μl PBS.

Article Title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis
Article Snippet: The ~ 450 bp PCR product was gel purified, and served as a template for digoxigenin-labeled cRNA probe synthesis. .. For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis). .. To visualize cellular expression sites of insl3 mRNA in zebrafish testis, whole mount in situ hybridization was performed on zebrafish testicular tissue, fixed in 4% paraformaldehyde in PBS (pH 7.4), as described by Westerfield (2000) http://zfin.org/zf_info/zfbook/chapt9/9.82.html with some modifications to the protocol.

Article Title: N. meningitidis 1681 is a member of the FinO family of RNA chaperones
Article Snippet: Standard deviations for the binding affinities were calculated based on three independent experiments. .. Strand exchange assays were performed as previously described on ice in 12 µL reaction mixtures containing 25 mM Tris (pH 8.1), 50 µg/mL bovine serum albumin (BSA), 5% (v/v) glycerol, 0.05% (v/v) β-mercaptoethanol and 10 mM NaCl, 2 units (1 µL) of RNAguard (Amersham), 10 µM protein, 5 nmoles of the labeled SIIA/B duplex (described above) to give a final duplex concentration of ∼400 pM and 2.5 µmoles of the unlabeled SIIA strand (to give a final concentration of ∼200 nM). .. Strand exchange assays were initiated by placing reaction tubes at 37°C and stopped at the indicated times by adding an equal volume of stop solution (5% (v/v) glycerol, 0.4% (w/v) SDS and 20 mM EDTA).

Titration:

Article Title: Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors
Article Snippet: IPs were carried out using 2 μg cellular RNA in 200 μl of the same buffer plus 1μl RNAguard (Amersham) for 1 hr at room temperature followed by three washes. .. RNA was purified from the IPs and inputs by phenol-chloroform extraction and precipitated using 4 μg of tRNA as a carrier.

Sequencing:

Article Title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis
Article Snippet: A zebrafish-specific insl3 -specific PCR product was generated with primer 2126 (5'- GGGCGGGTG TTATTAACCCTCACTAAA GGGAGTGAAGATGTGCGAGTGAAGC-3'; containing the T3 RNA polymerase promoter sequence [underlined]) and primer 2127 (5'-CCGGGGGGTG TAATACGACTCACTATA GGGGTACTGAATCAGTT CATTCATGGTGCA-3'; containing the T7 RNA polymerase promoter sequence [underlined]). .. For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis).

Article Title: Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins
Article Snippet: Paragraph title: RT-PCR and sequencing. ... The reaction mix (20 μl), which contained, in addition to pre-supplied buffer, 35 ng of primer, 10–100 ng of viral RNA, 1 mM dNTPs, 10 mM DTT, 25 U of RNAguard (Amersham, Little Chalfont, United Kingdom), and 200 U of reverse transcriptase, was incubated at 42 °C for 60 min and then at 94 °C for 2 min.

Article Title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein
Article Snippet: Twenty fmol of purified pre-mRNA that contained the WT NRS sequence, its reverse sequence or variant NRS sequences inserted in the C2 HIV-1 pre-mRNA were incubated for 2 h at 30°C in a 22 µl standard reaction containing 8 µl of HeLa cell nuclear extract ( ). .. Reverse transcriptions were performed by addition of 2 µl of this solution in 20 µl of 1 × MMLV buffer (Promega) containing 5 pmol of O-5825 primer, 1 mM of each dNTP, 1 µl of RNAguard (GE Healthcare) and 200 U of MMLV reverse transcriptase (Promega).

Selection:

Article Title: Maternal Argonaute 2 Is Essential for Early Mouse Development at the Maternal-Zygotic Transition
Article Snippet: Briefly, the embryo was lysed at 65°C for 1 min in lysis buffer (0.5% NP40, 1.25 mM each of dATP, dCTP, dGTP, and dTTP, 1 μl of 50 OD/ml oligo d(T)24 , 0.4 U primeRNase inhibitor [Roche] and 0.38 U RNAguard [Amersham Biotechnology, Piscataway, NJ [ in 1× MMLV (Moloney murine leukemia virus) buffer (Roche). .. All oligonucleotides used in RT-PCR analysis for Argonaute 1-4 and control primers used in the subtractive hybridization are listed in Supplementary Table S1.

