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Effect of RBP16 or RBP16(F14,16A) on in vitro insertion editing of A6 with natural gRNA. RBP16 ( A ) or RBP16(F14,16A) ( B ) was titrated into in vitro insertion reactions (20 μL final volume) containing [ 32 <t>P]pCp</t> 3′-labeled A6 <t>pre-mRNA</t> (∼10
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Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction

Journal:

doi: 10.1261/rna.2331506

Effect of RBP16 or RBP16(F14,16A) on in vitro insertion editing of A6 with natural gRNA. RBP16 ( A ) or RBP16(F14,16A) ( B ) was titrated into in vitro insertion reactions (20 μL final volume) containing [ 32 P]pCp 3′-labeled A6 pre-mRNA (∼10
Figure Legend Snippet: Effect of RBP16 or RBP16(F14,16A) on in vitro insertion editing of A6 with natural gRNA. RBP16 ( A ) or RBP16(F14,16A) ( B ) was titrated into in vitro insertion reactions (20 μL final volume) containing [ 32 P]pCp 3′-labeled A6 pre-mRNA (∼10

Techniques Used: In Vitro, Labeling

Related Articles

Clone Assay:

Article Title: Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol) and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. The single chromosomal gene (rnc ) encoding Aa-RNase III ( A) was amplified by PCR from a sample of A. aeolicus DNA (a generous gift of R. Huber), and cloned into the NdeI and BamHI sites of plasmid pET-15b (Novagen) as described ( ).

Article Title: Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior, and analysis of conserved base-pairs that function as reactivity epitopes for the Thermotoga 23S ribosomal RNA precursor. †
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol), and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. The gene for Tm-RNase III ( ) was amplified from a sample of T. maritima DNA (a generous gift of Francis Jenney, University of Georgia), and cloned into plasmid pET-15b (Novagen) as described ( ).

Amplification:

Article Title: E. coli RNase III(E38A) generates discrete-sized products from long dsRNA
Article Snippet: The 23-bp dsRNA product was electrophoresed, gel purified, and radiolabeled by the addition of 32 P pCp (PerkinElmer) to the 3′ end with T4 RNA Ligase I (New England Biolabs) according to the manufacturer's instructions. .. The dnmt1 insert DNA was amplified and digested with AciI and BpmI, electrophoresed on a 1.25% agarose gel, and transferred to nitrocellulose ( ).

Article Title: Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol) and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. The single chromosomal gene (rnc ) encoding Aa-RNase III ( A) was amplified by PCR from a sample of A. aeolicus DNA (a generous gift of R. Huber), and cloned into the NdeI and BamHI sites of plasmid pET-15b (Novagen) as described ( ).

Article Title: Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior, and analysis of conserved base-pairs that function as reactivity epitopes for the Thermotoga 23S ribosomal RNA precursor. †
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol), and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. The gene for Tm-RNase III ( ) was amplified from a sample of T. maritima DNA (a generous gift of Francis Jenney, University of Georgia), and cloned into plasmid pET-15b (Novagen) as described ( ).

Synthesized:

Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
Article Snippet: RNAs utilized in editing assays were synthesized in vitro using an Ambion T7 Megascript kit and purified by gel electrophoresis on 6% acrylamide/7 M urea. .. Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega).

Article Title: The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells
Article Snippet: Internally labeled XBP1 mRNA transcript was synthesized in the presence of 32 P-αGTP (Perkin Elmer). .. RNA 3′ end labeling was achieved by direct ligation to 32 P-pCp (Perkin Elmer) using RNA ligase I (NEB), according to manufacturer's protocol.

Quantitative RT-PCR:

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: For quantitative reverse transcription (qRT)-PCR analysis of heterodimeric RNAs, the shifted bands were extracted from excised gel using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s procedure. .. The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight.

Electrophoresis:

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: The reaction was immediately analyzed by electrophoresis in a 1% agarose gel supplemented with 0.1 mM MgCl2 in a TBM buffer (45 mM Tris-HCl (pH 8.3), 43 mM boric acid, and 0.1 mM MgCl2 ) at 160 V and 4 °C for 2.5 h, followed by staining with 0.01% (w /v ) ethidium bromide. .. The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight.

Incubation:

Article Title: CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage
Article Snippet: WT or ΔBS1 ΔBS2 flhDC leader RNA (+1 to +171) was 3’end-labeled using T4 RNA ligase (New England Biolabs) and [32 P]pCp (Perkin Elmer) using previously published reaction conditions ( ). .. Reaction mixtures were incubated for 30 min at 37°C to allow CsrA-RNA complex formation before adding RNase E. Incubation was continued at 37°C and 10 μl aliquots were removed at various times and quenched with 10 μl of footprint stop buffer, frozen in dry ice, and stored at −80°C.

Article Title: A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction
Article Snippet: Labeling of DNA and RNA substrates DNA and RNA substrates used in cleavage assays were 5΄ labeled with 32 P using T4 polynucleotide kinase (New England Biolabs) or 3΄ labeled with [32 P]pCp (PerkinElmer) using T4 RNA ligase (Invitrogen). .. For the annealing assay, 50 pmol of labeled substrate was mixed with an equivalent amount of unlabeled nucleic acid as indicated in the experiments and incubated at 90°C for 0.5 min.

Article Title: The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells
Article Snippet: The recovered transcripts were dissolved in 1× RNAi buffer (30 mM HEPES pH 7.4, 100 mM KCl, 5 μM MgCl2 , 0.5 μM DTT, 10 (v/v) % glycerol) and annealed to end-matching LNA oligos (1 μM) (to minimize exonucleolytic cleavage upon incubation with cell extracts) by 30 s incubation at 95°C followed by cooling to room temperature. .. RNA 3′ end labeling was achieved by direct ligation to 32 P-pCp (Perkin Elmer) using RNA ligase I (NEB), according to manufacturer's protocol.

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: The mixture was incubated for 3 h at 37 °C, followed by RNase-free DNase One treatment. .. The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight.

