Rna oligonucleotides

Manufactured by GE Healthcare

Sourced in France

RNA oligonucleotides are synthetic, single-stranded RNA molecules that can be used in various laboratory applications. They are typically used as probes, primers, or templates for research involving RNA analysis, detection, and manipulation.

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Lab products found in correlation

Molecular biology grade bovine serum albumin, by New England Biolabs (3 mentions) Storage phosphor imaging plates, by GE Healthcare (3 mentions) Typhoon 9400 phosphorimager, by GE Healthcare (3 mentions) The avian myeloblastosis virus reverse transcriptase, by Promega (3 mentions) Deoxynucleotide triphosphate dntp mix, by Promega (3 mentions) Dpn 1, by Thermo Fisher Scientific (3 mentions) Rnase inhibitor, by New England Biolabs (3 mentions) T4 polynucleotide kinase, by New England Biolabs (3 mentions) T4 dna ligase, by New England Biolabs (3 mentions) γ 32p atp, by PerkinElmer (3 mentions) Rq1 rnase free dnase, by Promega (3 mentions) Quickchange 2 xl mutagenesis kit, by Agilent Technologies (2 mentions) Dna oligonucleotide, by Integrated DNA Technologies (2 mentions) Victor x5, by PerkinElmer (1 mentions) Rnasin, by Promega (1 mentions) Dna oligonucleotides, by Merck Group (1 mentions) Synthetic dna oligonucleotides, by Integrated DNA Technologies (1 mentions)

8 protocols using rna oligonucleotides

Fluorescence Polarization Assay for RNA-Protein Interactions

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Sam68 and SLM2 STAR domains were produced as previously described (15 (link)). RNA oligonucleotides were purchased from Dharmacon, GE Healthcare, deprotected according to the manufacturer's instructions, lyophilized, and resuspended in ddH₂O. All RNAs used for fluorescence polarization contained a fluorescein tag and three cytosines at the 5΄ end of the sequence described in Table 1.
Fluorescence polarization experiments were carried out in black 96-well plates with a 50 μl sample volume per well in 10 mM Tris pH 7, 100 mM NaCl, 0.1% β-mercaptoethanol. Sam68 and SLM2 domains were serially diluted across the plate from 200 to 0 μM. Fluorescein-labeled RNA was then added at 0.2 μM final concentration. Plates were analyzed using a Perkin Elmer Victor X5 plate reader at excitation wavelength of 531 nm and emission at 595 nm, and experiments were carried out in triplicate.

Danilenko M., Dalgliesh C., Pagliarini V., Naro C., Ehrmann I., Feracci M., Kheirollahi-Chadegani M., Tyson-Capper A., Clowry G.J., Fort P., Dominguez C., Sette C, & Elliott D.J. (2016). Binding site density enables paralog-specific activity of SLM2 and Sam68 proteins in Neurexin2 AS4 splicing control. Nucleic Acids Research, 45(7), 4120-4130.

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Engineered miR-129 Mimics for Enhanced Stability

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miR-129 mimics 1 and 2 were modified by substituting uracil (U) with 5-fluorouracil (5-FU). Mimic-3 was modified with 2’-Flurouracil and Phosphorothioates in the guide strand to enhance stability as well as 2’-O-Methylation of the passenger strand to prevent RISC loading and enhance stability [22 (link)]. RNA oligonucleotides with these modifications as well as their corresponding passenger strands were purchased from Dharmacon, (GE Life Sciences). The modified miR-129 and passenger strands were annealed prior to use for transfection (Figure 1).

Wu N., Fesler A., Liu H, & Ju J. (2017). Development of novel miR-129 mimics with enhanced efficacy to eliminate chemoresistant colon cancer stem cells. Oncotarget, 9(10), 8887-8897.

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RNA Oligonucleotide Preparation and Quantification

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RNA oligonucleotides were purchased from Dharmacon (GE healthcare), de-protected according to manufacturer’s protocol and lyophilized. The RNA was re-suspended in either nuclease-free water or ITC (isothermal titration calorimetry) buffer containing 40–100 u ml⁻¹ (RNAsin, Promega) and the concentration determined by measuring absorbance at 260 nm and extinction coefficients calculated from the base composition of the sequence.

Dagil R., Ball N.J., Ogrodowicz R.W., Hobor F., Purkiss A.G., Kelly G., Martin S.R., Taylor I.A, & Ramos A. (2019). IMP1 KH1 and KH2 domains create a structural platform with unique RNA recognition and re-modelling properties. Nucleic Acids Research, 47(8), 4334-4348.

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Enzymatic Synthesis and Characterization of Oligonucleotides

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Unless otherwise stated, reagents were purchased from Fisher Scientific, Sigma-Aldrich, or Life Technologies. T4 polynucleotide kinase, T4 DNA ligase, molecular biology grade bovine serum albumin (BSA), and RNase inhibitor were purchased from New England Biolabs. γ-[³²P] ATP was purchased from Perkin-Elmer Life Sciences. The Avian Myeloblastosis Virus (AMV) reverse transcriptase, deoxynucleotide triphosphate (dNTP) mix and RQ1 RNase free DNase were purchased from Promega. Pfu Ultra II was purchased from Stratagene. Dpn 1 was purchased from Invitrogen. Quickchange XL II mutagenesis kit was purchased from Agilent Technologies. RNA oligonucleotides were synthesized at the University of Utah DNA/Peptide Core Facility or purchased from GE Healthcare Dharmacon, Inc. or Sigma Aldrich. DNA oligonucleotides were purchased from Integrated DNA Technologies. Storage phosphor imaging plates from Molecular Dynamics were imaged using Molecular Dynamics 9400 Typhoon phosphorimager. Data were analyzed using Molecular Dynamics ImageQuant 5.2 software. Electrospray Ionization (ESI) mass spectrometry of oligonucleotide samples was carried out at the Campus Mass Spectrometry Facilities, UC Davis. Oligonucleotide masses were determined using Mongo Oligo Mass Calculator v2.06.