Polyacrylamide Gel Electrophoresis:

Article Title: N. meningitidis 1681 is a member of the FinO family of RNA chaperones
Article Snippet: Strand exchange assays were performed as previously described on ice in 12 µL reaction mixtures containing 25 mM Tris (pH 8.1), 50 µg/mL bovine serum albumin (BSA), 5% (v/v) glycerol, 0.05% (v/v) β-mercaptoethanol and 10 mM NaCl, 2 units (1 µL) of RNAguard (Amersham), 10 µM protein, 5 nmoles of the labeled SIIA/B duplex (described above) to give a final duplex concentration of ∼400 pM and 2.5 µmoles of the unlabeled SIIA strand (to give a final concentration of ∼200 nM). .. Strand exchange assays were performed as previously described on ice in 12 µL reaction mixtures containing 25 mM Tris (pH 8.1), 50 µg/mL bovine serum albumin (BSA), 5% (v/v) glycerol, 0.05% (v/v) β-mercaptoethanol and 10 mM NaCl, 2 units (1 µL) of RNAguard (Amersham), 10 µM protein, 5 nmoles of the labeled SIIA/B duplex (described above) to give a final duplex concentration of ∼400 pM and 2.5 µmoles of the unlabeled SIIA strand (to give a final concentration of ∼200 nM).

Lysis:

Article Title: Maternal Argonaute 2 Is Essential for Early Mouse Development at the Maternal-Zygotic Transition
Article Snippet: Construction of cDNA libraries from single cells and the representational difference analysis (RDA) were done as previously described ( ; ; ) with a few modifications. .. Briefly, the embryo was lysed at 65°C for 1 min in lysis buffer (0.5% NP40, 1.25 mM each of dATP, dCTP, dGTP, and dTTP, 1 μl of 50 OD/ml oligo d(T)24 , 0.4 U primeRNase inhibitor [Roche] and 0.38 U RNAguard [Amersham Biotechnology, Piscataway, NJ [ in 1× MMLV (Moloney murine leukemia virus) buffer (Roche). .. The reaction was allowed to cool down at RT for 1 min and kept on ice before adding AMV-and MMLV-reverse transcriptases according to the manufacturer's instructions (Roche). cDNA libraries for subtractive hybridization were further treated with the tailing reaction using terminal deoxynucleotidyltransferase according the manufacturer's instructions (10533-065, Invitrogen) and subsequently amplified using the OL-1 primer to allow the insertion of restriction enzyme site.

Article Title: Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition
Article Snippet: Immunoprecipitations were performed using the FLAG Tagged Protein Immunoprecipitation Kit (FLAGIPT-1 Sigma), essentially following manufacturer’s recommendations. .. Typically, 1.5 mg of proteinase-K-treated mitochondria isolated from HEK293T cells expressing mtPABP1 or mtLUC were re-suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with Complete EDTA free Protease Inhibitor Cocktail (Roche) and RNAguard (GE Healthcare). .. Lysis was performed for 20 min rotating at 4°C, followed by centrifugation at 12 000g for 10 min to remove insoluble material.

Purification:

Article Title: Direct measurement of the mechanical work during translocation by the ribosome
Article Snippet: Ternary complexes were eluted with 600 µl elution buffer (TL + 250 mM imidazole) and dialyzed into Buffer TL for a total of 4 hr. .. Next, 50 µl purified ternary complexes (containing 0.2 U total aa-tRNA) were diluted with 390 µl (total 440 µl) buffer TL containing 1 mM GTP, 1 mM ATP, 40 U RNAguard (GE Healthcare, Piscataway, NJ) and 1 µM EF-G (final concentrations). .. Finally, the mixture was filtered with a 0.22 µm low protein- binding MILLEX-GV Durapore membrane, (Millipore, Billerica, MA) and kept on ice.

Article Title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis
Article Snippet: The ~ 450 bp PCR product was gel purified, and served as a template for digoxigenin-labeled cRNA probe synthesis. .. For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis).

