rnaseout recombinant ribonuclease inhibitor  (Thermo Fisher)


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    Structured Review

    Thermo Fisher rnaseout recombinant ribonuclease inhibitor
    Rnaseout Recombinant Ribonuclease Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnaseout recombinant ribonuclease inhibitor/product/Thermo Fisher
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    rnaseout recombinant ribonuclease inhibitor - by Bioz Stars, 2020-02
    99/100 stars

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    Clone Assay:

    Article Title: Defining nonsense-mediated mRNA decay intermediates in human cells
    Article Snippet: .. Baker, Catalog # JT9182–01) Ethanol (100% and 75%) (Koptec, Catalog # QDEV1001TP) 2-Propanol (Fisher Scientific, Catalog # A4514) Glycogen Solution (20 mg/ml) (Thermo Fisher Scienctific, Catalog # R0561) SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, Catalog # S11494) Phenol: Chloroform: Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris (pH 8.0), 1 mM EDTA (Sigma-Aldrich, Catalog # P2069) Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100×) (Thermo Fisher Scientific, Catalog # 78443) IgG from rabbit serum (Sigma-Aldrich, Catalog # I5006) DNase I (RQ1 RNase-Free DNase) (Promega, Catalog # M6101) RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific Invitrogen, Catalog # 10777019) Shrimp alkaline phosphatase (rSAP) (New England Biolabs, Catalog # M0371L) T4 RNA Ligase 2, truncated KQ (New England Biolabs, Catalog # M0373L) T4 polynucleotide kinase (T4 PNK) (New England Biolabs, Catalog # M0201L) T4 RNA Ligase, cloned, 5 U/μl (Thermo Fisher Scientific, Ambion, Catalog # AM2141) SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Invitrogen, Catalog # 18080085) Q5 High-Fidelity DNA Polymerase (New England Biolabs, Catalog # M0491S) Urea (EMD Millipore, Catalog # 9510) Acrylamide/bis-acrylamide 19:1 (40%) (Fisher scientific, Catalog # BP1406–1) Acrylamide/bis-acrylamide 29:1 (40%) (Fisher scientific, Catalog # BP1408–1) Ammonium persulfate, crystal (Mallinckrodt chemicals, Catalog # 3460–04) UltraPure TEMED (Thermo Fisher Scientific, Invitrogen, Catalog # 15524010) Sodium dodecyl sulfate (Sigma-Aldrich, Catalog # 75746) TWEEN 20 (Sigma-Aldrich, Catalog # P7949) TRITON X-100 (Sigma-Aldrich, Catalog # T9284) Nonidet P-40 (NP-40) (Sigma-Aldrich, Catalog # I3021) 2-Mercaptoethanol (Sigma-Aldrich, Catalog # M3148) RNase T1 (5 U/μl) (Thermo Fisher Scientific Ambion, AM2283) Halt Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Fisher Scientific, Catalog # PI78443) Bromophenol blue (Fisher Scientific, Catalog # B-392) 1 Kb Plus DNA Ladder (Thermo Fisher Scientific Invitrogen, Catalog # 10787018) Dynabeads Protein A (Thermo Fisher Scientific, Catalog # 10002D) Anti-phospho-UPF1 Ser1116 antibody (anti-phospho-UPF1 (Ser1127), EMD Millipore, Catalog # 07–1016) 3 M Sodium Acetate (pH 5.5) (Thermo Fisher Scientific Ambion, Catalog # AM9740) Trizma base (Sigma-Aldrich, Catalog # T6066) Boric acid (Millipore Sigma, Catalog # 2720–5KG) Ethylenediaminetetraacetic acid (EDTA), disodium salt (J.T. .. Baker, Catalog # 8993–01) Acrylamide:Bis-Acrylamide (19:1) 40% solution (Fisher Scientific, Catalog # BP1406–1) Ammonium persulfate (APS) (Acros Organics, Catalog # 401165000)

    Centrifugation:

    Article Title: The DEAD-box protein Dbp2p is linked to noncoding RNAs, the helicase Sen1p, and R-loops
    Article Snippet: .. The RNA was pelleted by centrifugation at 4°C for 25 min at 14,000 rpm and re-suspended in 50 μL of RNase-free H2 O. DNA was digested with DNase I (RNase-free; Invitrogen) and RNaseOUT (Invitrogen; 10777019) in 1× DNase I reaction buffer for 1 h at 37°C. .. Phenol/chloroform/LET extraction, precipitation, and re-suspension was then repeated as described above. cDNA libraries were prepared using Illumina ScriptSeq Complete Gold Kit for yeast (BGY1306) and ScriptSeq Index PCR Primers set 1 (RSBC10948) according to the manufacturer's protocol with the following adjustments.

    Luciferase:

    Article Title: RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome
    Article Snippet: .. Briefly, for each sample, 400-μl mixtures were prepared on ice containing 1 μg of cytoplasmic RNA, 0.2 ng of 5′-triphosphate luciferase RNA (Promega, L4561), and 0.2 ng of 5′-m7 G-capped mCherry RNA (TriLink) in a buffer containing 150 m m NaCl, 0.1% (v/v) IGEPAL CA-630, 10 m m Tris, pH 7.5, 1 m m DTT, and 0.2 units/μl RNaseOUT (Thermo Fisher Scientific, 10777019). .. From each mixture, 200 μl was set aside to be processed as input RNA, and the other 200 μl was incubated with bead-bound GST-eIF4E K119A and washed.

    Synthesized:

    Article Title: Transcription factor dimerization activates the p300 acetyltransferase
    Article Snippet: In vitro eRNA transcription eKlf6 eRNA corresponding to 496 nucleotides of the sense strand of human chr13:5802100-5802596 , was produced by in vitro transcription from a pMA plasmid containing a eKlf6 insert synthesized by GeneArt Gene Synthesis (Thermo Fisher). .. The in vitro transcription reaction was done in a final volume of 1 ml, using 1x T7 buffer, T7 RNA Polymerase and 1 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher).

    Article Title: miRNA Genetic Variants Alter Their Secondary Structure and Expression in Patients With RASopathies Syndromes
    Article Snippet: The miRNA cDNA was synthesized by adding 50 ng of total RNA and a specific stem loop primer at 0.5 µM in an initial volume of 10 µl, followed by an elongation step (70°C for 5 min). .. For the reverse transcription step, 1 U of SuperScript II reverse transcriptase (Life Technologies, Carlsbad, CA, USA); 1X First Strand Buffer, 0.5 mM dNTPs, and 40 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Life Technologies, Carlsbad, CA, USA) were included in a final volume of 20 µl.

    Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
    Article Snippet: Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.). .. The reaction was carried out at 37°C for 50 min and heat inactivated at 70°C for 15 min. One microliter of the synthesized cDNA solution was subjected to a semi-quantitative PCR of 25 (for GAPDH) or 30 (for TM4SF5) cycles consisting of denaturation for 40 sec at 95°C, annealing for 40 sec at 58°C, and extension for 40 sec at 72°C.

    Construct:

    Article Title: Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E
    Article Snippet: The lacY gene in each of the chromosomal lac fusion constructs was deleted, so that active transport of externally added IPTG could not occur into the cells. .. The reaction mixture was similar to that of Caruthers et al. , with enzyme activity being determined at 30°C in a 20-μl volume [containing 20 mM Tris–Cl (pH 8), 10 mM MgCl2 , 10 mM NaCl, 0.1 mM dithiothreitol and 20 U of RNaseOUT recombinant ribonuclease inhibitor (Thermo Fisher Scientific, USA)] after addition of 1 pmol of the protein preparation(s) and 2 pmol of substrate; at different time points, 6 μl aliquots were removed and the reactions were terminated by addition to each of an equal volume of loading dye [95% formamide, 18 mM EDTA and 0.025% each of sodium dodecyl sulphate (SDS), xylene cyanol, and bromophenol blue].

    SYBR Green Assay:

    Article Title: Genomic copy-number loss is rescued by self-limiting production of DNA circles
    Article Snippet: Samples for RT were incubated with Revert Aid (Fermentas, 1 L per sample), Revert Aid buffer (Fisher), Oligo-dT (100 M, Fisher, 1 L per sample) and RNaseOut recombinant ribonuclease inhibitor (Invitrogen, 1 L per sample) at 42°C for 1 hour in a 20 L reaction. .. For qPCR analysis, 8 L samples (diluted ChIP samples or diluted RT samples) and 1 L of each primer (20nM) were mixed with Quantifast SYBR green master mix (Qiagen, 10 L per reaction) and placed in a BioRad CFX96 Real-Time System.

