5′-Cy5-labeled miR-21-hp RNA was dissolved in 0.1 × PBS (pH 7.4) to a concentration of 100 μM and was annealed by briefly heating to 95 °C for 3 min, followed by slowly cooling to RT for 1 h.22 (link),23 (link) The annealed RNA was incubated at a final concentration of 3 μM in 50 mM Tris at pH 8.5, 10 mM KCl buffer with either a DMSO control (5% final concentration), or compound at a concentration of 3, 10, 30, 100, or 300 μM. MgCl2 was added at a final concentration of 1 mM, and the reactions were incubated at RT in darkness for 4 days. Alkaline hydrolysis was performed in 10 mM NaHCO3 at pH 9.0 and 95 °C for 5 min. Ribonuclease T1 digestion was carried out with 0.1 U of Ribonuclease T1 (Ambion) in 20 mM Tris at pH 7.5, 50 mM NaCl, and 0.1 mM MgCl2 at RT for 20 min. The RNase T1 reaction was stopped by adding 0.2 volumes of 5 mM EDTA. Equal volumes of loading buffer containing 7 M urea, 1 × TBE, and 0.01% direct red dye was added to each reaction, and the samples were heated to 95 °C for 5 min prior to analysis by electrophoresis on a denaturing polyacrylamide sequencing gel (20%, 19:1 cross-linking, 7 M urea). The gel was visualized by fluorescence of the 5′-Cy5 label (630 nm excitation, 670 emission) with a Typhoon FLA9500 gel imager (GE Healthcare Life Sciences) and was analyzed with ImageQuant software.
Ribonuclease t1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ribonuclease T1 is an enzyme that catalyzes the hydrolytic cleavage of single-stranded RNA at guanine residues. It is commonly used in molecular biology and biochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Calf intestinal phosphatase, by Thermo Fisher Scientific (2 mentions)
Nuclease p1, by Merck Group (2 mentions)
Neg019a, by New England Biolabs (2 mentions)
Rna gel loading dye, by New England Biolabs (2 mentions)
Typhoon fla 9500 fluorescence imager, by GE Healthcare (2 mentions)
T4 rna ligase 1, by New England Biolabs (2 mentions)
Ribonuclease a, by Merck Group (2 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (2 mentions)
Typhoon fla 9500 gel imager, by GE Healthcare (1 mentions)
Typhoon 9200 scanner, by Cytiva (1 mentions)
S1 nuclease, by Thermo Fisher Scientific (1 mentions)
α 32p ctp, by PerkinElmer (1 mentions)
α 32p gtp, by PerkinElmer (1 mentions)
α 32p utp, by PerkinElmer (1 mentions)
Molecular imager fx system, by Bio-Rad (1 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Personal molecular imager, by Bio-Rad (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
14 protocols using ribonuclease t1
1
Structural Analysis of miR-21 Hairpin RNA
2
In Vitro Probing of RNA Structures
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Templates for in vitro probing, containing a T7 promoter, were obtained by PCR amplification. Lead acetate degradation and inline probing assays were performed as previously described [46 (link)]. In brief, 0.2 μM of in vitro–generated gcrA+209 and P2-ctrA+143, 5′ end-labeled were incubated with or without 1 μM CcnA ncRNA. Radiolabeled RNA was incubated 5 min at 90°C with alkaline buffer or 5 min at 37°C with ribonuclease T1 (0.1 U; Ambion) to generate the Alkaline (OH) ladder and the T1 ladder, respectively. RNA was analyzed on an 8% acrylamide/7M urea gel.
3
Characterization of Labeled RNA Structure
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The precipitate of de-protected RNA (5′-labeled with Dy547) was dissolved in 0.1× PBS to a final concentration of 15 µM, then heated briefly to 95°C and allowed to cool slowly (over 2 h) to room temperature. The resulting RNA was incubated, at a final concentration of 3 µM, in 10 mM KCl, 50 mM Tris, pH 8.5. MgCl2 and peptoid were added to specified concentrations as stock solutions. Reaction mixtures were incubated at room temperature in darkness for 7 d, then precipitated with ethanol, sodium acetate, and 5 µg of yeast phenylalanine tRNA. Alkaline hydrolysis was carried out in 10 mM NaHCO3, pH 9.0, at 95°C for 4 min.
ribonuclease T1 digestion was carried out with 0.007 units of ribonuclease T1 (Ambion) in 20 mM Tris, pH 7.15, 50 mM NaCl, 0.1 mM MgCl2, at room temperature for 20 min. The reaction was stopped by addition of 0.2 volumes of 5 mM EDTA and precipitation with ethanol, sodium acetate, and 5 µg of yeast phenylalanine tRNA. The precipitates were heated briefly at 95°C in formamide prior to analysis by electrophoresis on a polyacrylamide gel (20%, 19:1 crosslinking, 8 M urea). The gel was visualized by fluorescence (532-nm excitation) with a Typhoon 9200 scanner (Amersham Biosciences) and analyzed with ImageQuant software.