Article Title: Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors
Article Snippet: 5 μl purified anti-m7G antibody or control antibody was prebound to 25 μl Protein A/G Sepharose in PBS, 0.01%Triton, 0.1 mg/ml BSA, 0.1 mg/ml polyU and 1 mM DTT for 30 min at room temperature followed by 2 washes. .. IPs were carried out using 2 μg cellular RNA in 200 μl of the same buffer plus 1μl RNAguard (Amersham) for 1 hr at room temperature followed by three washes.

Article Title: Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins
Article Snippet: The reaction mix (20 μl), which contained, in addition to pre-supplied buffer, 35 ng of primer, 10–100 ng of viral RNA, 1 mM dNTPs, 10 mM DTT, 25 U of RNAguard (Amersham, Little Chalfont, United Kingdom), and 200 U of reverse transcriptase, was incubated at 42 °C for 60 min and then at 94 °C for 2 min. .. The reaction mix (100 μl), which contained, in addition to pre-supplied buffer, 70 ng of primer pair, 1 μl of RT reaction product, 200 μM dNTPs, 2 mM MgCl2 , and 2.5 U of DNA polymerase, was incubated at 94 °C for 1 min, then 94 °C for 20 s, 50 °C for 20 s, 68 °C for 3 min, for a total of 35 cycles and a final 10-min extension at 68 °C.

Article Title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein
Article Snippet: To study the influence of protein 9G8 on NRS activity, splicing reactions were performed with a 1:1 mixture of HeLa cell cytoplasmic extract and HeLa cell nuclear extract and 180 or 270 ng of purified recombinant human 9G8 protein (produced in baculovirus) or 500 ng of commercial purified BSA used as a control. .. Reverse transcriptions were performed by addition of 2 µl of this solution in 20 µl of 1 × MMLV buffer (Promega) containing 5 pmol of O-5825 primer, 1 mM of each dNTP, 1 µl of RNAguard (GE Healthcare) and 200 U of MMLV reverse transcriptase (Promega).

SDS Page:

Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
Article Snippet: The 32 P-labeled PHGPx SECIS or U40C mutant RNAs (10 fmol) were incubated in buffer containing 0.7X PBS, 11 mM DTT, 250 ng/μL yeast tRNA and RNAguard (Amersham). .. The 32 P-labeled PHGPx SECIS or U40C mutant RNAs (10 fmol) were incubated in buffer containing 0.7X PBS, 11 mM DTT, 250 ng/μL yeast tRNA and RNAguard (Amersham).

In Vitro:

Article Title: Yeast targets for mRNA methylation
Article Snippet: The probes were DNA fragments identical to the in vitro transcribed sequences ( Supplementary Data 2 ) and were radio labeled with [α-32 P] dCTP using Rediprime Kit (Amersham). .. Following the washes 3.8 μg Ab and 3 μl RNAguard (Amersham Pharmacia) was added to the oligo(dT) bound mRNA, in a total volume of 500 μl PBS.

Article Title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis
Article Snippet: The ~ 450 bp PCR product was gel purified, and served as a template for digoxigenin-labeled cRNA probe synthesis. .. For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis). .. To visualize cellular expression sites of insl3 mRNA in zebrafish testis, whole mount in situ hybridization was performed on zebrafish testicular tissue, fixed in 4% paraformaldehyde in PBS (pH 7.4), as described by Westerfield (2000) http://zfin.org/zf_info/zfbook/chapt9/9.82.html with some modifications to the protocol.

Article Title: Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors
Article Snippet: G-mRNA and m7G-mRNA was in vitro transcribed from pBS c-Myc using the MaxiScript kit (Ambion). .. IPs were carried out using 2 μg cellular RNA in 200 μl of the same buffer plus 1μl RNAguard (Amersham) for 1 hr at room temperature followed by three washes.

Article Title: Characterization of the Mechanisms Controlling Greatwall Activity
Article Snippet: Extracts were prepared 50 min after ionophore addition by the same procedure as that described for CSF extracts. mRNAs encoding different wild-type (Wt) and mutant forms of Gwl were transcribed in vitro with SP6 RNA polymerase. .. For mRNA translation, CSF egg extracts were supplemented with dithiothreitol (DTT; 1 mM), RNAguard (0.4 U/μl extract; Amersham Biosciences), tRNA (0.1 μg/μl), and the corresponding mRNA (0.05 μg/μl extract) and incubated for 2 h at 20°C ( ).