    Article Title: Prenatal Bisphenol A Exposure in Mice Induces Multitissue Multiomics Disruptions Linking to Cardiometabolic Disorders
    Article Snippet: Concentration and quality of the RNA were measured using the Thermo Fisher Scientific NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Waltham, MA) following the manufacturer’s protocol with minor modification by adding RNaseOUT Recombinant Ribonuclease Inhibitor (20 U/μL) from Invitrogen (Carlsbad, CA). .. Upon completion, the cDNA was stored at −20°C until quantitative RT-PCR (qPCR) was performed. qPCR was done using the PowerUP SYBR Green Master Mix from Applied Biosystems.

    Incubation:

    Article Title: Transcription factor dimerization activates the p300 acetyltransferase
    Article Snippet: 50 μg of pMA_Klf6 plasmid was linearized with 80 U of Kpn I-HF in a final volume of 100 μl and incubated at 37 °C for 14-16h. .. The in vitro transcription reaction was done in a final volume of 1 ml, using 1x T7 buffer, T7 RNA Polymerase and 1 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher).

    Article Title: RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome
    Article Snippet: Briefly, for each sample, 400-μl mixtures were prepared on ice containing 1 μg of cytoplasmic RNA, 0.2 ng of 5′-triphosphate luciferase RNA (Promega, L4561), and 0.2 ng of 5′-m7 G-capped mCherry RNA (TriLink) in a buffer containing 150 m m NaCl, 0.1% (v/v) IGEPAL CA-630, 10 m m Tris, pH 7.5, 1 m m DTT, and 0.2 units/μl RNaseOUT (Thermo Fisher Scientific, 10777019). .. From each mixture, 200 μl was set aside to be processed as input RNA, and the other 200 μl was incubated with bead-bound GST-eIF4E K119A and washed.

    Article Title: Genomic copy-number loss is rescued by self-limiting production of DNA circles
    Article Snippet: .. Samples for RT were incubated with Revert Aid (Fermentas, 1 L per sample), Revert Aid buffer (Fisher), Oligo-dT (100 M, Fisher, 1 L per sample) and RNaseOut recombinant ribonuclease inhibitor (Invitrogen, 1 L per sample) at 42°C for 1 hour in a 20 L reaction. ..

    Article Title: The effect of estradiol on the expression of estrogen, progesterone, androgen, and prolactin receptors in human peritoneal fibroblasts
    Article Snippet: .. A master mixture containing 4 μl 5× First Strand Buffer, 2 μl 0.1 M DTT, 1 μl RNaseOut Recombinant Ribonuclease Inhibitor (40 units/μl) (Invitrogen, CA) was added, and incubated at 42°C for 2 min. One microliter (200 units) of SuperScript II (GIBCO BRL) was added to each reaction and incubated for 50 min at 42°C, to inactivate the enzyme by heating at 70°C for 15 min. Oligonucleotide primers for real-time reverse transcriptase polymerase chain reaction (RT-PCR) amplification of reverse-transcribed complimentary DNA (cDNA) were selected with the aid of software program Oligo 4.0 (National Bioscience, Inc., Plymouth, MN). .. Sequences of the oligonucleotides used for amplification of estradiol-α receptor (ER-α), estradiol-β receptor (ER-β), progesterone receptor (P-R), androgen receptor (A-R), prolactin receptor (PRL-R), and β-actin mRNA are shown in Table .

    Article Title: Fragile X mental retardation protein modulates the stability of its m6A-marked messenger RNA targets
    Article Snippet: .. The fresh cell pellet was re-suspended with 2 mL of RIP lysis buffer (300 m m NaCl, 50 m m Tris•HCl pH 7.4, 30 m m EDTA and 0.5% Triton X-100) supplemented with protease inhibitor cocktail and 200 U/ml RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher) and incubated on ice for 10 min. .. The cell lysate was passed through a small needle (e.g. 26G) several times and centrifuged at 20000 g, 4°C for 15 min. 80 μL of supernatant was saved as input (10%) for RNA extraction, and another 80 μL of supernatant was set aside for protein analysis.

    Article Title: Prenatal Bisphenol A Exposure in Mice Induces Multitissue Multiomics Disruptions Linking to Cardiometabolic Disorders
    Article Snippet: Concentration and quality of the RNA were measured using the Thermo Fisher Scientific NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Waltham, MA) following the manufacturer’s protocol with minor modification by adding RNaseOUT Recombinant Ribonuclease Inhibitor (20 U/μL) from Invitrogen (Carlsbad, CA). .. Following the addition of reverse-transcription components, samples were incubated at the following thermocycler conditions: 10 minutes at 25°C, 120 minutes at 37°C, and then 5 minutes at 85°C.

    Amplification:

    Article Title: The effect of estradiol on the expression of estrogen, progesterone, androgen, and prolactin receptors in human peritoneal fibroblasts
    Article Snippet: .. A master mixture containing 4 μl 5× First Strand Buffer, 2 μl 0.1 M DTT, 1 μl RNaseOut Recombinant Ribonuclease Inhibitor (40 units/μl) (Invitrogen, CA) was added, and incubated at 42°C for 2 min. One microliter (200 units) of SuperScript II (GIBCO BRL) was added to each reaction and incubated for 50 min at 42°C, to inactivate the enzyme by heating at 70°C for 15 min. Oligonucleotide primers for real-time reverse transcriptase polymerase chain reaction (RT-PCR) amplification of reverse-transcribed complimentary DNA (cDNA) were selected with the aid of software program Oligo 4.0 (National Bioscience, Inc., Plymouth, MN). .. Sequences of the oligonucleotides used for amplification of estradiol-α receptor (ER-α), estradiol-β receptor (ER-β), progesterone receptor (P-R), androgen receptor (A-R), prolactin receptor (PRL-R), and β-actin mRNA are shown in Table .

    Activity Assay:

    Article Title: Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E
    Article Snippet: .. The reaction mixture was similar to that of Caruthers et al. , with enzyme activity being determined at 30°C in a 20-μl volume [containing 20 mM Tris–Cl (pH 8), 10 mM MgCl2 , 10 mM NaCl, 0.1 mM dithiothreitol and 20 U of RNaseOUT recombinant ribonuclease inhibitor (Thermo Fisher Scientific, USA)] after addition of 1 pmol of the protein preparation(s) and 2 pmol of substrate; at different time points, 6 μl aliquots were removed and the reactions were terminated by addition to each of an equal volume of loading dye [95% formamide, 18 mM EDTA and 0.025% each of sodium dodecyl sulphate (SDS), xylene cyanol, and bromophenol blue]. .. The samples were denatured at 85°C for 3 min and then subjected to electrophoresis on an 8 M urea–15% polyacrylamide gel ( ).

    Expressing:

    Article Title: Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E
    Article Snippet: When rne-lac expression was measured in strains also carrying the rne+ shelter plasmid pHYD1613 or its derivatives, the lacZ gene on the plasmid was disrupted with the lacZ4526 ::Tn10 dTet insertion that was sourced from strain GJ2278 ( ). .. The reaction mixture was similar to that of Caruthers et al. , with enzyme activity being determined at 30°C in a 20-μl volume [containing 20 mM Tris–Cl (pH 8), 10 mM MgCl2 , 10 mM NaCl, 0.1 mM dithiothreitol and 20 U of RNaseOUT recombinant ribonuclease inhibitor (Thermo Fisher Scientific, USA)] after addition of 1 pmol of the protein preparation(s) and 2 pmol of substrate; at different time points, 6 μl aliquots were removed and the reactions were terminated by addition to each of an equal volume of loading dye [95% formamide, 18 mM EDTA and 0.025% each of sodium dodecyl sulphate (SDS), xylene cyanol, and bromophenol blue].