Article Title: Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity
Article Snippet: The 3′-end labelling was carried out at 4 °C overnight in 10 μl using RNA ligase I (NEB) according to the manufacturer’s instructions with 3,000 Ci mmol−1 32 P PcP (Perkin Elmer). .. Blots were prehybridized in PerfectHyb (Sigma) for 1 h at 42 °C before overnight incubation with DNA probes (Invitrogen) that were perfectly complementary to the miRNA or Y RNA: miR-100: 5′-ACACAAGTTCGGATCTACGGGTT-3′, YRNA-5P: 5′-ACCCTACGACTCCGGACCAAGCGCG-3′, YRNA-3P: 5p-GCGCCGGTCGAGCTTTTGTCGAAGGGAAT-3p, Y RNA-loop: 5p-AAGGGAATTCGAGACATTGTTGATAAC-3p.

Expressing:

Article Title: Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol) and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. Production of recombinant Aa-RNase III used the E. coli expression strain BL21(DE3)rnc 105rec A ( ) that carries an inactivating mutation (rnc105 ) of the chromosomal RNase III gene.

Article Title: Purification of radiolabeled RNA products using denaturing gel electrophoresis
Article Snippet: 10 mM ATP (Thermo) 5’ [32 P]-pCp (3000 Ci/mmol, 10 µCi/µL) (Perkin Elmer) T4 RNA ligase and 10× buffer (Thermo) G50 buffer (see recipe) Ethanol Assemble all reaction components below on ice. .. 30 pmol tRNA 2 µL 10× buffer for T4 RNA ligase 1 µL 10 mM ATP 10 µL 5’ [32 P]-pCp (3000 Ci/mmol, 10 µCi/µL) 10 Units T4 RNA ligase Bring up to 20 µL with ddH2 O Note: If a 30-pmol RNA is not possible due to low levels of expression, keep a small amount of RNA; it’s not necessary to proportionally adjust the other reagents.

Article Title: Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior, and analysis of conserved base-pairs that function as reactivity epitopes for the Thermotoga 23S ribosomal RNA precursor. †
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol), and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. Production of recombinant Tm-RNase III used the E. coli expression strain, BL21(DE3) rnc 105 rec A, that carries an inactivating mutation ( rnc105 ) of the chromosomal RNase III gene ( ).

Hybridization:

Article Title: E. coli RNase III(E38A) generates discrete-sized products from long dsRNA
Article Snippet: Paragraph title: Southern blotting and RNA/DNA hybridization ... The 23-bp dsRNA product was electrophoresed, gel purified, and radiolabeled by the addition of 32 P pCp (PerkinElmer) to the 3′ end with T4 RNA Ligase I (New England Biolabs) according to the manufacturer's instructions.

Article Title: Mode of Action of RNase BN/RNase Z on tRNA Precursors
Article Snippet: .. [α-32 P]ATP, [γ-32 P]ATP, [5′-32 P]pCp, and GeneScreen Plus hybridization transfer membrane were obtained from PerkinElmer Life Sciences. .. The GenEluteTM PCR clean-up kit was purchased from Sigma.

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: .. The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight. .. Unincorporated 32 P was removed by exclusion chromatography (Illustra Microspin G-25 column, GE Healthcare, Chicago, IL, USA).

Chromatography:

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight. .. Unincorporated 32 P was removed by exclusion chromatography (Illustra Microspin G-25 column, GE Healthcare, Chicago, IL, USA).

Ligation:

Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
Article Snippet: .. Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega). .. RNAs used in gel retardation assays were synthesized and internally radiolabeled with [α-32 P] UTP using an Ambion T7 Maxiscript kit as described ( ) and were identical in sequence to those used in editing assays.

Article Title: The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells
Article Snippet: .. RNA 3′ end labeling was achieved by direct ligation to 32 P-pCp (Perkin Elmer) using RNA ligase I (NEB), according to manufacturer's protocol. .. All labeled transcripts were purified by preparative PAGE and dissolved in 1× RNAi buffer.

In Vitro:

Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
Article Snippet: RNAs utilized in editing assays were synthesized in vitro using an Ambion T7 Megascript kit and purified by gel electrophoresis on 6% acrylamide/7 M urea. .. Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega).

Article Title: CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage
Article Snippet: The resulting material was stored in buffer B containing 50% glycerol (vol/vol) at –20° C. RNase E assays were performed in vitro by modifying published procedures ( ). .. WT or ΔBS1 ΔBS2 flhDC leader RNA (+1 to +171) was 3’end-labeled using T4 RNA ligase (New England Biolabs) and [32 P]pCp (Perkin Elmer) using previously published reaction conditions ( ).

Article Title: Mutually exclusive RNA secondary structures regulate translation initiation of DinQ in Escherichia coli
Article Snippet: .. In vitro transcribed +1 dinQ , +44 dinQ , and agrB RNA was 3′ end labeled with T4 RNA Ligase (Ambion) and equimolar amounts of [32 P]pCp (PerkinElmer, NEG019A) according to the protocol. ..

Northern Blot:

Article Title: Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity
Article Snippet: Paragraph title: pCp end labelling and northern blot ... The 3′-end labelling was carried out at 4 °C overnight in 10 μl using RNA ligase I (NEB) according to the manufacturer’s instructions with 3,000 Ci mmol−1 32 P PcP (Perkin Elmer).

Generated:

Article Title: A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction
Article Snippet: Labeling of DNA and RNA substrates DNA and RNA substrates used in cleavage assays were 5΄ labeled with 32 P using T4 polynucleotide kinase (New England Biolabs) or 3΄ labeled with [32 P]pCp (PerkinElmer) using T4 RNA ligase (Invitrogen). .. Double strand DNA was generated by annealing of labeled SS1 ssDNA with unlabeled SS1T ssDNA; bubble DNA was made by annealing of labeled SS1 ssDNA and unlabeled S32T ssDNA; R-loop DNA was made by annealing of labeled SS1 ssDNA, unlabeled S32* RNA and unlabeled S32T ssDNA ( ).

Sequencing:

Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
Article Snippet: Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega). .. RNAs used in gel retardation assays were synthesized and internally radiolabeled with [α-32 P] UTP using an Ambion T7 Maxiscript kit as described ( ) and were identical in sequence to those used in editing assays.