Article Title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein
Article Snippet: Paragraph title: In vitro splicing assays ... Reverse transcriptions were performed by addition of 2 µl of this solution in 20 µl of 1 × MMLV buffer (Promega) containing 5 pmol of O-5825 primer, 1 mM of each dNTP, 1 µl of RNAguard (GE Healthcare) and 200 U of MMLV reverse transcriptase (Promega).

Electrophoresis:

Article Title: N1303K (c.3909C > G) Mutation and Splicing: Implication of Its c.[744-33GATT(6); 869+11C > T] Complex Allele in CFTR Exon 7 Aberrant Splicing
Article Snippet: Total mRNA was extracted from cell lysates using the RNeasy Mini Kit (Qiagen, Germany) and dissolved in 30 μ L of sterile water. cDNA synthesis was carried out at 37°C for 1 h after adjustment of the mixture to contain 5 μ L of 5x buffer (Gibco-BRL, France; 250 mmol/L of Tris-HCl pH 8.3, 375 mmol/L of KCl, 15 mmol/L of MgCl2 ), 10 mmol/L of dithiothreitol (Gibco-BRL, France), 1 mmol/L of dNTPs (Roche Diagnostics, France), 2.4 μ g of random hexamer primers, 10 μ L of RNA, 40 U RNAguard (Amersham Biosciences, Orsay, France), and 400 U Moloney murine leukemia virus (MMLV) reverse transcriptase. .. PCRs were performed using a 9700 GeneAmp Thermo Cycler (Perkin Elmer) with the following cycling conditions: initial denaturation (94°C, 2 min), followed by 30 cycles (94°C, 30 sec; 58°C, 30 sec; 72°C, 30 sec), and a final extension step (72°C, 5 min).

RNA Extraction:

Article Title: Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice
Article Snippet: Paragraph title: RNA extraction, cDNA preparation and QPCR analysis ... For reverse transcription 2 μg total RNA was incubated for 1 h at 37°C in buffer containing 50mM Tris-HCl pH 8.3, 75 mM KCl, 3mM MgCl2 , 10mM DTT and 1mM dNTPs, supplemented with 400 U M-MLV-reverse transcriptase (Thermo Fisher Scientific, Waltham, MA), 80 U RNAguard (Amersham Biosciences, Little Chalfont, UK) and 1 μg oligodT primer.

Recombinant:

Article Title: Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition
Article Snippet: Typically, 1.5 mg of proteinase-K-treated mitochondria isolated from HEK293T cells expressing mtPABP1 or mtLUC were re-suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with Complete EDTA free Protease Inhibitor Cocktail (Roche) and RNAguard (GE Healthcare). .. Typically, 1.5 mg of proteinase-K-treated mitochondria isolated from HEK293T cells expressing mtPABP1 or mtLUC were re-suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with Complete EDTA free Protease Inhibitor Cocktail (Roche) and RNAguard (GE Healthcare).

Article Title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein
Article Snippet: To study the influence of protein 9G8 on NRS activity, splicing reactions were performed with a 1:1 mixture of HeLa cell cytoplasmic extract and HeLa cell nuclear extract and 180 or 270 ng of purified recombinant human 9G8 protein (produced in baculovirus) or 500 ng of commercial purified BSA used as a control. .. Reverse transcriptions were performed by addition of 2 µl of this solution in 20 µl of 1 × MMLV buffer (Promega) containing 5 pmol of O-5825 primer, 1 mM of each dNTP, 1 µl of RNAguard (GE Healthcare) and 200 U of MMLV reverse transcriptase (Promega).