    Modification:

    Article Title: Defining nonsense-mediated mRNA decay intermediates in human cells
    Article Snippet: Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific Gibco, Catalog # 11995) Fetal bovine serum (FBS) (VWR, Catalog # 97068–085) Okadaic acid, free acid > 98% (LC Laboratories, Catalog # O-2220) TRIzol Reagent (Thermo Fisher Scientific, Catalog # 15596018) Chloroform (J.T. .. Baker, Catalog # JT9182–01) Ethanol (100% and 75%) (Koptec, Catalog # QDEV1001TP) 2-Propanol (Fisher Scientific, Catalog # A4514) Glycogen Solution (20 mg/ml) (Thermo Fisher Scienctific, Catalog # R0561) SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, Catalog # S11494) Phenol: Chloroform: Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris (pH 8.0), 1 mM EDTA (Sigma-Aldrich, Catalog # P2069) Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100×) (Thermo Fisher Scientific, Catalog # 78443) IgG from rabbit serum (Sigma-Aldrich, Catalog # I5006) DNase I (RQ1 RNase-Free DNase) (Promega, Catalog # M6101) RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific Invitrogen, Catalog # 10777019) Shrimp alkaline phosphatase (rSAP) (New England Biolabs, Catalog # M0371L) T4 RNA Ligase 2, truncated KQ (New England Biolabs, Catalog # M0373L) T4 polynucleotide kinase (T4 PNK) (New England Biolabs, Catalog # M0201L) T4 RNA Ligase, cloned, 5 U/μl (Thermo Fisher Scientific, Ambion, Catalog # AM2141) SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Invitrogen, Catalog # 18080085) Q5 High-Fidelity DNA Polymerase (New England Biolabs, Catalog # M0491S) Urea (EMD Millipore, Catalog # 9510) Acrylamide/bis-acrylamide 19:1 (40%) (Fisher scientific, Catalog # BP1406–1) Acrylamide/bis-acrylamide 29:1 (40%) (Fisher scientific, Catalog # BP1408–1) Ammonium persulfate, crystal (Mallinckrodt chemicals, Catalog # 3460–04) UltraPure TEMED (Thermo Fisher Scientific, Invitrogen, Catalog # 15524010) Sodium dodecyl sulfate (Sigma-Aldrich, Catalog # 75746) TWEEN 20 (Sigma-Aldrich, Catalog # P7949) TRITON X-100 (Sigma-Aldrich, Catalog # T9284) Nonidet P-40 (NP-40) (Sigma-Aldrich, Catalog # I3021) 2-Mercaptoethanol (Sigma-Aldrich, Catalog # M3148) RNase T1 (5 U/μl) (Thermo Fisher Scientific Ambion, AM2283) Halt Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Fisher Scientific, Catalog # PI78443) Bromophenol blue (Fisher Scientific, Catalog # B-392) 1 Kb Plus DNA Ladder (Thermo Fisher Scientific Invitrogen, Catalog # 10787018) Dynabeads Protein A (Thermo Fisher Scientific, Catalog # 10002D) Anti-phospho-UPF1 Ser1116 antibody (anti-phospho-UPF1 (Ser1127), EMD Millipore, Catalog # 07–1016) 3 M Sodium Acetate (pH 5.5) (Thermo Fisher Scientific Ambion, Catalog # AM9740) Trizma base (Sigma-Aldrich, Catalog # T6066) Boric acid (Millipore Sigma, Catalog # 2720–5KG) Ethylenediaminetetraacetic acid (EDTA), disodium salt (J.T.

    Article Title: Prenatal Bisphenol A Exposure in Mice Induces Multitissue Multiomics Disruptions Linking to Cardiometabolic Disorders
    Article Snippet: .. Concentration and quality of the RNA were measured using the Thermo Fisher Scientific NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Waltham, MA) following the manufacturer’s protocol with minor modification by adding RNaseOUT Recombinant Ribonuclease Inhibitor (20 U/μL) from Invitrogen (Carlsbad, CA). .. Following the addition of reverse-transcription components, samples were incubated at the following thermocycler conditions: 10 minutes at 25°C, 120 minutes at 37°C, and then 5 minutes at 85°C.

    Western Blot:

    Article Title: RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome
    Article Snippet: From each culture, cytoplasmic RNA was prepared and qualified as described , and reduced levels of CE upon geldanamycin treatment were confirmed by Western blotting. .. Briefly, for each sample, 400-μl mixtures were prepared on ice containing 1 μg of cytoplasmic RNA, 0.2 ng of 5′-triphosphate luciferase RNA (Promega, L4561), and 0.2 ng of 5′-m7 G-capped mCherry RNA (TriLink) in a buffer containing 150 m m NaCl, 0.1% (v/v) IGEPAL CA-630, 10 m m Tris, pH 7.5, 1 m m DTT, and 0.2 units/μl RNaseOUT (Thermo Fisher Scientific, 10777019).

    Real-time Polymerase Chain Reaction:

    Article Title: RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome
    Article Snippet: Briefly, for each sample, 400-μl mixtures were prepared on ice containing 1 μg of cytoplasmic RNA, 0.2 ng of 5′-triphosphate luciferase RNA (Promega, L4561), and 0.2 ng of 5′-m7 G-capped mCherry RNA (TriLink) in a buffer containing 150 m m NaCl, 0.1% (v/v) IGEPAL CA-630, 10 m m Tris, pH 7.5, 1 m m DTT, and 0.2 units/μl RNaseOUT (Thermo Fisher Scientific, 10777019). .. Input and eIF4E-recovered RNA were purified using the Direct-zol RNA MiniPrep kit (Zymo Research, R2053), eluting with 25 μl of RNase-free water. cDNA synthesis and quantitative PCR were performed as described , using the same primers described therein.

    Article Title: Genomic copy-number loss is rescued by self-limiting production of DNA circles
    Article Snippet: Paragraph title: RNA preparation and qPCR analysis ... Samples for RT were incubated with Revert Aid (Fermentas, 1 L per sample), Revert Aid buffer (Fisher), Oligo-dT (100 M, Fisher, 1 L per sample) and RNaseOut recombinant ribonuclease inhibitor (Invitrogen, 1 L per sample) at 42°C for 1 hour in a 20 L reaction.

    Article Title: Prenatal Bisphenol A Exposure in Mice Induces Multitissue Multiomics Disruptions Linking to Cardiometabolic Disorders
    Article Snippet: Concentration and quality of the RNA were measured using the Thermo Fisher Scientific NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Waltham, MA) following the manufacturer’s protocol with minor modification by adding RNaseOUT Recombinant Ribonuclease Inhibitor (20 U/μL) from Invitrogen (Carlsbad, CA). .. Upon completion, the cDNA was stored at −20°C until quantitative RT-PCR (qPCR) was performed. qPCR was done using the PowerUP SYBR Green Master Mix from Applied Biosystems.

    Sequencing:

    Article Title: The single cell transcriptional landscape of mammalian organogenesis
    Article Snippet: Paragraph title: sci-RNA-seq3 library preparation and sequencing ... 14 μL of first-strand reaction mix, containing 8 μL 5× Superscript IV First-Strand Buffer (Invitrogen), 2 μl 100 mM DTT (Invitrogen), 2 μl SuperScript IV reverse transcriptase (200 U/μl, Invitrogen), 2 μL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), was then added to each well.

    Ligation:

    Article Title: The single cell transcriptional landscape of mammalian organogenesis
    Article Snippet: 14 μL of first-strand reaction mix, containing 8 μL 5× Superscript IV First-Strand Buffer (Invitrogen), 2 μl 100 mM DTT (Invitrogen), 2 μl SuperScript IV reverse transcriptase (200 U/μl, Invitrogen), 2 μL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), was then added to each well. .. After ligation reaction, 60 μL nuclei dilution buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 1% BSA) was added into each well.

    Protease Inhibitor:

    Article Title: Fragile X mental retardation protein modulates the stability of its m6A-marked messenger RNA targets
    Article Snippet: .. The fresh cell pellet was re-suspended with 2 mL of RIP lysis buffer (300 m m NaCl, 50 m m Tris•HCl pH 7.4, 30 m m EDTA and 0.5% Triton X-100) supplemented with protease inhibitor cocktail and 200 U/ml RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher) and incubated on ice for 10 min. .. The cell lysate was passed through a small needle (e.g. 26G) several times and centrifuged at 20000 g, 4°C for 15 min. 80 μL of supernatant was saved as input (10%) for RNA extraction, and another 80 μL of supernatant was set aside for protein analysis.