Recombinant:

Article Title: Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol) and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. Production of recombinant Aa-RNase III used the E. coli expression strain BL21(DE3)rnc 105rec A ( ) that carries an inactivating mutation (rnc105 ) of the chromosomal RNase III gene.

Article Title: Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior, and analysis of conserved base-pairs that function as reactivity epitopes for the Thermotoga 23S ribosomal RNA precursor. †
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol), and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. Production of recombinant Tm-RNase III used the E. coli expression strain, BL21(DE3) rnc 105 rec A, that carries an inactivating mutation ( rnc105 ) of the chromosomal RNase III gene ( ).

DNA Extraction:

Article Title: Mode of Action of RNase BN/RNase Z on tRNA Precursors
Article Snippet: The genomic DNA isolation kit was obtained from Promega (Madison, WI). .. [α-32 P]ATP, [γ-32 P]ATP, [5′-32 P]pCp, and GeneScreen Plus hybridization transfer membrane were obtained from PerkinElmer Life Sciences.

Nucleic Acid Electrophoresis:

Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
Article Snippet: RNAs utilized in editing assays were synthesized in vitro using an Ambion T7 Megascript kit and purified by gel electrophoresis on 6% acrylamide/7 M urea. .. Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega).

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: The integrity of the transcribed RNA was checked by denaturing gel electrophoresis on a 1% agarose gel with formaldehyde (10 mL of 10× MOPS running buffer) and 18 mL of 37% formaldehyde (12 M) on a pH 7.0 1× MOPS running buffer (0.4 M MOPS, 1 M sodium acetate, and 0.01 M ethylenediaminetetraacetic acid (EDTA)). .. The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight.

Radioactivity:

Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
Article Snippet: .. Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega). .. RNAs used in gel retardation assays were synthesized and internally radiolabeled with [α-32 P] UTP using an Ambion T7 Maxiscript kit as described ( ) and were identical in sequence to those used in editing assays.

Mutagenesis:

Article Title: Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol) and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. Production of recombinant Aa-RNase III used the E. coli expression strain BL21(DE3)rnc 105rec A ( ) that carries an inactivating mutation (rnc105 ) of the chromosomal RNase III gene.

Article Title: Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior, and analysis of conserved base-pairs that function as reactivity epitopes for the Thermotoga 23S ribosomal RNA precursor. †
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol), and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. Production of recombinant Tm-RNase III used the E. coli expression strain, BL21(DE3) rnc 105 rec A, that carries an inactivating mutation ( rnc105 ) of the chromosomal RNase III gene ( ).

Labeling:

Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
Article Snippet: Paragraph title: Preparation and labeling of RNAs ... Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega).

Article Title: Intermolecular domain docking in the hairpin ribozyme
Article Snippet: .. Ribozyme kinetics Loop A (~20 pmol) was labeled at the 3′ end with (5′-32 P)pCp (Perkin-Elmer Life Sciences) and T4 RNA ligase (New England Biolabs Inc.) using standard methods and purified on 20% polyacrylamide gels. .. Cleavage reactions were performed in HPLS buffer at 30°C and were initiated by addition of Co(NH3 )6 3+ to the desired total concentration.

Article Title: The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities ▿
Article Snippet: .. To prepare the 3′-labeled RNA substrate, phosphates were removed from the 5′ end with Antarctic phosphatase and the RNA was labeled using T4 RNA ligase (New England BioLabs) and [5′-32 P]pCp (Perkin Elmer). .. The reaction mixture contained the manufacturer's supplied buffer, the phosphatase-treated RNA, 6 μl [5′-32 P]pCp, and 10 μl T4 RNA ligase (New England BioLabs) in a final volume of 100 μl.

Article Title: A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction
Article Snippet: .. Labeling of DNA and RNA substrates DNA and RNA substrates used in cleavage assays were 5΄ labeled with 32 P using T4 polynucleotide kinase (New England Biolabs) or 3΄ labeled with [32 P]pCp (PerkinElmer) using T4 RNA ligase (Invitrogen). .. Double strand DNA was generated by annealing of labeled SS1 ssDNA with unlabeled SS1T ssDNA; bubble DNA was made by annealing of labeled SS1 ssDNA and unlabeled S32T ssDNA; R-loop DNA was made by annealing of labeled SS1 ssDNA, unlabeled S32* RNA and unlabeled S32T ssDNA ( ).

Article Title: The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells
Article Snippet: For 5′ end labeling, RNA was dephosphorylated using calf intestinal phosphatase (NEB), treated with proteinase K and subsequently labeled using T4 polynucleotide kinase (NEB) and 32 P-γATP (Perkin Elmer). .. RNA 3′ end labeling was achieved by direct ligation to 32 P-pCp (Perkin Elmer) using RNA ligase I (NEB), according to manufacturer's protocol.

Article Title: Mutually exclusive RNA secondary structures regulate translation initiation of DinQ in Escherichia coli
Article Snippet: .. In vitro transcribed +1 dinQ , +44 dinQ , and agrB RNA was 3′ end labeled with T4 RNA Ligase (Ambion) and equimolar amounts of [32 P]pCp (PerkinElmer, NEG019A) according to the protocol. ..

Purification:

Article Title: E. coli RNase III(E38A) generates discrete-sized products from long dsRNA
Article Snippet: .. The 23-bp dsRNA product was electrophoresed, gel purified, and radiolabeled by the addition of 32 P pCp (PerkinElmer) to the 3′ end with T4 RNA Ligase I (New England Biolabs) according to the manufacturer's instructions. .. The dnmt1 insert DNA was amplified and digested with AciI and BpmI, electrophoresed on a 1.25% agarose gel, and transferred to nitrocellulose ( ).

Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
Article Snippet: RNAs utilized in editing assays were synthesized in vitro using an Ambion T7 Megascript kit and purified by gel electrophoresis on 6% acrylamide/7 M urea. .. Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega).