Agarose Gel Electrophoresis:

Article Title: N1303K (c.3909C > G) Mutation and Splicing: Implication of Its c.[744-33GATT(6); 869+11C > T] Complex Allele in CFTR Exon 7 Aberrant Splicing
Article Snippet: Total mRNA was extracted from cell lysates using the RNeasy Mini Kit (Qiagen, Germany) and dissolved in 30 μ L of sterile water. cDNA synthesis was carried out at 37°C for 1 h after adjustment of the mixture to contain 5 μ L of 5x buffer (Gibco-BRL, France; 250 mmol/L of Tris-HCl pH 8.3, 375 mmol/L of KCl, 15 mmol/L of MgCl2 ), 10 mmol/L of dithiothreitol (Gibco-BRL, France), 1 mmol/L of dNTPs (Roche Diagnostics, France), 2.4 μ g of random hexamer primers, 10 μ L of RNA, 40 U RNAguard (Amersham Biosciences, Orsay, France), and 400 U Moloney murine leukemia virus (MMLV) reverse transcriptase. .. PCRs were performed using a 9700 GeneAmp Thermo Cycler (Perkin Elmer) with the following cycling conditions: initial denaturation (94°C, 2 min), followed by 30 cycles (94°C, 30 sec; 58°C, 30 sec; 72°C, 30 sec), and a final extension step (72°C, 5 min).

In Situ:

Article Title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis
Article Snippet: Paragraph title: In situ hybridization using insl3 on zebrafish testis ... For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis).

Ethanol Precipitation:

Article Title: Yeast targets for mRNA methylation
Article Snippet: After ethanol precipitation, the mRNA samples from the Ab bound fraction and from the Ab depleted fraction were subjected to northern blot analysis. .. Following the washes 3.8 μg Ab and 3 μl RNAguard (Amersham Pharmacia) was added to the oligo(dT) bound mRNA, in a total volume of 500 μl PBS.

Article Title: Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins
Article Snippet: The reaction mix (20 μl), which contained, in addition to pre-supplied buffer, 35 ng of primer, 10–100 ng of viral RNA, 1 mM dNTPs, 10 mM DTT, 25 U of RNAguard (Amersham, Little Chalfont, United Kingdom), and 200 U of reverse transcriptase, was incubated at 42 °C for 60 min and then at 94 °C for 2 min. .. The reaction mix (100 μl), which contained, in addition to pre-supplied buffer, 70 ng of primer pair, 1 μl of RT reaction product, 200 μM dNTPs, 2 mM MgCl2 , and 2.5 U of DNA polymerase, was incubated at 94 °C for 1 min, then 94 °C for 20 s, 50 °C for 20 s, 68 °C for 3 min, for a total of 35 cycles and a final 10-min extension at 68 °C.

Laser Capture Microdissection:

Article Title: Identifying single-cell molecular programs by stochastic profiling
Article Snippet: Samples were eluted from the microdissection caps by adding 4 μl digestion buffer (1.25× MMLV RT buffer [Invitrogen], 100 μM dNTPs [Roche], 0.08 OD ml−1 oligo(dT)24 , and 250 μg ml−1 proteinase K [Sigma]) and incubating at 42°C for 1 hr. .. Digested samples were spun into PCR tubes and quenched with 1 μl of digestion stop buffer (1.5 U ml−1 Prime RNAse inhibitor [Eppendorf], 1.5 U ml−1 RNAguard [Amersham], and 5 mM freshly prepared PMSF).

Incubation:

Article Title: Following translation by single ribosomes one codon at a time
Article Snippet: In general, the following components were mixed in 50 μl of Buffer TL: 2 mM ATP, 1 mM GTP, 5 mM PEP (phosphoenol pyruvate), 1 OD total tRNA mixture, 24 μM EF-Tu, 4 μM EF-G, 0.02 mg ml-1 pyruvate kinase (Roche), 1 μl DEAE-purified S-100 enzymes, 40 U RNAguard (RNase inhibitor; Amersham) and amino-acid mixture containing 0.1 mM of each amino acid. .. In general, the following components were mixed in 50 μl of Buffer TL: 2 mM ATP, 1 mM GTP, 5 mM PEP (phosphoenol pyruvate), 1 OD total tRNA mixture, 24 μM EF-Tu, 4 μM EF-G, 0.02 mg ml-1 pyruvate kinase (Roche), 1 μl DEAE-purified S-100 enzymes, 40 U RNAguard (RNase inhibitor; Amersham) and amino-acid mixture containing 0.1 mM of each amino acid.