    Polymerase Chain Reaction:

    Article Title: The DEAD-box protein Dbp2p is linked to noncoding RNAs, the helicase Sen1p, and R-loops
    Article Snippet: The RNA was pelleted by centrifugation at 4°C for 25 min at 14,000 rpm and re-suspended in 50 μL of RNase-free H2 O. DNA was digested with DNase I (RNase-free; Invitrogen) and RNaseOUT (Invitrogen; 10777019) in 1× DNase I reaction buffer for 1 h at 37°C. .. Phenol/chloroform/LET extraction, precipitation, and re-suspension was then repeated as described above. cDNA libraries were prepared using Illumina ScriptSeq Complete Gold Kit for yeast (BGY1306) and ScriptSeq Index PCR Primers set 1 (RSBC10948) according to the manufacturer's protocol with the following adjustments.

    Article Title: The effect of estradiol on the expression of estrogen, progesterone, androgen, and prolactin receptors in human peritoneal fibroblasts
    Article Snippet: .. A master mixture containing 4 μl 5× First Strand Buffer, 2 μl 0.1 M DTT, 1 μl RNaseOut Recombinant Ribonuclease Inhibitor (40 units/μl) (Invitrogen, CA) was added, and incubated at 42°C for 2 min. One microliter (200 units) of SuperScript II (GIBCO BRL) was added to each reaction and incubated for 50 min at 42°C, to inactivate the enzyme by heating at 70°C for 15 min. Oligonucleotide primers for real-time reverse transcriptase polymerase chain reaction (RT-PCR) amplification of reverse-transcribed complimentary DNA (cDNA) were selected with the aid of software program Oligo 4.0 (National Bioscience, Inc., Plymouth, MN). .. Sequences of the oligonucleotides used for amplification of estradiol-α receptor (ER-α), estradiol-β receptor (ER-β), progesterone receptor (P-R), androgen receptor (A-R), prolactin receptor (PRL-R), and β-actin mRNA are shown in Table .

    Article Title: Differential Regulation of Root Arginine Catabolism and Polyamine Metabolism in Clubroot-Susceptible and Partially Resistant Arabidopsis Genotypes
    Article Snippet: To check for genomic DNA contamination, a PCR reaction was carried out on the RNA samples using Actin8 primers. .. First-strand complementary DNA (cDNA) synthesis was performed in a 20- μ L total reaction volume using 250 ng of DNAse-digested total RNA, 1 μ m oligo(dT) primer, 1 m m dNTPs, 1× first-strand buffer (Invitrogen), 20 m m dithiothreitol (Invitrogen), 40 units RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 200 units SuperScript II reverse transcriptase (Invitrogen) by incubating for 2 h at 42°C.

    Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
    Article Snippet: Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.). .. The reaction was carried out at 37°C for 50 min and heat inactivated at 70°C for 15 min. One microliter of the synthesized cDNA solution was subjected to a semi-quantitative PCR of 25 (for GAPDH) or 30 (for TM4SF5) cycles consisting of denaturation for 40 sec at 95°C, annealing for 40 sec at 58°C, and extension for 40 sec at 72°C.

    Sonication:

    Article Title: The single cell transcriptional landscape of mammalian organogenesis
    Article Snippet: sci-RNA-seq3 library preparation and sequencing Thawed nuclei were permeabilized with 0.2% TritonX-100 (in nuclei wash buffer) for 3 min on ice, and briefly sonicated (Diagenode, 12 sec on low power mode) to reduce nuclei clumping. .. 14 μL of first-strand reaction mix, containing 8 μL 5× Superscript IV First-Strand Buffer (Invitrogen), 2 μl 100 mM DTT (Invitrogen), 2 μl SuperScript IV reverse transcriptase (200 U/μl, Invitrogen), 2 μL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), was then added to each well.

    Recombinant:

    Article Title: Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E
    Article Snippet: .. The reaction mixture was similar to that of Caruthers et al. , with enzyme activity being determined at 30°C in a 20-μl volume [containing 20 mM Tris–Cl (pH 8), 10 mM MgCl2 , 10 mM NaCl, 0.1 mM dithiothreitol and 20 U of RNaseOUT recombinant ribonuclease inhibitor (Thermo Fisher Scientific, USA)] after addition of 1 pmol of the protein preparation(s) and 2 pmol of substrate; at different time points, 6 μl aliquots were removed and the reactions were terminated by addition to each of an equal volume of loading dye [95% formamide, 18 mM EDTA and 0.025% each of sodium dodecyl sulphate (SDS), xylene cyanol, and bromophenol blue]. .. The samples were denatured at 85°C for 3 min and then subjected to electrophoresis on an 8 M urea–15% polyacrylamide gel ( ).

    Article Title: The single cell transcriptional landscape of mammalian organogenesis
    Article Snippet: .. 14 μL of first-strand reaction mix, containing 8 μL 5× Superscript IV First-Strand Buffer (Invitrogen), 2 μl 100 mM DTT (Invitrogen), 2 μl SuperScript IV reverse transcriptase (200 U/μl, Invitrogen), 2 μL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), was then added to each well. .. After ligation reaction, 60 μL nuclei dilution buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 1% BSA) was added into each well.

    Article Title: Defining nonsense-mediated mRNA decay intermediates in human cells
    Article Snippet: .. Baker, Catalog # JT9182–01) Ethanol (100% and 75%) (Koptec, Catalog # QDEV1001TP) 2-Propanol (Fisher Scientific, Catalog # A4514) Glycogen Solution (20 mg/ml) (Thermo Fisher Scienctific, Catalog # R0561) SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, Catalog # S11494) Phenol: Chloroform: Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris (pH 8.0), 1 mM EDTA (Sigma-Aldrich, Catalog # P2069) Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100×) (Thermo Fisher Scientific, Catalog # 78443) IgG from rabbit serum (Sigma-Aldrich, Catalog # I5006) DNase I (RQ1 RNase-Free DNase) (Promega, Catalog # M6101) RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific Invitrogen, Catalog # 10777019) Shrimp alkaline phosphatase (rSAP) (New England Biolabs, Catalog # M0371L) T4 RNA Ligase 2, truncated KQ (New England Biolabs, Catalog # M0373L) T4 polynucleotide kinase (T4 PNK) (New England Biolabs, Catalog # M0201L) T4 RNA Ligase, cloned, 5 U/μl (Thermo Fisher Scientific, Ambion, Catalog # AM2141) SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Invitrogen, Catalog # 18080085) Q5 High-Fidelity DNA Polymerase (New England Biolabs, Catalog # M0491S) Urea (EMD Millipore, Catalog # 9510) Acrylamide/bis-acrylamide 19:1 (40%) (Fisher scientific, Catalog # BP1406–1) Acrylamide/bis-acrylamide 29:1 (40%) (Fisher scientific, Catalog # BP1408–1) Ammonium persulfate, crystal (Mallinckrodt chemicals, Catalog # 3460–04) UltraPure TEMED (Thermo Fisher Scientific, Invitrogen, Catalog # 15524010) Sodium dodecyl sulfate (Sigma-Aldrich, Catalog # 75746) TWEEN 20 (Sigma-Aldrich, Catalog # P7949) TRITON X-100 (Sigma-Aldrich, Catalog # T9284) Nonidet P-40 (NP-40) (Sigma-Aldrich, Catalog # I3021) 2-Mercaptoethanol (Sigma-Aldrich, Catalog # M3148) RNase T1 (5 U/μl) (Thermo Fisher Scientific Ambion, AM2283) Halt Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Fisher Scientific, Catalog # PI78443) Bromophenol blue (Fisher Scientific, Catalog # B-392) 1 Kb Plus DNA Ladder (Thermo Fisher Scientific Invitrogen, Catalog # 10787018) Dynabeads Protein A (Thermo Fisher Scientific, Catalog # 10002D) Anti-phospho-UPF1 Ser1116 antibody (anti-phospho-UPF1 (Ser1127), EMD Millipore, Catalog # 07–1016) 3 M Sodium Acetate (pH 5.5) (Thermo Fisher Scientific Ambion, Catalog # AM9740) Trizma base (Sigma-Aldrich, Catalog # T6066) Boric acid (Millipore Sigma, Catalog # 2720–5KG) Ethylenediaminetetraacetic acid (EDTA), disodium salt (J.T. .. Baker, Catalog # 8993–01) Acrylamide:Bis-Acrylamide (19:1) 40% solution (Fisher Scientific, Catalog # BP1406–1) Ammonium persulfate (APS) (Acros Organics, Catalog # 401165000)

    Article Title: Transcription factor dimerization activates the p300 acetyltransferase
    Article Snippet: .. The in vitro transcription reaction was done in a final volume of 1 ml, using 1x T7 buffer, T7 RNA Polymerase and 1 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher). .. After incubation for 2 h at 37°C, 0.5 U of TURBO™ DNase (2 U/μl) (Thermo Fisher) and 1μM CaCl2 was added to the reaction and incubated for 30 min at 37 °C.