Matthews M.M., Thomas J.M., Zheng Y., Tran K., Phelps K.J., Scott A.I., Havel J., Fisher A.J, & Beal P.A. (2016). Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nature structural & molecular biology, 23(5), 426-433.

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Oligonucleotide Synthesis and Characterization

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Unless otherwise stated, reagents were purchased from Fisher Scientific, Sigma-Aldrich, or Life Technologies. Brown Centrex columns were purchased from VWR. T4 polynucleotide kinase, T4 DNA ligase, molecular biology grade bovine serum albumin (BSA), chemically competent DH5α Escherichia coli and RNase inhibitor were purchased from New England Biolabs. γ-[³²P] ATP was purchased from Perkin-Elmer Life Sciences. The Avian Myeloblastosis Virus (AMV) reverse transcriptase, deoxynucleotide triphosphate (dNTP) mix and RQ1 RNase free DNase were purchased from Promega. Nuclease T1 and nuclease V1 were purchased from Ambion. Pfu Ultra II was purchased from Stratagene. Dpn 1 was purchased from Invitrogen. RNA oligonucleotides were synthesized at the University of Utah DNA/Peptide Core Facility or purchased from GE Healthcare Dharmacon, Inc. or Sigma Aldrich. DNA oligonucleotides were purchased from Integrated DNA Technologies or Sigma Aldrich. Storage phosphor imaging plates from Molecular Dynamics were imaged using Molecular Dynamics 9400 Typhoon phosphorimager. Data were analyzed using Molecular Dynamics ImageQuant 5.2 software. Electrospray Ionization (ESI) mass spectrometry of oligonucleotide samples was carried out at either the Campus Mass Spectrometry Facilities, UC Davis or at Novatia, LLC. Oligonucleotide masses were determined using Mongo Oligo Mass Calculator v2.06.

Phelps K.J., Tran K., Eifler T., Erickson A.I., Fisher A.J, & Beal P.A. (2015). Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2. Nucleic Acids Research, 43(2), 1123-1132.

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Purification and Structure Prediction of RNA Oligonucleotides

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RNA oligonucleotides were purchased from Dharmacon (GE Healthcare Europe GmbH, Vélizy-Villacoublay, France) purified by electrophoresis in 7 M urea 20 % polyacrylamide gels, and desalted on Sephadex G-25 spin columns prepared in 1 mL syringes. The secondary structure predictions were performed using the Mfold web server at http://mfold.rna.albany.edu/?q=node/60 [23 (link)].

Masante C., Jaubert C., Palau W., Plissonneau J., Besnard L., Ventura M, & Di Primo C. (2015). Mutations of the SL2 dimerization sequence of the hepatitis C genome abrogate viral replication. Cellular and Molecular Life Sciences, 72(17), 3375-3385.

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Enzymatic Synthesis and Characterization of Oligonucleotides

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Reconstitution of Mycobacterial Transcription

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Synthetic DNA oligonucleotides were obtained from Integrated DNA
Technologies (Coralville, IA) and RNA oligonucleotides were obtained
from GE Healthcare Dharmacon Inc. (Lafayette, CO). The nucleic acids for
the EC and PEC scaffolds were resuspended in annealing buffer (10 mM
HEPES pH 7.5, 1 mM EDTA, 50 mM NaCl). Respective template DNA (t-DNA)
and RNA were annealed in a 1:3 ratio for 5 min at 95 °C in a
covered heat block, then cooled to room temperature, then immediately
moved to ice. The annealed RNA-DNA hybrids were stored at – 80
°C until use. Purified Mtb core RNAP was
dialyzed overnight into cryo-EM buffer (20 mM HEPES pH 7.5, 150 mM KGlu,
5 mM MgOAc, 2.5 mM DTT). To prepare complexes, annealed t-DNA:RNA was
combined with core at 1.3:1 molar excess and incubated at room
temperature for 15 min. Next, non-template DNA (nt-DNA) was added to the
complexes at 1.5:1 molar excess with the t-DNA:RNA and incubated for 10
min at room temperature. The complexes were then injected onto the
Superose 6 Increase 10/300 GL column (Cytiva) for purification.
Mtb NusG or Eco NusG was buffer
exchanged and purified on the Superose 6 column. Purified NusG was added
in 3x molar excess to purified core RNAP and incubated for 10 min at
room temperature. Complexes were concentrated by centrifugal filtration
(Amicon Ultra) to 4–6.5 mg RNAP/mL before grid preparation.

Delbeau M., Omollo E.O., Froom R., Koh S., Mooney R.A., Lilic M., Brewer J.J., Rock J., Darst S.A., Campbell E.A, & Landick R. (2023). Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis. Molecular cell, 83(9), 1474-1488.e8.

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