Article Title: Intermolecular domain docking in the hairpin ribozyme
Article Snippet: .. Ribozyme kinetics Loop A (~20 pmol) was labeled at the 3′ end with (5′-32 P)pCp (Perkin-Elmer Life Sciences) and T4 RNA ligase (New England Biolabs Inc.) using standard methods and purified on 20% polyacrylamide gels. .. Cleavage reactions were performed in HPLS buffer at 30°C and were initiated by addition of Co(NH3 )6 3+ to the desired total concentration.

Article Title: The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities ▿
Article Snippet: A 43-nucleotide RNA (5′-GGAAUCUCUCUCUCUCUCUAUGCUCUCUCUCUCUCUCUCUCUC-3′) was transcribed and purified as previously described ( , ). .. To prepare the 3′-labeled RNA substrate, phosphates were removed from the 5′ end with Antarctic phosphatase and the RNA was labeled using T4 RNA ligase (New England BioLabs) and [5′-32 P]pCp (Perkin Elmer).

Article Title: CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage
Article Snippet: Purified, denatured RNase E was subjected to a series of refolding dialysis steps against buffer B (50 mM Tris-HCl, pH 7.9, 200 mM KCl, 10 mM MgCl2 , 1 mM DTT, and 20% [vol/vol] glycerol) containing 4 M guanidine-HCl, 2 M guanidine-HCl, and no denaturant. .. WT or ΔBS1 ΔBS2 flhDC leader RNA (+1 to +171) was 3’end-labeled using T4 RNA ligase (New England Biolabs) and [32 P]pCp (Perkin Elmer) using previously published reaction conditions ( ).

Article Title: A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction
Article Snippet: Labeling of DNA and RNA substrates DNA and RNA substrates used in cleavage assays were 5΄ labeled with 32 P using T4 polynucleotide kinase (New England Biolabs) or 3΄ labeled with [32 P]pCp (PerkinElmer) using T4 RNA ligase (Invitrogen). .. All nucleic acids were purified by recovering the corresponding bands from either a native polyacrylamide gel (dsDNA) or denaturing polyacrylamide gel (ssDNA/RNA) PAGE.

Article Title: The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells
Article Snippet: RNA 3′ end labeling was achieved by direct ligation to 32 P-pCp (Perkin Elmer) using RNA ligase I (NEB), according to manufacturer's protocol. .. All labeled transcripts were purified by preparative PAGE and dissolved in 1× RNAi buffer.

Article Title: Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol) and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. Bacteriophage T7 RNA polymerase ( ) and E. coli RNase III ( ) were purified as described.

Article Title: Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior, and analysis of conserved base-pairs that function as reactivity epitopes for the Thermotoga 23S ribosomal RNA precursor. †
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol), and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. E. coli bulk stripped tRNA was purchased from Sigma-Aldrich and was further purified by repeated phenol extraction followed by ethanol precipitation.

Polymerase Chain Reaction:

Article Title: Mode of Action of RNase BN/RNase Z on tRNA Precursors
Article Snippet: [α-32 P]ATP, [γ-32 P]ATP, [5′-32 P]pCp, and GeneScreen Plus hybridization transfer membrane were obtained from PerkinElmer Life Sciences. .. The GenEluteTM PCR clean-up kit was purchased from Sigma.

Article Title: The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells
Article Snippet: Templates for the spliced and unspliced form of human XBP1 mRNA were obtained by PCR on genomic DNA and cDNA, respectively, using the following primers: T7-hXBP118FW, 5′-TAA TAC GAC TCA CTA TAG GGG AAT GAA GTG AGG CCA GT-3′ and hXBP118RV, 5′-AAT CCA TGG GGA GAT GTT CTG GAG-3′. .. RNA 3′ end labeling was achieved by direct ligation to 32 P-pCp (Perkin Elmer) using RNA ligase I (NEB), according to manufacturer's protocol.

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: For quantitative reverse transcription (qRT)-PCR analysis of heterodimeric RNAs, the shifted bands were extracted from excised gel using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s procedure. .. The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight.

Electrophoretic Mobility Shift Assay:

Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
Article Snippet: Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega). .. RNAs used in gel retardation assays were synthesized and internally radiolabeled with [α-32 P] UTP using an Ambion T7 Maxiscript kit as described ( ) and were identical in sequence to those used in editing assays.

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: Paragraph title: 2.3. Electrophoretic Mobility Shift Assay (EMSA) for RNA–RNA Interaction ... The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight.

Polyacrylamide Gel Electrophoresis:

Article Title: A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction
Article Snippet: Labeling of DNA and RNA substrates DNA and RNA substrates used in cleavage assays were 5΄ labeled with 32 P using T4 polynucleotide kinase (New England Biolabs) or 3΄ labeled with [32 P]pCp (PerkinElmer) using T4 RNA ligase (Invitrogen). .. All nucleic acids were purified by recovering the corresponding bands from either a native polyacrylamide gel (dsDNA) or denaturing polyacrylamide gel (ssDNA/RNA) PAGE.

Article Title: The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells
Article Snippet: RNA 3′ end labeling was achieved by direct ligation to 32 P-pCp (Perkin Elmer) using RNA ligase I (NEB), according to manufacturer's protocol. .. All labeled transcripts were purified by preparative PAGE and dissolved in 1× RNAi buffer.

Article Title: Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity
Article Snippet: The 3′-end labelling was carried out at 4 °C overnight in 10 μl using RNA ligase I (NEB) according to the manufacturer’s instructions with 3,000 Ci mmol−1 32 P PcP (Perkin Elmer). .. Reactions were quenched by the addition of 2 × loading buffer (8 M urea, 0.5% TBE) and 4 μl run on an 18% PAGE at 350 V for 8 h, which was then visualized by phosphorimaging using a Typhoon Scanner (GE Healthcare).

SDS Page:

Article Title: CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage
Article Snippet: The elution peaks were established by SDS-PAGE. .. WT or ΔBS1 ΔBS2 flhDC leader RNA (+1 to +171) was 3’end-labeled using T4 RNA ligase (New England Biolabs) and [32 P]pCp (Perkin Elmer) using previously published reaction conditions ( ).