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation
    Article Snippet: .. Pellets were lysed on ice for 15 min in TBS containing 1% DDM (Thermso Fisher), 0.4 U/μl of RNaseOUT™ Recombinant Ribonuclease Inhibitor (Thermo Fisher) and 1 mM DTT (Sigma). .. Supernatants were transferred to microcentrifuge tubes containing 50 μl of washed Pierce® Anti‐HA Agarose beads (Thermo scientific).

    Article Title: miRNA Genetic Variants Alter Their Secondary Structure and Expression in Patients With RASopathies Syndromes
    Article Snippet: .. For the reverse transcription step, 1 U of SuperScript II reverse transcriptase (Life Technologies, Carlsbad, CA, USA); 1X First Strand Buffer, 0.5 mM dNTPs, and 40 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Life Technologies, Carlsbad, CA, USA) were included in a final volume of 20 µl. ..

    Article Title: RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome
    Article Snippet: Cap status analysis was performed by binding of cytoplasmic RNA to recombinant GST-eIF4E K119A as described ( ). .. Briefly, for each sample, 400-μl mixtures were prepared on ice containing 1 μg of cytoplasmic RNA, 0.2 ng of 5′-triphosphate luciferase RNA (Promega, L4561), and 0.2 ng of 5′-m7 G-capped mCherry RNA (TriLink) in a buffer containing 150 m m NaCl, 0.1% (v/v) IGEPAL CA-630, 10 m m Tris, pH 7.5, 1 m m DTT, and 0.2 units/μl RNaseOUT (Thermo Fisher Scientific, 10777019).

    Article Title: Genomic copy-number loss is rescued by self-limiting production of DNA circles
    Article Snippet: .. Samples for RT were incubated with Revert Aid (Fermentas, 1 L per sample), Revert Aid buffer (Fisher), Oligo-dT (100 M, Fisher, 1 L per sample) and RNaseOut recombinant ribonuclease inhibitor (Invitrogen, 1 L per sample) at 42°C for 1 hour in a 20 L reaction. ..

    Article Title: The effect of estradiol on the expression of estrogen, progesterone, androgen, and prolactin receptors in human peritoneal fibroblasts
    Article Snippet: .. A master mixture containing 4 μl 5× First Strand Buffer, 2 μl 0.1 M DTT, 1 μl RNaseOut Recombinant Ribonuclease Inhibitor (40 units/μl) (Invitrogen, CA) was added, and incubated at 42°C for 2 min. One microliter (200 units) of SuperScript II (GIBCO BRL) was added to each reaction and incubated for 50 min at 42°C, to inactivate the enzyme by heating at 70°C for 15 min. Oligonucleotide primers for real-time reverse transcriptase polymerase chain reaction (RT-PCR) amplification of reverse-transcribed complimentary DNA (cDNA) were selected with the aid of software program Oligo 4.0 (National Bioscience, Inc., Plymouth, MN). .. Sequences of the oligonucleotides used for amplification of estradiol-α receptor (ER-α), estradiol-β receptor (ER-β), progesterone receptor (P-R), androgen receptor (A-R), prolactin receptor (PRL-R), and β-actin mRNA are shown in Table .

    Article Title: Fragile X mental retardation protein modulates the stability of its m6A-marked messenger RNA targets
    Article Snippet: .. The fresh cell pellet was re-suspended with 2 mL of RIP lysis buffer (300 m m NaCl, 50 m m Tris•HCl pH 7.4, 30 m m EDTA and 0.5% Triton X-100) supplemented with protease inhibitor cocktail and 200 U/ml RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher) and incubated on ice for 10 min. .. The cell lysate was passed through a small needle (e.g. 26G) several times and centrifuged at 20000 g, 4°C for 15 min. 80 μL of supernatant was saved as input (10%) for RNA extraction, and another 80 μL of supernatant was set aside for protein analysis.

    Article Title: Prenatal Bisphenol A Exposure in Mice Induces Multitissue Multiomics Disruptions Linking to Cardiometabolic Disorders
    Article Snippet: .. Concentration and quality of the RNA were measured using the Thermo Fisher Scientific NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Waltham, MA) following the manufacturer’s protocol with minor modification by adding RNaseOUT Recombinant Ribonuclease Inhibitor (20 U/μL) from Invitrogen (Carlsbad, CA). .. Following the addition of reverse-transcription components, samples were incubated at the following thermocycler conditions: 10 minutes at 25°C, 120 minutes at 37°C, and then 5 minutes at 85°C.

    Article Title: Differential Regulation of Root Arginine Catabolism and Polyamine Metabolism in Clubroot-Susceptible and Partially Resistant Arabidopsis Genotypes
    Article Snippet: .. First-strand complementary DNA (cDNA) synthesis was performed in a 20- μ L total reaction volume using 250 ng of DNAse-digested total RNA, 1 μ m oligo(dT) primer, 1 m m dNTPs, 1× first-strand buffer (Invitrogen), 20 m m dithiothreitol (Invitrogen), 40 units RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 200 units SuperScript II reverse transcriptase (Invitrogen) by incubating for 2 h at 42°C. .. The cDNAs were diluted by 1/40 and their quality was confirmed by conventional RT-PCR with Actin8 primers ( ).

    Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
    Article Snippet: .. Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.). .. The reaction was carried out at 37°C for 50 min and heat inactivated at 70°C for 15 min. One microliter of the synthesized cDNA solution was subjected to a semi-quantitative PCR of 25 (for GAPDH) or 30 (for TM4SF5) cycles consisting of denaturation for 40 sec at 95°C, annealing for 40 sec at 58°C, and extension for 40 sec at 72°C.

    Molecular Weight:

    Article Title: Transcription factor dimerization activates the p300 acetyltransferase
    Article Snippet: The in vitro transcription reaction was done in a final volume of 1 ml, using 1x T7 buffer, T7 RNA Polymerase and 1 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher). .. Buffer was exchanged into 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM TCEP using Amicon Ultra-0.5ml Centrifugal Filters (Molecular weight cut off = 3kDa; EMD Millipore).

    Staining:

    Article Title: Defining nonsense-mediated mRNA decay intermediates in human cells
    Article Snippet: .. Baker, Catalog # JT9182–01) Ethanol (100% and 75%) (Koptec, Catalog # QDEV1001TP) 2-Propanol (Fisher Scientific, Catalog # A4514) Glycogen Solution (20 mg/ml) (Thermo Fisher Scienctific, Catalog # R0561) SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, Catalog # S11494) Phenol: Chloroform: Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris (pH 8.0), 1 mM EDTA (Sigma-Aldrich, Catalog # P2069) Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100×) (Thermo Fisher Scientific, Catalog # 78443) IgG from rabbit serum (Sigma-Aldrich, Catalog # I5006) DNase I (RQ1 RNase-Free DNase) (Promega, Catalog # M6101) RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific Invitrogen, Catalog # 10777019) Shrimp alkaline phosphatase (rSAP) (New England Biolabs, Catalog # M0371L) T4 RNA Ligase 2, truncated KQ (New England Biolabs, Catalog # M0373L) T4 polynucleotide kinase (T4 PNK) (New England Biolabs, Catalog # M0201L) T4 RNA Ligase, cloned, 5 U/μl (Thermo Fisher Scientific, Ambion, Catalog # AM2141) SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Invitrogen, Catalog # 18080085) Q5 High-Fidelity DNA Polymerase (New England Biolabs, Catalog # M0491S) Urea (EMD Millipore, Catalog # 9510) Acrylamide/bis-acrylamide 19:1 (40%) (Fisher scientific, Catalog # BP1406–1) Acrylamide/bis-acrylamide 29:1 (40%) (Fisher scientific, Catalog # BP1408–1) Ammonium persulfate, crystal (Mallinckrodt chemicals, Catalog # 3460–04) UltraPure TEMED (Thermo Fisher Scientific, Invitrogen, Catalog # 15524010) Sodium dodecyl sulfate (Sigma-Aldrich, Catalog # 75746) TWEEN 20 (Sigma-Aldrich, Catalog # P7949) TRITON X-100 (Sigma-Aldrich, Catalog # T9284) Nonidet P-40 (NP-40) (Sigma-Aldrich, Catalog # I3021) 2-Mercaptoethanol (Sigma-Aldrich, Catalog # M3148) RNase T1 (5 U/μl) (Thermo Fisher Scientific Ambion, AM2283) Halt Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Fisher Scientific, Catalog # PI78443) Bromophenol blue (Fisher Scientific, Catalog # B-392) 1 Kb Plus DNA Ladder (Thermo Fisher Scientific Invitrogen, Catalog # 10787018) Dynabeads Protein A (Thermo Fisher Scientific, Catalog # 10002D) Anti-phospho-UPF1 Ser1116 antibody (anti-phospho-UPF1 (Ser1127), EMD Millipore, Catalog # 07–1016) 3 M Sodium Acetate (pH 5.5) (Thermo Fisher Scientific Ambion, Catalog # AM9740) Trizma base (Sigma-Aldrich, Catalog # T6066) Boric acid (Millipore Sigma, Catalog # 2720–5KG) Ethylenediaminetetraacetic acid (EDTA), disodium salt (J.T. .. Baker, Catalog # 8993–01) Acrylamide:Bis-Acrylamide (19:1) 40% solution (Fisher Scientific, Catalog # BP1406–1) Ammonium persulfate (APS) (Acros Organics, Catalog # 401165000)