ribonuclease T1 digestion was carried out with 0.007 units of ribonuclease T1 (Ambion) in 20 mM Tris, pH 7.15, 50 mM NaCl, 0.1 mM MgCl2, at room temperature for 20 min. The reaction was stopped by addition of 0.2 volumes of 5 mM EDTA and precipitation with ethanol, sodium acetate, and 5 µg of yeast phenylalanine tRNA. The precipitates were heated briefly at 95°C in formamide prior to analysis by electrophoresis on a polyacrylamide gel (20%, 19:1 crosslinking, 8 M urea). The gel was visualized by fluorescence (532-nm excitation) with a Typhoon 9200 scanner (Amersham Biosciences) and analyzed with ImageQuant software.
4
In-line probing of hdeD transcript
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Lead acetate degradation and In-line probing assays were performed as described by Lalaouna et al. (18 (link)). In brief, 0.2 μM of in vitro-generated hdeD+195 5′-end-labeled was incubated with or without 1 μM CyaR or RprA sRNA. Radiolabeled RNA was incubated 5 min at 90°C with alkaline buffer or 5 min at 37°C with ribonuclease T1 (0.1 U; Ambion) to generate the alkaline (OH) ladder and the T1 ladder, respectively. RNA was analyzed on an 8% acrylamide/7 M urea gel.
5
Characterization of the TPP Riboswitch
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The TPP riboswitch, the P3a::4U* RNA and its variants rep (A6C) and derep (G12A/C23U) were obtained by in vitro transcription. The RNA was purified and dephosphorylated with CIP (Calf intestinal phosphatase, Thermo Scientific, Waltham, USA). RNA was labeled at the 5′ end as described (31 (link)). Partial digestions with ribonuclease T1 (0.00125 U) (Ambion, Austin, USA) and nuclease S1 (0.125 U) (Thermo Scientific, Waltham, USA) were performed according to (19 (link)) at indicated temperatures in absence or presence of 100 μM TPP. For digestion with RNase T1, 5x TN buffer (100 mM Tris acetate, pH 7, 500 mM NaCl) was used. Digestion with nuclease S1 was performed using the supplied 5× reaction buffer. An alkaline hydrolysis ladder (31 (link)) and a T1-ladder were prepared. For the T1-ladder 30,000 cpm labeled RNA was incubated with 1 μl sequencing buffer (provided with RNase T1) at 90°C followed by incubation with the enzyme at 45°C for 5 min.
6
Radiolabeled Nucleotide Synthesis Assays
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NTPs (100 mM LiCl salts) used
for enzyme assays were purchased from Roche; [γ-32P]GTP (6000 Ci/mmol), [α-32P]CTP (3000 Ci/mmol),
[α-32P]GTP (3000 Ci/mmol), and [α-32P]UTP (3000 Ci/mmol) were purchased from Perkin-Elmer. For Thg1 transient
kinetic assays, ribonuclease A (RNase A) and ribonuclease T1 (RNase
T1) were purchased from Ambion; calf alkaline intestinal phosphatase
(CIP) was purchased from Invitrogen, and P1 nuclease was purchased
from Sigma.
for enzyme assays were purchased from Roche; [γ-32P]GTP (6000 Ci/mmol), [α-32P]CTP (3000 Ci/mmol),
[α-32P]GTP (3000 Ci/mmol), and [α-32P]UTP (3000 Ci/mmol) were purchased from Perkin-Elmer. For Thg1 transient
kinetic assays, ribonuclease A (RNase A) and ribonuclease T1 (RNase
T1) were purchased from Ambion; calf alkaline intestinal phosphatase
(CIP) was purchased from Invitrogen, and P1 nuclease was purchased
from Sigma.
7
m6A Levels Quantification by 2D TLC
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Total RNA was isolated using hot phenol extration39 (link). Poly(A) RNA was prepared according to the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, NEB). m6A levels were measured by two-dimensional thin layer chromatography (TLC) analysis as described in Zhong et al.6 (link). Fifty nanogram of poly(A) RNA was digested by 1 μL of Ribonuclease T1 (1000 U μL−1; Thermo Fisher Scientific) in 1× polynucleotide kinase (PNK) buffer A at 37 °C for 1 h. 5′ ends of digested RNA fragments were labeled with 0.5 μL of [γ-32P]ATP (6000 Ci mmol−1; PerkinElmer) using 10 U of T4 PNK. After overnight ethanol precipitation, the labeled RNA pellet was resuspended in 10 μL of 50 mM sodium acetate (pH 5.5) and digested by nuclease P1 (Sigma-Aldrich) at 37 °C for at least 1 h to produce mononucleotides. One microliter of the digested sample was loaded onto the cellulose F TLC plate (20 × 20 cm; Merck) and developed in a solvent system, with isobutyric acid:0.5 M NH4OH (5:3, v/v) as the first dimension buffer and isopropanol:HCl:water (70:15:15, v/v/v) as the second dimension buffer. The labeled nucleotides were identified and quantified by using a storage phosphor screen (Fuji-Screen) and Bio-Rad Molecular Imager FX system in combination with Quantity One software (v4.6.2.70).