Plasmid Preparation:

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: Electrophoretic Mobility Shift Assay (EMSA) for RNA–RNA Interaction RNA segments were co-transcribed using 150 ng of linearized plasmid (S8–S11) in a buffer containing 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 20 mM NaCl2 , 3 mM spermidine, 50 mM dithiothreitol (DTT), 5 mM each of rATP, rUTP, rCTP, and rGTP, 10 U of RNase inhibitor and 40 U of T7 RNA polymerase (Thermo Scientific, Waltham, MA, USA). .. The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight.

Article Title: Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol) and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. The single chromosomal gene (rnc ) encoding Aa-RNase III ( A) was amplified by PCR from a sample of A. aeolicus DNA (a generous gift of R. Huber), and cloned into the NdeI and BamHI sites of plasmid pET-15b (Novagen) as described ( ).

Article Title: Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior, and analysis of conserved base-pairs that function as reactivity epitopes for the Thermotoga 23S ribosomal RNA precursor. †
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol), and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. The gene for Tm-RNase III ( ) was amplified from a sample of T. maritima DNA (a generous gift of Francis Jenney, University of Georgia), and cloned into plasmid pET-15b (Novagen) as described ( ).

Software:

Article Title: E. coli RNase III(E38A) generates discrete-sized products from long dsRNA
Article Snippet: The 23-bp dsRNA product was electrophoresed, gel purified, and radiolabeled by the addition of 32 P pCp (PerkinElmer) to the 3′ end with T4 RNA Ligase I (New England Biolabs) according to the manufacturer's instructions. .. The washed blot was autoradiographed and hybridizated, along with the EtBr-stained gel, and quantitated by densitometry using Quantity 1 Software (Bio-Rad).

Article Title: Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing
Article Snippet: In brief, T4 RNA ligase was used to 3′-end label total RNA (5–10 μg) with [32 P]-pCp (cytidine 3′,5′-bisphosphate) (Perkin Elmer), followed by digestion with RNase A and T1. .. Gel images were obtained using a Typhoon phosphorimager and quantified using ImageQuant software.

Positron Emission Tomography:

Article Title: Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol) and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. The single chromosomal gene (rnc ) encoding Aa-RNase III ( A) was amplified by PCR from a sample of A. aeolicus DNA (a generous gift of R. Huber), and cloned into the NdeI and BamHI sites of plasmid pET-15b (Novagen) as described ( ).

Article Title: Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior, and analysis of conserved base-pairs that function as reactivity epitopes for the Thermotoga 23S ribosomal RNA precursor. †
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol), and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. The gene for Tm-RNase III ( ) was amplified from a sample of T. maritima DNA (a generous gift of Francis Jenney, University of Georgia), and cloned into plasmid pET-15b (Novagen) as described ( ).

Agarose Gel Electrophoresis:

Article Title: E. coli RNase III(E38A) generates discrete-sized products from long dsRNA
Article Snippet: The 23-bp dsRNA product was electrophoresed, gel purified, and radiolabeled by the addition of 32 P pCp (PerkinElmer) to the 3′ end with T4 RNA Ligase I (New England Biolabs) according to the manufacturer's instructions. .. The dnmt1 insert DNA was amplified and digested with AciI and BpmI, electrophoresed on a 1.25% agarose gel, and transferred to nitrocellulose ( ).

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: The integrity of the transcribed RNA was checked by denaturing gel electrophoresis on a 1% agarose gel with formaldehyde (10 mL of 10× MOPS running buffer) and 18 mL of 37% formaldehyde (12 M) on a pH 7.0 1× MOPS running buffer (0.4 M MOPS, 1 M sodium acetate, and 0.01 M ethylenediaminetetraacetic acid (EDTA)). .. The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight.

Southern Blot:

Article Title: E. coli RNase III(E38A) generates discrete-sized products from long dsRNA
Article Snippet: Paragraph title: Southern blotting and RNA/DNA hybridization ... The 23-bp dsRNA product was electrophoresed, gel purified, and radiolabeled by the addition of 32 P pCp (PerkinElmer) to the 3′ end with T4 RNA Ligase I (New England Biolabs) according to the manufacturer's instructions.

Ethanol Precipitation:

Article Title: Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior, and analysis of conserved base-pairs that function as reactivity epitopes for the Thermotoga 23S ribosomal RNA precursor. †
Article Snippet: [γ-32 P]ATP (3000 Ci/mmol), [α-32 P]UTP (3000 Ci/mmol), and [5′-32 P]pCp (3000 Ci/mmol) were purchased from Perkin-Elmer. .. E. coli bulk stripped tRNA was purchased from Sigma-Aldrich and was further purified by repeated phenol extraction followed by ethanol precipitation.

Produced:

Article Title: E. coli RNase III(E38A) generates discrete-sized products from long dsRNA
Article Snippet: As described above, 23-bp dsRNA was produced from a 1112-bp clone of the human DNA methylase, dnmt1 ( ). .. The 23-bp dsRNA product was electrophoresed, gel purified, and radiolabeled by the addition of 32 P pCp (PerkinElmer) to the 3′ end with T4 RNA Ligase I (New England Biolabs) according to the manufacturer's instructions.

Concentration Assay:

Article Title: Intermolecular domain docking in the hairpin ribozyme
Article Snippet: Ribozyme kinetics Loop A (~20 pmol) was labeled at the 3′ end with (5′-32 P)pCp (Perkin-Elmer Life Sciences) and T4 RNA ligase (New England Biolabs Inc.) using standard methods and purified on 20% polyacrylamide gels. .. Cleavage reactions were performed in HPLS buffer at 30°C and were initiated by addition of Co(NH3 )6 3+ to the desired total concentration.

Article Title: Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity
Article Snippet: pCp end labelling and northern blot For 3′ end-labelling, total RNA was extracted from the life stages and secretion product using the miRNAeasy kit (Qiagen): 1 μg total RNA was used from life stages and RNA extracted from a volume of secretion product equating to 15 μg protein (the total RNA concentration was too low to detect by nanodrop or qubit). .. The 3′-end labelling was carried out at 4 °C overnight in 10 μl using RNA ligase I (NEB) according to the manufacturer’s instructions with 3,000 Ci mmol−1 32 P PcP (Perkin Elmer).