    RNA Sequencing Assay:

    Article Title: The DEAD-box protein Dbp2p is linked to noncoding RNAs, the helicase Sen1p, and R-loops
    Article Snippet: Paragraph title: RNA-seq ... The RNA was pelleted by centrifugation at 4°C for 25 min at 14,000 rpm and re-suspended in 50 μL of RNase-free H2 O. DNA was digested with DNase I (RNase-free; Invitrogen) and RNaseOUT (Invitrogen; 10777019) in 1× DNase I reaction buffer for 1 h at 37°C.

    Magnetic Beads:

    Article Title: Fragile X mental retardation protein modulates the stability of its m6A-marked messenger RNA targets
    Article Snippet: The fresh cell pellet was re-suspended with 2 mL of RIP lysis buffer (300 m m NaCl, 50 m m Tris•HCl pH 7.4, 30 m m EDTA and 0.5% Triton X-100) supplemented with protease inhibitor cocktail and 200 U/ml RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher) and incubated on ice for 10 min. .. 10 μg of rabbit anti-FMRP polyclonal antibody (abcam, ab17722) or 10 μg of normal rabbit IgG (as a control; Cell Signaling Technology) was added to the re-suspended magnetic beads, and the mixture was rotated continuously at room temperature for 30 min. After incubation, beads-antibody complex was washed four times with 0.7 mL of RIP lysis buffer, mixed with 0.8 mL of cleared cell lysate and then rotated continuously at 4°C for 3 h. The tubes were centrifuged briefly and placed on a magnetic separator.

    Isolation:

    Article Title: The DEAD-box protein Dbp2p is linked to noncoding RNAs, the helicase Sen1p, and R-loops
    Article Snippet: The aqueous phase was isolated and vortexed with 500 μL phenol/chloroform/LET. .. The RNA was pelleted by centrifugation at 4°C for 25 min at 14,000 rpm and re-suspended in 50 μL of RNase-free H2 O. DNA was digested with DNase I (RNase-free; Invitrogen) and RNaseOUT (Invitrogen; 10777019) in 1× DNase I reaction buffer for 1 h at 37°C.

    Article Title: miRNA Genetic Variants Alter Their Secondary Structure and Expression in Patients With RASopathies Syndromes
    Article Snippet: The quantity and quality of the isolated RNA were evaluated using a NanoDrop ND-2000c (Fisher Scientific, Ottawa, Ontario, Canada). .. For the reverse transcription step, 1 U of SuperScript II reverse transcriptase (Life Technologies, Carlsbad, CA, USA); 1X First Strand Buffer, 0.5 mM dNTPs, and 40 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Life Technologies, Carlsbad, CA, USA) were included in a final volume of 20 µl.

    Article Title: Differential Regulation of Root Arginine Catabolism and Polyamine Metabolism in Clubroot-Susceptible and Partially Resistant Arabidopsis Genotypes
    Article Snippet: Total RNA was extracted from approximately 30 mg of frozen root samples using the SV Total RNA Isolation kit (Promega). .. First-strand complementary DNA (cDNA) synthesis was performed in a 20- μ L total reaction volume using 250 ng of DNAse-digested total RNA, 1 μ m oligo(dT) primer, 1 m m dNTPs, 1× first-strand buffer (Invitrogen), 20 m m dithiothreitol (Invitrogen), 40 units RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 200 units SuperScript II reverse transcriptase (Invitrogen) by incubating for 2 h at 42°C.

    Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
    Article Snippet: Reverse transcription (RT) PCR Total RNA was isolated with the TRI Reagent® according to the manufacturer's instructions (MRC). .. Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.).

    Size-exclusion Chromatography:

    Article Title: The single cell transcriptional landscape of mammalian organogenesis
    Article Snippet: sci-RNA-seq3 library preparation and sequencing Thawed nuclei were permeabilized with 0.2% TritonX-100 (in nuclei wash buffer) for 3 min on ice, and briefly sonicated (Diagenode, 12 sec on low power mode) to reduce nuclei clumping. .. 14 μL of first-strand reaction mix, containing 8 μL 5× Superscript IV First-Strand Buffer (Invitrogen), 2 μl 100 mM DTT (Invitrogen), 2 μl SuperScript IV reverse transcriptase (200 U/μl, Invitrogen), 2 μL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), was then added to each well.

    Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
    Article Snippet: Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.). .. The reaction was carried out at 37°C for 50 min and heat inactivated at 70°C for 15 min. One microliter of the synthesized cDNA solution was subjected to a semi-quantitative PCR of 25 (for GAPDH) or 30 (for TM4SF5) cycles consisting of denaturation for 40 sec at 95°C, annealing for 40 sec at 58°C, and extension for 40 sec at 72°C.

    Purification:

    Article Title: The DEAD-box protein Dbp2p is linked to noncoding RNAs, the helicase Sen1p, and R-loops
    Article Snippet: The RNA was pelleted by centrifugation at 4°C for 25 min at 14,000 rpm and re-suspended in 50 μL of RNase-free H2 O. DNA was digested with DNase I (RNase-free; Invitrogen) and RNaseOUT (Invitrogen; 10777019) in 1× DNase I reaction buffer for 1 h at 37°C. .. To avoid depletion of short RNAs including snoRNAs, purification of the rRNA-depleted samples was performed by ethanol precipitation instead of AMPure bead purification. cDNA was purified by the MinElute PCR Purification kit (Qiagen) in place of the Agencourt AMPure XP system.

    Article Title: RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome
    Article Snippet: Briefly, for each sample, 400-μl mixtures were prepared on ice containing 1 μg of cytoplasmic RNA, 0.2 ng of 5′-triphosphate luciferase RNA (Promega, L4561), and 0.2 ng of 5′-m7 G-capped mCherry RNA (TriLink) in a buffer containing 150 m m NaCl, 0.1% (v/v) IGEPAL CA-630, 10 m m Tris, pH 7.5, 1 m m DTT, and 0.2 units/μl RNaseOUT (Thermo Fisher Scientific, 10777019). .. Input and eIF4E-recovered RNA were purified using the Direct-zol RNA MiniPrep kit (Zymo Research, R2053), eluting with 25 μl of RNase-free water. cDNA synthesis and quantitative PCR were performed as described , using the same primers described therein.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The effect of estradiol on the expression of estrogen, progesterone, androgen, and prolactin receptors in human peritoneal fibroblasts
    Article Snippet: .. A master mixture containing 4 μl 5× First Strand Buffer, 2 μl 0.1 M DTT, 1 μl RNaseOut Recombinant Ribonuclease Inhibitor (40 units/μl) (Invitrogen, CA) was added, and incubated at 42°C for 2 min. One microliter (200 units) of SuperScript II (GIBCO BRL) was added to each reaction and incubated for 50 min at 42°C, to inactivate the enzyme by heating at 70°C for 15 min. Oligonucleotide primers for real-time reverse transcriptase polymerase chain reaction (RT-PCR) amplification of reverse-transcribed complimentary DNA (cDNA) were selected with the aid of software program Oligo 4.0 (National Bioscience, Inc., Plymouth, MN). .. Sequences of the oligonucleotides used for amplification of estradiol-α receptor (ER-α), estradiol-β receptor (ER-β), progesterone receptor (P-R), androgen receptor (A-R), prolactin receptor (PRL-R), and β-actin mRNA are shown in Table .

