The RNA probe (5’-CGAUCCUCGGCCAGGAC CAGCCUUCCCCAG-3’) derived from Hsp70 5’UTR was commercially synthesized in vitro (Thermo Fisher Scientific). Fusion proteins of M3M14, M14M3 and their catalytically inactive forms were purified from bacteria. The in vitro methylation assay was performed in a 50 µl reaction mixture containing 400 nM RNA probe, 20 mM Tris (pH 7.5), 50 µM ZnCl2, 1 mM DTT, 0.01% Triton-X, 0.2 U/µL RNaseOUT, 1% glycerol, 0.5 μCi [methyl-3H]AdoMet (PerkinElmer) and 100 nM purified protein. The reaction was incubated at 30°C for 1 h and then stopped by adding Trizol reagent (Invitrogen). RNA after reaction was precipitated and purified using sodium acetate at −20°C for at least 2 h. The precipitated RNA was subjected to radioactivity measurement using scintillation counting (Beckman). Levels of 3H-methyl-incorporated RNA are shown as disintegrations per minute (DPM). The methylation assay data are shown as mean ± s.e.m. from three independent replicates.
Rna probe
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RNA probe is a laboratory instrument used to detect and analyze specific RNA molecules. It functions by hybridizing with target RNA sequences, allowing for their identification and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Scintillation counting, by Beckman Coulter (2 mentions)
Methyl 3h adomet, by PerkinElmer (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Viewrna cell plus, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Clariostar microplate reader, by BMG Labtech (1 mentions)
4 protocols using rna probe
1
Hsp70 RNA Methylation Assay
2
Hsp70 RNA Methylation Assay
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The RNA probe (5’-CGAUCCUCGGCCAGGAC CAGCCUUCCCCAG-3’) derived from Hsp70 5’UTR was commercially synthesized in vitro (Thermo Fisher Scientific). Fusion proteins of M3M14, M14M3 and their catalytically inactive forms were purified from bacteria. The in vitro methylation assay was performed in a 50 µl reaction mixture containing 400 nM RNA probe, 20 mM Tris (pH 7.5), 50 µM ZnCl2, 1 mM DTT, 0.01% Triton-X, 0.2 U/µL RNaseOUT, 1% glycerol, 0.5 μCi [methyl-3H]AdoMet (PerkinElmer) and 100 nM purified protein. The reaction was incubated at 30°C for 1 h and then stopped by adding Trizol reagent (Invitrogen). RNA after reaction was precipitated and purified using sodium acetate at −20°C for at least 2 h. The precipitated RNA was subjected to radioactivity measurement using scintillation counting (Beckman). Levels of 3H-methyl-incorporated RNA are shown as disintegrations per minute (DPM). The methylation assay data are shown as mean ± s.e.m. from three independent replicates.
3
Visualizing Nuclear mRNA by FISH
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poly(A)RNA FISH was performed as described earlier66 (link). In brief, cells were treated with 1 mM auxin for 8 h, washed with PBS, fixed for 15 min in 4% PFA at RT, incubated in 70% ethanol for 16 h at 4 °C, washed with PBS, and incubated with wash buffer for 5 min at RT. Cells were then incubated with oligo(dT)-Quasar 670 probes (LGC Biosearch Technologies) for 4 h at 37 °C, followed by incubation with wash buffer at 37 °C for 30 min and then washed with PBS. Cells were then stained with 1.5 μg/ml Hoechst 33258 (Molecular Probes/Life Technologies) for 10 min, washed with PBS, and finally mounted in ProLong Gold antifade reagent (Life Technologies).
Individual RNAs were detected by FISH using ViewRNA Cell Plus (Thermo Fisher Scientific, Cat#88-19000-99) and corresponding RNA probes (Thermo Fisher Scientific, Cat# VX-01), according to the manufacturer’s instructions. Briefly, cells were seeded on 24-well plates with coverslips, processed once they reach 80% confluency, and fixed according to the ViewRNA Cell Plus protocol. Mab414 antibodies were used to detect NPC.
Cytoplasmic-to-nucleus (Cyt/Nu) ratio of mRNA foci was plotted with scatter plot function in Prism software. Data are expressed as mean value with standard deviations as error bars. Unpaired two-tailed Student’s t test has been applied for comparison of untreated and auxin-treated cells.
Individual RNAs were detected by FISH using ViewRNA Cell Plus (Thermo Fisher Scientific, Cat#88-19000-99) and corresponding RNA probes (Thermo Fisher Scientific, Cat# VX-01), according to the manufacturer’s instructions. Briefly, cells were seeded on 24-well plates with coverslips, processed once they reach 80% confluency, and fixed according to the ViewRNA Cell Plus protocol. Mab414 antibodies were used to detect NPC.
Cytoplasmic-to-nucleus (Cyt/Nu) ratio of mRNA foci was plotted with scatter plot function in Prism software. Data are expressed as mean value with standard deviations as error bars. Unpaired two-tailed Student’s t test has been applied for comparison of untreated and auxin-treated cells.
4
Characterizing SARS-CoV-2 N Protein-RNA Interactions
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Chemically synthesized RNA probes 5’-labelled with fluorescein isothiocyanate (FITC) and purified by HPLC were obtained from Thermo Scientific (USA). Probe sequences were as follows: RNA1 (5’-CACUCACUGUCUUUUUUGAUGGUAGAGU-3’), RNA2 (5’-CACUCACUGUCAAAAAAGAUGGUAGAGU-3’), RNA3 (5’-CACUCACUGUCUUGUUUGAUGGUAGAGU-3’), RNA4 (5’-CACUCACUGUCUUUUUU-3’), RNA5 (5’-CACUCACUGUC-3’) and scramble control RNA6 (5’-AUAUAGCUAC-3’).
Fluorescence polarization (FP) assays were used to determine the binding affinity of the N protein to the RNA probes, solubilized in 50 mM sodium phosphate buffer (pH, 7.6). Purified N protein from 2.5 nM to 15 µM in 50 mM sodium phosphate buffer, 100 mM NaCl (pH, 7.6) was mixed with each RNA at 10 nM final concentration, in 384-well plates. FP data was acquired using a ClarioStar microplate reader (BMG LabTech), with excitation and emission wavelengths set to 485 and 530 nm, respectively. Affinity binding curves were fitted to a Hill1 model using the OriginPro software.
Fluorescence polarization (FP) assays were used to determine the binding affinity of the N protein to the RNA probes, solubilized in 50 mM sodium phosphate buffer (pH, 7.6). Purified N protein from 2.5 nM to 15 µM in 50 mM sodium phosphate buffer, 100 mM NaCl (pH, 7.6) was mixed with each RNA at 10 nM final concentration, in 384-well plates. FP data was acquired using a ClarioStar microplate reader (BMG LabTech), with excitation and emission wavelengths set to 485 and 530 nm, respectively. Affinity binding curves were fitted to a Hill1 model using the OriginPro software.
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