End Labeling:

Article Title: The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells
Article Snippet: .. RNA 3′ end labeling was achieved by direct ligation to 32 P-pCp (Perkin Elmer) using RNA ligase I (NEB), according to manufacturer's protocol. .. All labeled transcripts were purified by preparative PAGE and dissolved in 1× RNAi buffer.

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: .. The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight. .. Unincorporated 32 P was removed by exclusion chromatography (Illustra Microspin G-25 column, GE Healthcare, Chicago, IL, USA).

Article Title: Mutually exclusive RNA secondary structures regulate translation initiation of DinQ in Escherichia coli
Article Snippet: Paragraph title: 3′ end labeling and RNase III cleavage ... In vitro transcribed +1 dinQ , +44 dinQ , and agrB RNA was 3′ end labeled with T4 RNA Ligase (Ambion) and equimolar amounts of [32 P]pCp (PerkinElmer, NEG019A) according to the protocol.

CTG Assay:

Article Title: The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells
Article Snippet: Templates for the spliced and unspliced form of human XBP1 mRNA were obtained by PCR on genomic DNA and cDNA, respectively, using the following primers: T7-hXBP118FW, 5′-TAA TAC GAC TCA CTA TAG GGG AAT GAA GTG AGG CCA GT-3′ and hXBP118RV, 5′-AAT CCA TGG GGA GAT GTT CTG GAG-3′. .. RNA 3′ end labeling was achieved by direct ligation to 32 P-pCp (Perkin Elmer) using RNA ligase I (NEB), according to manufacturer's protocol.

Staining:

Article Title: Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity
Article Snippet: The reaction was immediately analyzed by electrophoresis in a 1% agarose gel supplemented with 0.1 mM MgCl2 in a TBM buffer (45 mM Tris-HCl (pH 8.3), 43 mM boric acid, and 0.1 mM MgCl2 ) at 160 V and 4 °C for 2.5 h, followed by staining with 0.01% (w /v ) ethidium bromide. .. The RNA–ORN hybridization assay was carried out by the 3′ end labeling of 10 pmol of S10 and S11 ORNs (10.2, 10.3 and 11.2 ORNs) including scrambled (Scr) with 10 μCi [32 P]pCp (Perkin Elmer, Waltham, MA, USA) with T4 RNA ligase (Thermo Scientific, Waltham, MA, USA) in a T4 RNA ligase buffer, and incubating at 4 °C overnight.

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    PerkinElmer p pcp
    Schematic representation of radiolabeling of <t>RNA</t> at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 <t>P]pCp</t> is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule
    P Pcp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    Article Title: Synthesis and Labeling of RNA In Vitro

    doi: 10.1002/0471142727.mb0415s102

    Figure Lengend Snippet: Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Article Snippet: 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase.

    Techniques: Radioactivity, Ligation

    Enzymatic determination of the new 3′-end of HCV and CSFV RNA end-groups produced by UV-C-induced self-cleavage. ( a ) T4 RNA ligase treatment of gel-purified HCV RNA 1–130 (left panel) and CSFV RNA 1–218 (right panel) cleavage product band B2. B2 RNAs [4000 dpm (10 5 dpm/µg)] were incubated with T4 RNA ligase and [5′- 32 P]pCp. Lane 1: control reaction with B2 RNA incubated in SAP phosphatase buffer, then in ligase buffer and [5′- 32 P]pCp in the absence of any enzyme; Lane 2: control reaction of B2 RNA treated the same as in lane 1 but incubated with the phosphatase; Lane 3: B2 RNA incubated with T4 RNA ligase without previous dephosphorylation; Lane 4: complete reaction of B2 RNA incubated with the ligase after being treated with the phosphatase. ( b ) Addition of [ 32 P]-labeled poly (A) or poly (U) to bands B2 of HCV (left panel) and CSFV (right panel) with E. coli poly (A) polymerase or Schizosaccharomyces pombe poly (U) polymerase.A total of 4000 dpm RNA (10 5 dpm/µg) was used for both viral RNAs. A total of 20 µCi of the labeled nucleotide (ATP or UTP) was distributed for the four reactions. Lanes 1 and 2: B2 RNA incubated with the poly (A) polymerase after being treated or not with shrimp alkaline phosphatase, respectively. Lanes 3 and 4: control reactions of B2 RNA treated or not with the phosphatase but without incubation with the polymerase. Lanes 1′ 2′ 3′ and 4′ same as above, but using poly (U) polymerase. MW is a molecular weight marker.

    Journal: Nucleic Acids Research

    Article Title: RNA self-cleavage activated by ultraviolet light-induced oxidation

    doi: 10.1093/nar/gkr822

    Figure Lengend Snippet: Enzymatic determination of the new 3′-end of HCV and CSFV RNA end-groups produced by UV-C-induced self-cleavage. ( a ) T4 RNA ligase treatment of gel-purified HCV RNA 1–130 (left panel) and CSFV RNA 1–218 (right panel) cleavage product band B2. B2 RNAs [4000 dpm (10 5 dpm/µg)] were incubated with T4 RNA ligase and [5′- 32 P]pCp. Lane 1: control reaction with B2 RNA incubated in SAP phosphatase buffer, then in ligase buffer and [5′- 32 P]pCp in the absence of any enzyme; Lane 2: control reaction of B2 RNA treated the same as in lane 1 but incubated with the phosphatase; Lane 3: B2 RNA incubated with T4 RNA ligase without previous dephosphorylation; Lane 4: complete reaction of B2 RNA incubated with the ligase after being treated with the phosphatase. ( b ) Addition of [ 32 P]-labeled poly (A) or poly (U) to bands B2 of HCV (left panel) and CSFV (right panel) with E. coli poly (A) polymerase or Schizosaccharomyces pombe poly (U) polymerase.A total of 4000 dpm RNA (10 5 dpm/µg) was used for both viral RNAs. A total of 20 µCi of the labeled nucleotide (ATP or UTP) was distributed for the four reactions. Lanes 1 and 2: B2 RNA incubated with the poly (A) polymerase after being treated or not with shrimp alkaline phosphatase, respectively. Lanes 3 and 4: control reactions of B2 RNA treated or not with the phosphatase but without incubation with the polymerase. Lanes 1′ 2′ 3′ and 4′ same as above, but using poly (U) polymerase. MW is a molecular weight marker.