    Article Title: Differential Regulation of Root Arginine Catabolism and Polyamine Metabolism in Clubroot-Susceptible and Partially Resistant Arabidopsis Genotypes
    Article Snippet: First-strand complementary DNA (cDNA) synthesis was performed in a 20- μ L total reaction volume using 250 ng of DNAse-digested total RNA, 1 μ m oligo(dT) primer, 1 m m dNTPs, 1× first-strand buffer (Invitrogen), 20 m m dithiothreitol (Invitrogen), 40 units RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 200 units SuperScript II reverse transcriptase (Invitrogen) by incubating for 2 h at 42°C. .. The cDNAs were diluted by 1/40 and their quality was confirmed by conventional RT-PCR with Actin8 primers ( ).

    Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
    Article Snippet: Paragraph title: Reverse transcription (RT) PCR ... Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.).

    Immunoprecipitation:

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation
    Article Snippet: Paragraph title: Immunoprecipitation, RNA extraction and RT‐PCRs ... Pellets were lysed on ice for 15 min in TBS containing 1% DDM (Thermso Fisher), 0.4 U/μl of RNaseOUT™ Recombinant Ribonuclease Inhibitor (Thermo Fisher) and 1 mM DTT (Sigma).

    Quantitative RT-PCR:

    Article Title: Prenatal Bisphenol A Exposure in Mice Induces Multitissue Multiomics Disruptions Linking to Cardiometabolic Disorders
    Article Snippet: Paragraph title: Quantitative RT-PCR analysis ... Concentration and quality of the RNA were measured using the Thermo Fisher Scientific NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Waltham, MA) following the manufacturer’s protocol with minor modification by adding RNaseOUT Recombinant Ribonuclease Inhibitor (20 U/μL) from Invitrogen (Carlsbad, CA).

    Article Title: Differential Regulation of Root Arginine Catabolism and Polyamine Metabolism in Clubroot-Susceptible and Partially Resistant Arabidopsis Genotypes
    Article Snippet: Paragraph title: Real-Time RT-PCR ... First-strand complementary DNA (cDNA) synthesis was performed in a 20- μ L total reaction volume using 250 ng of DNAse-digested total RNA, 1 μ m oligo(dT) primer, 1 m m dNTPs, 1× first-strand buffer (Invitrogen), 20 m m dithiothreitol (Invitrogen), 40 units RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 200 units SuperScript II reverse transcriptase (Invitrogen) by incubating for 2 h at 42°C.

    Lysis:

    Article Title: Fragile X mental retardation protein modulates the stability of its m6A-marked messenger RNA targets
    Article Snippet: .. The fresh cell pellet was re-suspended with 2 mL of RIP lysis buffer (300 m m NaCl, 50 m m Tris•HCl pH 7.4, 30 m m EDTA and 0.5% Triton X-100) supplemented with protease inhibitor cocktail and 200 U/ml RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher) and incubated on ice for 10 min. .. The cell lysate was passed through a small needle (e.g. 26G) several times and centrifuged at 20000 g, 4°C for 15 min. 80 μL of supernatant was saved as input (10%) for RNA extraction, and another 80 μL of supernatant was set aside for protein analysis.

    Article Title: Overcoming Steric Restrictions of VRC01 HIV-1 Neutralizing Antibodies through Immunization
    Article Snippet: .. Individual B cells were sorted using the FACS ARIA II into a 96 well plate containing 20 ul of lysis buffer (20 U of RNase out (Thermo Fisher Scientific, Cat#: 10777019), 5 ul 5X Superscript IV RT buffer, 1.25 ul of 0.1M DTT, 0.625 ul of 10% Igepal, 13 ul nuclease free H2 0) in each well. .. PCR amplification and sequencing of VH and VL genes RNA was reverse transcribed to cDNA.

    Chromatin Immunoprecipitation:

    Article Title: Genomic copy-number loss is rescued by self-limiting production of DNA circles
    Article Snippet: Samples for RT were incubated with Revert Aid (Fermentas, 1 L per sample), Revert Aid buffer (Fisher), Oligo-dT (100 M, Fisher, 1 L per sample) and RNaseOut recombinant ribonuclease inhibitor (Invitrogen, 1 L per sample) at 42°C for 1 hour in a 20 L reaction. .. For qPCR analysis, 8 L samples (diluted ChIP samples or diluted RT samples) and 1 L of each primer (20nM) were mixed with Quantifast SYBR green master mix (Qiagen, 10 L per reaction) and placed in a BioRad CFX96 Real-Time System.

    Plasmid Preparation:

    Article Title: Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E
    Article Snippet: When rne-lac expression was measured in strains also carrying the rne+ shelter plasmid pHYD1613 or its derivatives, the lacZ gene on the plasmid was disrupted with the lacZ4526 ::Tn10 dTet insertion that was sourced from strain GJ2278 ( ). .. The reaction mixture was similar to that of Caruthers et al. , with enzyme activity being determined at 30°C in a 20-μl volume [containing 20 mM Tris–Cl (pH 8), 10 mM MgCl2 , 10 mM NaCl, 0.1 mM dithiothreitol and 20 U of RNaseOUT recombinant ribonuclease inhibitor (Thermo Fisher Scientific, USA)] after addition of 1 pmol of the protein preparation(s) and 2 pmol of substrate; at different time points, 6 μl aliquots were removed and the reactions were terminated by addition to each of an equal volume of loading dye [95% formamide, 18 mM EDTA and 0.025% each of sodium dodecyl sulphate (SDS), xylene cyanol, and bromophenol blue].

    Article Title: Transcription factor dimerization activates the p300 acetyltransferase
    Article Snippet: 50 μg of pMA_Klf6 plasmid was linearized with 80 U of Kpn I-HF in a final volume of 100 μl and incubated at 37 °C for 14-16h. .. The in vitro transcription reaction was done in a final volume of 1 ml, using 1x T7 buffer, T7 RNA Polymerase and 1 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher).

    Software:

    Article Title: The effect of estradiol on the expression of estrogen, progesterone, androgen, and prolactin receptors in human peritoneal fibroblasts
    Article Snippet: .. A master mixture containing 4 μl 5× First Strand Buffer, 2 μl 0.1 M DTT, 1 μl RNaseOut Recombinant Ribonuclease Inhibitor (40 units/μl) (Invitrogen, CA) was added, and incubated at 42°C for 2 min. One microliter (200 units) of SuperScript II (GIBCO BRL) was added to each reaction and incubated for 50 min at 42°C, to inactivate the enzyme by heating at 70°C for 15 min. Oligonucleotide primers for real-time reverse transcriptase polymerase chain reaction (RT-PCR) amplification of reverse-transcribed complimentary DNA (cDNA) were selected with the aid of software program Oligo 4.0 (National Bioscience, Inc., Plymouth, MN). .. Sequences of the oligonucleotides used for amplification of estradiol-α receptor (ER-α), estradiol-β receptor (ER-β), progesterone receptor (P-R), androgen receptor (A-R), prolactin receptor (PRL-R), and β-actin mRNA are shown in Table .

    Electrophoresis:

    Article Title: Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E
    Article Snippet: The reaction mixture was similar to that of Caruthers et al. , with enzyme activity being determined at 30°C in a 20-μl volume [containing 20 mM Tris–Cl (pH 8), 10 mM MgCl2 , 10 mM NaCl, 0.1 mM dithiothreitol and 20 U of RNaseOUT recombinant ribonuclease inhibitor (Thermo Fisher Scientific, USA)] after addition of 1 pmol of the protein preparation(s) and 2 pmol of substrate; at different time points, 6 μl aliquots were removed and the reactions were terminated by addition to each of an equal volume of loading dye [95% formamide, 18 mM EDTA and 0.025% each of sodium dodecyl sulphate (SDS), xylene cyanol, and bromophenol blue]. .. The samples were denatured at 85°C for 3 min and then subjected to electrophoresis on an 8 M urea–15% polyacrylamide gel ( ).