Article Title: Direct measurement of the mechanical work during translocation by the ribosome
Article Snippet: Briefly: The above reaction was bound to a Ni-NTA resin (30 min incubation at 4°C), and the resin washed three times with TL, with the addition of 20 mM imidazole. .. Next, 50 µl purified ternary complexes (containing 0.2 U total aa-tRNA) were diluted with 390 µl (total 440 µl) buffer TL containing 1 mM GTP, 1 mM ATP, 40 U RNAguard (GE Healthcare, Piscataway, NJ) and 1 µM EF-G (final concentrations).

Article Title: Identifying single-cell molecular programs by stochastic profiling
Article Snippet: Digested samples were spun into PCR tubes and quenched with 1 μl of digestion stop buffer (1.5 U ml−1 Prime RNAse inhibitor [Eppendorf], 1.5 U ml−1 RNAguard [Amersham], and 5 mM freshly prepared PMSF). .. Digested samples were spun into PCR tubes and quenched with 1 μl of digestion stop buffer (1.5 U ml−1 Prime RNAse inhibitor [Eppendorf], 1.5 U ml−1 RNAguard [Amersham], and 5 mM freshly prepared PMSF).

Article Title: Yeast targets for mRNA methylation
Article Snippet: Subsequently 30 μl proteinG magnetic beads (NEB) was added and incubated at 4°C for 1 h. The magnetic beads were separated together with the Ab bound mRNA. .. Following the washes 3.8 μg Ab and 3 μl RNAguard (Amersham Pharmacia) was added to the oligo(dT) bound mRNA, in a total volume of 500 μl PBS.

Article Title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis
Article Snippet: The ~ 450 bp PCR product was gel purified, and served as a template for digoxigenin-labeled cRNA probe synthesis. .. For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis). .. To visualize cellular expression sites of insl3 mRNA in zebrafish testis, whole mount in situ hybridization was performed on zebrafish testicular tissue, fixed in 4% paraformaldehyde in PBS (pH 7.4), as described by Westerfield (2000) http://zfin.org/zf_info/zfbook/chapt9/9.82.html with some modifications to the protocol.

Article Title: Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition
Article Snippet: Typically, 1.5 mg of proteinase-K-treated mitochondria isolated from HEK293T cells expressing mtPABP1 or mtLUC were re-suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with Complete EDTA free Protease Inhibitor Cocktail (Roche) and RNAguard (GE Healthcare). .. Typically, 1.5 mg of proteinase-K-treated mitochondria isolated from HEK293T cells expressing mtPABP1 or mtLUC were re-suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with Complete EDTA free Protease Inhibitor Cocktail (Roche) and RNAguard (GE Healthcare).

Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
Article Snippet: The samples were frozen on dry ice and combined with an equal volume of denaturing loading buffer (10 M urea, 1X TBE). .. The 32 P-labeled PHGPx SECIS or U40C mutant RNAs (10 fmol) were incubated in buffer containing 0.7X PBS, 11 mM DTT, 250 ng/μL yeast tRNA and RNAguard (Amersham). .. The indicated amounts of L30 were added last and the reactions incubated at 37°C for 30 min.

Article Title: Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice
Article Snippet: RNA quality was assessed using the RNA 6000 Nano kit in a 2100 Bioanalyser (Agilent, Santa Clara, CA). .. For reverse transcription 2 μg total RNA was incubated for 1 h at 37°C in buffer containing 50mM Tris-HCl pH 8.3, 75 mM KCl, 3mM MgCl2 , 10mM DTT and 1mM dNTPs, supplemented with 400 U M-MLV-reverse transcriptase (Thermo Fisher Scientific, Waltham, MA), 80 U RNAguard (Amersham Biosciences, Little Chalfont, UK) and 1 μg oligodT primer. .. The procedure for QPCR analysis was described earlier [ ].

Article Title: Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors
Article Snippet: IPs were carried out using 2 μg cellular RNA in 200 μl of the same buffer plus 1μl RNAguard (Amersham) for 1 hr at room temperature followed by three washes. .. When used, 0.001 ng in vitro transcribed RNA was added to the IPs.