8
Qβ Replicase Replication Assay
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Replication reactions were carried out in 10 μl reaction buffer (100 mM Tris-HCl, рН 7.6, 10 mM MgCl2, 1 mM EDTA, 0.1 v/v% Triton X-100) at 30°C in the presence of 1 mM of each rNTP, 1 MBq/ml [α-32P] UTP, 0.01 μM Qβ (+)-RNA, 0.01 μM Hfq and 0.3 μM ribosomal protein S1. Where indicated, 50 mM (NH4)2SO4 was included in the reaction. After 1 min incubation at 30°C, 0.1 μM wild-type or mutant Qβ replicase core complex was added and replication continued for 10 min. Where indicated, single-stranded RNA product was digested by addition of 100 U ribonuclease T1 (Thermo Scientific) for 15 s at 30°C. Reactions were stopped by addition of 4 μl 60 mM EDTA on ice and the RNA was isolated by phenol/chloroform extraction (1:1, pH 4.5). The RNA was recovered by centrifugation and analysed by electrophoresis using a 1% agarose gel stained with ethidium bromide. Next, the gel was dried onto a Biodyne B transfer membrane (Pall Life Sciences) and visualized by autoradiography on a Personal Molecular Imager™ (Bio-Rad). Escherichia coli Hfq was overproduced using the intein system (Impact-CN™, New England Biolabs) as described by Link et al. (29 (link)).
9
Structure Probing of cnfY 5'-UTR
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Structure probing of the 5’-UTR and 80 nt of cnfY was performed with in vitro transcribed RNA using pBO4465 or pBO4466 as template. The in vitro transcribed RNA was purified and dephosphorylated using CIP enzyme (Calf intestinal phosphatase, Thermo Scientific, Waltham, USA). The RNA was labeled with [32P] at the 5′ end as described elsewhere [31 (link)]. Partial digestions of radiolabeled RNA with ribonuclease T1 (0.0025 U) (Thermo Scientific, Waltham, USA) and T2 (0.056 U) (MoBiTec, Göttingen, Germany) were performed according to [32 (link)] at 25, 37, and 42°C. For digestion with RNase T1 and RNase T2 a 5 x TN buffer (100 mM Tris acetate, pH 7, 500 mM NaCl) was used. An alkaline hydrolysis ladder was prepared as described before [31 (link)]. The T1-ladder was generated by using 30000 cpm labeled RNA. The RNA was heated with 1 μl sequencing buffer (provided with RNase T1) at 90°C. Afterwards, the RNA was incubated with the enzyme at 37°C for 5 min.
10
Bulk Poly(A) Tail Length Profiling
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Bulk poly(A) tail assay was conducted as previously described. Total RNA (2 µg) was labeled with [5′-32P] pCp (cytidine 3′,5′-bis[phosphate]) (0.11 pmol/μL in a total reaction volume of 30 μL) (PerkinElmer; NEG019A) using T4 RNA ligase 1 (NEB, M0204S) at 16 °C overnight. Labeled RNAs were incubated at 85 °C for 5 min and placed on ice. Then, labeled RNAs were digested with Ribonuclease A (50 ng/μL, Sigma) and Ribonuclease T1 (1.25 U/μL, Thermo Fisher Scientific) at 37 °C for 2 h in digestion buffer (100 mM Tris–HCl [pH 7.5], 3 M NaCl, 0.5 μg/mL yeast tRNA). Reactions were stopped by adding 5× stop solution (10 mg/mL Proteinase K, 0.125 M EDTA, 2.5% SDS) and subsequently incubating at 37 °C for 30 min. After adding 400 μL of RNA precipitation buffer (0.5 M NH4OAc, 10 mM EDTA), digested RNA samples were purified by phenol–chloroform extraction and isopropanol precipitation. Final products (10 μL) were mixed with RNA Gel loading Dye (NEB, R0641) and incubated at 95 °C for 2 min. Samples were fractionated on an 8 M urea-10% polyacrylamide denaturing gel (0.8 mm thick). Marker (Prestain Marker for small RNA Plus, BioDynamics Laboratory DM253) was also loaded. The gel was analyzed with a Typhoon FLA 9500 Fluorescence Imager (GE Healthcare). Band intensity was quantified using ImageJ.
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