    Article Snippet: RNA ligase was used to treat cleavage product bands, either previously phosphatase treated or not, as follows: RNAs were incubated at 4°C for 4 days in a buffer containing 10 mM MgCl2 , 50 mM HEPES, pH 8.3, 5 mM DTT, 0.12 mM ATP, 4 U of T4 RNA ligase (Promega) and [5′-32 P]pCp (Perkin-Elmer) ( , ).

    Techniques: Produced, Purification, Incubation, De-Phosphorylation Assay, Labeling, Molecular Weight, Marker

    Characterization of UV-C cleavage of viral RNAs by fingerprinting and electrophoretic methods. ( a ) The RNA fingerprints of internally [α- 32 P] HCV (panels 1–3) and CSFV RNA (panels 4–6). Labeled SM, band B1 and band B2 were exhaustively digested with RNase T1 and the products subjected to 2D separation. ( 1 ) RNA fingerprint of HCV 1–249. A total of 500 000 dpm of HCV SM was fingerprinted. Spot 1: the HCV oligonucleotide 5′ 78 UCUAG 82 3′ within which cleavage takes place; Spot 2: this has the characteristic mobility of the 5′-terminal nucleotide 5′pppGp3′. ( 2 ) RNA fingerprint of HCV B1. A total of 300 000 dpm of RNA was fingerprinted as above. Spot 1 has disappeared, while the absence of the 5′-end (spot 2) shows that HCV B1 contains the 3′-portion of SM. ( 3 ) Fingerprint of HCV B2 (100 000 dpm). ‘1’ indicates the loss of spot 1, while the presence of the HCV 5′-end (‘2’) shows that HCV B2 contains the 5′-portion of SM. ( 4–6 ): RNA fingerprints of CSFV 1-218. SM (500 000 dpm), B1 (300 000 dpm) and B2 (100 000 dpm, transcribed with all four [α- 32 P]-labeled rNTPs. ‘1’: the CSFV oligonucleotide 5′ 38 AUACACUAAAUUUCG 52 3′, which is present in SM but absent from B1 and B2; ‘X’ (a new CSFV B1 oligonucleotide) and ‘Y’ (the other new CSFV oligonucleotide) arise from cleavage within spot 1 (see text) . ‘2’: the 5′-end of CSFV, present in SM and B2, but not B1. Numbering according to Wang et al. ( 62 ) for HCV genotype 1b and Gene Bank J04358 for CSFV Alfort Isolate. The sequence of the spot numbered as 1 was identified by secondary RNase analysis and high voltage electrophoresis on DEAE and Whatmann paper by Hugh D. Robertson (data not available), as well as by superposition with previously resolved HCV fingerprints using secondary analysis and on the basis of the rules for RNA oligonucleotide mobility during 2D TLC. Briefly, these rules are: the larger the oligonucleotide, the slower the migration of the corresponding spot to the bottom. As far as composition is concerned, Us displace the spot to the left, Cs to the right, and As cause a slight delay, thus meaning that several As in the same oligo may cause it to behave as an oligo containing one or even two additional bases ( 37 ). As far as sequence is concerned, as HCV RNA was transcribed in the presence of [α- 32 P]GTP here, those T1 oligonucleotides in the original sequence that are followed by a (pG) carry a double label. In the case of HCV RNA several RNase T1 oligonucelotides are indicated as mobility reference: a: UCCUUUCUUGp(G); b: UCUUCAGp(C) 61:68; c: CUCAAUGp(G) 211–217; d: AUUUGp(G) 225–229. Spot 1 locates in the border of the triangle that can be formed by spots i, f and g. In CSFV, spot 1 is the slow migrating spot, and thus corresponded to the largest RNase T1 oligonucleotide. In both HCV and CSFV, band B2 contains the original 5′-terminal nucleotide, pppGp, of the substrate RNA transcript (indicated by ‘2’). The disappearance of spot 1 from both product band fingerprints (see Figure 1 a, images 2, 3 and images 5, 6) suggests that self-cleavage occurs within this oligonucleotide and that this event is specific. Moreover, in the case of CSFV RNA two smaller oligomers (X and Y) that represent the fragment products of spot 1 are observed within the fingerprints of both product bands (B1 and B2) for CSFV. Indicated at the bottom is the sequence surrounding RNase T1 cleavage sites. ( b ) Electrophoresis analysis: Autoradiogram showing a parallel run of HCV RNA 1–130 and CSFV RNA 1–218 UV-cleavage reaction, with RNase T1 treated samples and control reactions for transcripts labeled at either the 5′-extreme with [γ- 32 P]GTP during transcription or the 3′-extreme with [5′- 32 P]pCp and T4 RNA ligase. HCV (lanes 1–6) and CSFV (lanes 1–9). HCV: Lanes 1 and 1′: RNAs incubated in standard buffer; lanes 2 and 2′: RNAs treated with RNase T1 under denaturing conditions; lanes 3 and 3′: RNA irradiated with UV-C light for 180 s. CSFV: Lanes 1 and 1′: RNAs incubated under standard conditions; lanes 2, 3 and 4′: RNA treated with RNase T1; lanes 4 and 3′: RNA irradiated with UV-C light: Lanes 5 and 2′ RNAs partially degraded with alkali. ‘G’ positions of a relevant size are indicated on either side of the gels. Lines indicate SM, and products B1 and B2.