Article Title: New functionally-enhanced soy proteins as food ingredients with anti-viral activity
Article Snippet: Equal volumes (300 μl) of allantoic virus, 108 EID50 , and 2 % of preparations were mixed and incubated for 30 min at 4 °C. .. The RNA was converted to full-length cDNA in the following reaction: 2.5 μl of DMPC (di-methyl pyrocarbonate) water, 5 μl of 5× First Strand buffer (Invitrogen), 0.5 μl of 10 mMdNTP mix (Amersham Biosciences), 2 μl of 50 mM UNi12 primer, 32 U of RNAguard (Amersham Biosciences), 200 U of MMLV reverse transcriptase (Invitrogen) and 5 μl RNA solution in total volume of 25 μl.

Article Title: Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins
Article Snippet: Five oligonucleotides, P17, P31, P46, P61, and P65, were used to prime the RT of viral RNA with Superscript II RT (Invitrogen, Carlsbad, California, United States). .. The reaction mix (20 μl), which contained, in addition to pre-supplied buffer, 35 ng of primer, 10–100 ng of viral RNA, 1 mM dNTPs, 10 mM DTT, 25 U of RNAguard (Amersham, Little Chalfont, United Kingdom), and 200 U of reverse transcriptase, was incubated at 42 °C for 60 min and then at 94 °C for 2 min. .. The five cDNA templates were then amplified using eight primer pairs, P1/P16, P2/P22, P3/P30, P4/P38, P5/P45, P6/P53, P7/P60, and P8/P64, and thermostable, recombinant Taq DNA polymerase.

Article Title: Characterization of the Mechanisms Controlling Greatwall Activity
Article Snippet: Extracts were prepared 50 min after ionophore addition by the same procedure as that described for CSF extracts. mRNAs encoding different wild-type (Wt) and mutant forms of Gwl were transcribed in vitro with SP6 RNA polymerase. .. For mRNA translation, CSF egg extracts were supplemented with dithiothreitol (DTT; 1 mM), RNAguard (0.4 U/μl extract; Amersham Biosciences), tRNA (0.1 μg/μl), and the corresponding mRNA (0.05 μg/μl extract) and incubated for 2 h at 20°C ( ). .. Immunoprecipitations/immunodepletions were performed using 10 μl of extracts, 10 μl of magnetic protein G-Dynabeads (Dynal), and 2 μg of each antibody.

Article Title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein
Article Snippet: Twenty fmol of purified pre-mRNA that contained the WT NRS sequence, its reverse sequence or variant NRS sequences inserted in the C2 HIV-1 pre-mRNA were incubated for 2 h at 30°C in a 22 µl standard reaction containing 8 µl of HeLa cell nuclear extract ( ). .. Reverse transcriptions were performed by addition of 2 µl of this solution in 20 µl of 1 × MMLV buffer (Promega) containing 5 pmol of O-5825 primer, 1 mM of each dNTP, 1 µl of RNAguard (GE Healthcare) and 200 U of MMLV reverse transcriptase (Promega).

Article Title: Molecular analysis of human cancer cells infected by an oncolytic HSV-1 reveals multiple upregulated cellular genes and a role for SOCS1 in virus replication
Article Snippet: A total of 2 µg total RNA was combined with 1 µg of oligo-dT (Invitrogen) and incubated at 70 °C for 10 min, and then chilled on ice. .. A total of 4 µl of 5 × first-strand synthesis buffer (Invitrogen), 2 µl of 0.1 M dithiothreotol, 0.5 µl of 0.1M dNTP, 0.5 µl of RNAguard (Amersham Biosciences, Piscataway, NJ) and 1 µl of M-MLV RT (Promega, San Luis Obispo, CA) were added and incubated at 37 °C for 1 h. The reaction was stopped by 10 min incubation at 70 °C. .. A total of 1 µl of synthesized cDNA was combined with primers (5 pmol) and 10 µl of 2 × DyNamo HS SYBR Green qPCR master mix (New England BioLabs, Ipswich, MA).