    RNA Extraction:

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation
    Article Snippet: Paragraph title: Immunoprecipitation, RNA extraction and RT‐PCRs ... Pellets were lysed on ice for 15 min in TBS containing 1% DDM (Thermso Fisher), 0.4 U/μl of RNaseOUT™ Recombinant Ribonuclease Inhibitor (Thermo Fisher) and 1 mM DTT (Sigma).

    Article Title: miRNA Genetic Variants Alter Their Secondary Structure and Expression in Patients With RASopathies Syndromes
    Article Snippet: Paragraph title: DNA and RNA Extraction and cDNA Synthesis ... For the reverse transcription step, 1 U of SuperScript II reverse transcriptase (Life Technologies, Carlsbad, CA, USA); 1X First Strand Buffer, 0.5 mM dNTPs, and 40 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Life Technologies, Carlsbad, CA, USA) were included in a final volume of 20 µl.

    Article Title: Fragile X mental retardation protein modulates the stability of its m6A-marked messenger RNA targets
    Article Snippet: The fresh cell pellet was re-suspended with 2 mL of RIP lysis buffer (300 m m NaCl, 50 m m Tris•HCl pH 7.4, 30 m m EDTA and 0.5% Triton X-100) supplemented with protease inhibitor cocktail and 200 U/ml RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher) and incubated on ice for 10 min. .. The cell lysate was passed through a small needle (e.g. 26G) several times and centrifuged at 20000 g, 4°C for 15 min. 80 μL of supernatant was saved as input (10%) for RNA extraction, and another 80 μL of supernatant was set aside for protein analysis.

    Binding Assay:

    Article Title: RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome
    Article Snippet: Cap status analysis was performed by binding of cytoplasmic RNA to recombinant GST-eIF4E K119A as described ( ). .. Briefly, for each sample, 400-μl mixtures were prepared on ice containing 1 μg of cytoplasmic RNA, 0.2 ng of 5′-triphosphate luciferase RNA (Promega, L4561), and 0.2 ng of 5′-m7 G-capped mCherry RNA (TriLink) in a buffer containing 150 m m NaCl, 0.1% (v/v) IGEPAL CA-630, 10 m m Tris, pH 7.5, 1 m m DTT, and 0.2 units/μl RNaseOUT (Thermo Fisher Scientific, 10777019).

    Agarose Gel Electrophoresis:

    Article Title: Differential Regulation of Root Arginine Catabolism and Polyamine Metabolism in Clubroot-Susceptible and Partially Resistant Arabidopsis Genotypes
    Article Snippet: The total RNA was quantified with a spectrophotometer and electrophoresed on a 2% agarose gel to check the concentration and integrity. .. First-strand complementary DNA (cDNA) synthesis was performed in a 20- μ L total reaction volume using 250 ng of DNAse-digested total RNA, 1 μ m oligo(dT) primer, 1 m m dNTPs, 1× first-strand buffer (Invitrogen), 20 m m dithiothreitol (Invitrogen), 40 units RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 200 units SuperScript II reverse transcriptase (Invitrogen) by incubating for 2 h at 42°C.

    In Vitro:

    Article Title: Transcription factor dimerization activates the p300 acetyltransferase
    Article Snippet: .. The in vitro transcription reaction was done in a final volume of 1 ml, using 1x T7 buffer, T7 RNA Polymerase and 1 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher). .. After incubation for 2 h at 37°C, 0.5 U of TURBO™ DNase (2 U/μl) (Thermo Fisher) and 1μM CaCl2 was added to the reaction and incubated for 30 min at 37 °C.

    Ethanol Precipitation:

    Article Title: The DEAD-box protein Dbp2p is linked to noncoding RNAs, the helicase Sen1p, and R-loops
    Article Snippet: The RNA was pelleted by centrifugation at 4°C for 25 min at 14,000 rpm and re-suspended in 50 μL of RNase-free H2 O. DNA was digested with DNase I (RNase-free; Invitrogen) and RNaseOUT (Invitrogen; 10777019) in 1× DNase I reaction buffer for 1 h at 37°C. .. To avoid depletion of short RNAs including snoRNAs, purification of the rRNA-depleted samples was performed by ethanol precipitation instead of AMPure bead purification. cDNA was purified by the MinElute PCR Purification kit (Qiagen) in place of the Agencourt AMPure XP system.

    Spectrophotometry:

    Article Title: Differential Regulation of Root Arginine Catabolism and Polyamine Metabolism in Clubroot-Susceptible and Partially Resistant Arabidopsis Genotypes
    Article Snippet: The total RNA was quantified with a spectrophotometer and electrophoresed on a 2% agarose gel to check the concentration and integrity. .. First-strand complementary DNA (cDNA) synthesis was performed in a 20- μ L total reaction volume using 250 ng of DNAse-digested total RNA, 1 μ m oligo(dT) primer, 1 m m dNTPs, 1× first-strand buffer (Invitrogen), 20 m m dithiothreitol (Invitrogen), 40 units RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 200 units SuperScript II reverse transcriptase (Invitrogen) by incubating for 2 h at 42°C.

    Produced:

    Article Title: Transcription factor dimerization activates the p300 acetyltransferase
    Article Snippet: In vitro eRNA transcription eKlf6 eRNA corresponding to 496 nucleotides of the sense strand of human chr13:5802100-5802596 , was produced by in vitro transcription from a pMA plasmid containing a eKlf6 insert synthesized by GeneArt Gene Synthesis (Thermo Fisher). .. The in vitro transcription reaction was done in a final volume of 1 ml, using 1x T7 buffer, T7 RNA Polymerase and 1 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher).

    Concentration Assay:

    Article Title: Prenatal Bisphenol A Exposure in Mice Induces Multitissue Multiomics Disruptions Linking to Cardiometabolic Disorders
    Article Snippet: .. Concentration and quality of the RNA were measured using the Thermo Fisher Scientific NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Waltham, MA) following the manufacturer’s protocol with minor modification by adding RNaseOUT Recombinant Ribonuclease Inhibitor (20 U/μL) from Invitrogen (Carlsbad, CA). .. Following the addition of reverse-transcription components, samples were incubated at the following thermocycler conditions: 10 minutes at 25°C, 120 minutes at 37°C, and then 5 minutes at 85°C.

    Article Title: Differential Regulation of Root Arginine Catabolism and Polyamine Metabolism in Clubroot-Susceptible and Partially Resistant Arabidopsis Genotypes
    Article Snippet: The total RNA was quantified with a spectrophotometer and electrophoresed on a 2% agarose gel to check the concentration and integrity. .. First-strand complementary DNA (cDNA) synthesis was performed in a 20- μ L total reaction volume using 250 ng of DNAse-digested total RNA, 1 μ m oligo(dT) primer, 1 m m dNTPs, 1× first-strand buffer (Invitrogen), 20 m m dithiothreitol (Invitrogen), 40 units RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 200 units SuperScript II reverse transcriptase (Invitrogen) by incubating for 2 h at 42°C.

    FACS:

    Article Title: Overcoming Steric Restrictions of VRC01 HIV-1 Neutralizing Antibodies through Immunization
    Article Snippet: .. Individual B cells were sorted using the FACS ARIA II into a 96 well plate containing 20 ul of lysis buffer (20 U of RNase out (Thermo Fisher Scientific, Cat#: 10777019), 5 ul 5X Superscript IV RT buffer, 1.25 ul of 0.1M DTT, 0.625 ul of 10% Igepal, 13 ul nuclease free H2 0) in each well. .. PCR amplification and sequencing of VH and VL genes RNA was reverse transcribed to cDNA.

    Gradient Centrifugation:

    Article Title: miRNA Genetic Variants Alter Their Secondary Structure and Expression in Patients With RASopathies Syndromes
    Article Snippet: Peripheral blood mononuclear cells (PBMC) were extracted by density gradient centrifugation process using Ficoll-Paque. .. For the reverse transcription step, 1 U of SuperScript II reverse transcriptase (Life Technologies, Carlsbad, CA, USA); 1X First Strand Buffer, 0.5 mM dNTPs, and 40 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Life Technologies, Carlsbad, CA, USA) were included in a final volume of 20 µl.

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    Thermo Fisher rnaseout recombinant ribonuclease inhibitor
    Rnaseout Recombinant Ribonuclease Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rnaseout recombinant ribonuclease inhibitor - by Bioz Stars, 2020-02
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