    Journal: Nucleic Acids Research

    Article Title: RNA self-cleavage activated by ultraviolet light-induced oxidation

    doi: 10.1093/nar/gkr822

    Figure Lengend Snippet: Characterization of UV-C cleavage of viral RNAs by fingerprinting and electrophoretic methods. ( a ) The RNA fingerprints of internally [α- 32 P] HCV (panels 1–3) and CSFV RNA (panels 4–6). Labeled SM, band B1 and band B2 were exhaustively digested with RNase T1 and the products subjected to 2D separation. ( 1 ) RNA fingerprint of HCV 1–249. A total of 500 000 dpm of HCV SM was fingerprinted. Spot 1: the HCV oligonucleotide 5′ 78 UCUAG 82 3′ within which cleavage takes place; Spot 2: this has the characteristic mobility of the 5′-terminal nucleotide 5′pppGp3′. ( 2 ) RNA fingerprint of HCV B1. A total of 300 000 dpm of RNA was fingerprinted as above. Spot 1 has disappeared, while the absence of the 5′-end (spot 2) shows that HCV B1 contains the 3′-portion of SM. ( 3 ) Fingerprint of HCV B2 (100 000 dpm). ‘1’ indicates the loss of spot 1, while the presence of the HCV 5′-end (‘2’) shows that HCV B2 contains the 5′-portion of SM. ( 4–6 ): RNA fingerprints of CSFV 1-218. SM (500 000 dpm), B1 (300 000 dpm) and B2 (100 000 dpm, transcribed with all four [α- 32 P]-labeled rNTPs. ‘1’: the CSFV oligonucleotide 5′ 38 AUACACUAAAUUUCG 52 3′, which is present in SM but absent from B1 and B2; ‘X’ (a new CSFV B1 oligonucleotide) and ‘Y’ (the other new CSFV oligonucleotide) arise from cleavage within spot 1 (see text) . ‘2’: the 5′-end of CSFV, present in SM and B2, but not B1. Numbering according to Wang et al. ( 62 ) for HCV genotype 1b and Gene Bank J04358 for CSFV Alfort Isolate. The sequence of the spot numbered as 1 was identified by secondary RNase analysis and high voltage electrophoresis on DEAE and Whatmann paper by Hugh D. Robertson (data not available), as well as by superposition with previously resolved HCV fingerprints using secondary analysis and on the basis of the rules for RNA oligonucleotide mobility during 2D TLC. Briefly, these rules are: the larger the oligonucleotide, the slower the migration of the corresponding spot to the bottom. As far as composition is concerned, Us displace the spot to the left, Cs to the right, and As cause a slight delay, thus meaning that several As in the same oligo may cause it to behave as an oligo containing one or even two additional bases ( 37 ). As far as sequence is concerned, as HCV RNA was transcribed in the presence of [α- 32 P]GTP here, those T1 oligonucleotides in the original sequence that are followed by a (pG) carry a double label. In the case of HCV RNA several RNase T1 oligonucelotides are indicated as mobility reference: a: UCCUUUCUUGp(G); b: UCUUCAGp(C) 61:68; c: CUCAAUGp(G) 211–217; d: AUUUGp(G) 225–229. Spot 1 locates in the border of the triangle that can be formed by spots i, f and g. In CSFV, spot 1 is the slow migrating spot, and thus corresponded to the largest RNase T1 oligonucleotide. In both HCV and CSFV, band B2 contains the original 5′-terminal nucleotide, pppGp, of the substrate RNA transcript (indicated by ‘2’). The disappearance of spot 1 from both product band fingerprints (see Figure 1 a, images 2, 3 and images 5, 6) suggests that self-cleavage occurs within this oligonucleotide and that this event is specific. Moreover, in the case of CSFV RNA two smaller oligomers (X and Y) that represent the fragment products of spot 1 are observed within the fingerprints of both product bands (B1 and B2) for CSFV. Indicated at the bottom is the sequence surrounding RNase T1 cleavage sites. ( b ) Electrophoresis analysis: Autoradiogram showing a parallel run of HCV RNA 1–130 and CSFV RNA 1–218 UV-cleavage reaction, with RNase T1 treated samples and control reactions for transcripts labeled at either the 5′-extreme with [γ- 32 P]GTP during transcription or the 3′-extreme with [5′- 32 P]pCp and T4 RNA ligase. HCV (lanes 1–6) and CSFV (lanes 1–9). HCV: Lanes 1 and 1′: RNAs incubated in standard buffer; lanes 2 and 2′: RNAs treated with RNase T1 under denaturing conditions; lanes 3 and 3′: RNA irradiated with UV-C light for 180 s. CSFV: Lanes 1 and 1′: RNAs incubated under standard conditions; lanes 2, 3 and 4′: RNA treated with RNase T1; lanes 4 and 3′: RNA irradiated with UV-C light: Lanes 5 and 2′ RNAs partially degraded with alkali. ‘G’ positions of a relevant size are indicated on either side of the gels. Lines indicate SM, and products B1 and B2.

    Article Snippet: RNA ligase was used to treat cleavage product bands, either previously phosphatase treated or not, as follows: RNAs were incubated at 4°C for 4 days in a buffer containing 10 mM MgCl2 , 50 mM HEPES, pH 8.3, 5 mM DTT, 0.12 mM ATP, 4 U of T4 RNA ligase (Promega) and [5′-32 P]pCp (Perkin-Elmer) ( , ).

    Techniques: Labeling, Sequencing, Electrophoresis, Thin Layer Chromatography, Migration, Incubation, Irradiation

    Effect of RBP16 or RBP16(F14,16A) on in vitro insertion editing of A6 with natural gRNA. RBP16 ( A ) or RBP16(F14,16A) ( B ) was titrated into in vitro insertion reactions (20 μL final volume) containing [ 32 P]pCp 3′-labeled A6 pre-mRNA (∼10

    Journal:

    Article Title: RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction

    doi: 10.1261/rna.2331506

    Figure Lengend Snippet: Effect of RBP16 or RBP16(F14,16A) on in vitro insertion editing of A6 with natural gRNA. RBP16 ( A ) or RBP16(F14,16A) ( B ) was titrated into in vitro insertion reactions (20 μL final volume) containing [ 32 P]pCp 3′-labeled A6 pre-mRNA (∼10

    Article Snippet: Radiolabeling of pre-mRNA at the 3′ end was performed by ligation of [5′-32 P]pCp (Perkin Elmer) by T4 RNA ligase (Promega).

    Techniques: In Vitro, Labeling