Irradiation:

Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
Article Snippet: The 32 P-labeled PHGPx SECIS or U40C mutant RNAs (10 fmol) were incubated in buffer containing 0.7X PBS, 11 mM DTT, 250 ng/μL yeast tRNA and RNAguard (Amersham). .. The indicated amounts of L30 were added last and the reactions incubated at 37°C for 30 min.

Produced:

Article Title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein
Article Snippet: To study the influence of protein 9G8 on NRS activity, splicing reactions were performed with a 1:1 mixture of HeLa cell cytoplasmic extract and HeLa cell nuclear extract and 180 or 270 ng of purified recombinant human 9G8 protein (produced in baculovirus) or 500 ng of commercial purified BSA used as a control. .. Reverse transcriptions were performed by addition of 2 µl of this solution in 20 µl of 1 × MMLV buffer (Promega) containing 5 pmol of O-5825 primer, 1 mM of each dNTP, 1 µl of RNAguard (GE Healthcare) and 200 U of MMLV reverse transcriptase (Promega).

Concentration Assay:

Article Title: Yeast targets for mRNA methylation
Article Snippet: Following the washes 3.8 μg Ab and 3 μl RNAguard (Amersham Pharmacia) was added to the oligo(dT) bound mRNA, in a total volume of 500 μl PBS. .. At this point all mRNA, both Ab bound and unbound, was eluted from the oligo(dT) magnetic beads by adding 50 μl water and incubated for 5 min.

Article Title: N. meningitidis 1681 is a member of the FinO family of RNA chaperones
Article Snippet: Standard deviations for the binding affinities were calculated based on three independent experiments. .. Strand exchange assays were performed as previously described on ice in 12 µL reaction mixtures containing 25 mM Tris (pH 8.1), 50 µg/mL bovine serum albumin (BSA), 5% (v/v) glycerol, 0.05% (v/v) β-mercaptoethanol and 10 mM NaCl, 2 units (1 µL) of RNAguard (Amersham), 10 µM protein, 5 nmoles of the labeled SIIA/B duplex (described above) to give a final duplex concentration of ∼400 pM and 2.5 µmoles of the unlabeled SIIA strand (to give a final concentration of ∼200 nM). .. Strand exchange assays were initiated by placing reaction tubes at 37°C and stopped at the indicated times by adding an equal volume of stop solution (5% (v/v) glycerol, 0.4% (w/v) SDS and 20 mM EDTA).

Staining:

Article Title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis
Article Snippet: For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis). .. For digoxigenin-RNA labeling by in vitro transcription, 500 ng PCR product was incubated at 37°C for 2.5 h in a 20 μl reaction volume containing 4 μl 5 × T3/T7 RNA buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl (29.7 units/μl) RNAguard (GE Healthcare, Fairfield, CT, USA) and 2 μl 10 × DIG RNA labeling mix (Roche), and either 2 μl (50 units/μl) T3 RNA polymerase (Epicentre; for sense cRNA probe synthesis) or 2 μl (50 units/μl) T7 RNA polymerase (Epicentre; for antisense cRNA probe synthesis).

Variant Assay:

Article Title: Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein
Article Snippet: Twenty fmol of purified pre-mRNA that contained the WT NRS sequence, its reverse sequence or variant NRS sequences inserted in the C2 HIV-1 pre-mRNA were incubated for 2 h at 30°C in a 22 µl standard reaction containing 8 µl of HeLa cell nuclear extract ( ). .. Reverse transcriptions were performed by addition of 2 µl of this solution in 20 µl of 1 × MMLV buffer (Promega) containing 5 pmol of O-5825 primer, 1 mM of each dNTP, 1 µl of RNAguard (GE Healthcare) and 200 U of MMLV reverse transcriptase (Promega).

Immunoaffinity Purification:

Article Title: Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition
Article Snippet: Paragraph title: Immunoaffinity purification of FLAG tagged fusion proteins and RNA isolation ... Typically, 1.5 mg of proteinase-K-treated mitochondria isolated from HEK293T cells expressing mtPABP1 or mtLUC were re-suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with Complete EDTA free Protease Inhibitor Cocktail (Roche) and RNAguard (GE Healthcare).

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