Riboprobe in vitro transcription system

Manufactured by Promega

Sourced in United States, Germany

The Riboprobe In Vitro Transcription Systems are laboratory equipment designed for the in vitro synthesis of RNA molecules. These systems provide the necessary components, including RNA polymerases, ribonucleotides, and other essential reagents, to facilitate the production of labeled or unlabeled RNA transcripts from DNA templates.

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Lab products found in correlation

Probequant g 50 micro column, by GE Healthcare (3 mentions) Prime it 2 random primer labeling kit, by Agilent Technologies (2 mentions) Pgem t easy vector, by Promega (2 mentions) Dig labeling mix, by Roche (2 mentions) Anti dig pod fab fragments, by Roche (2 mentions) Tsa plus cy5 fluorescence system, by PerkinElmer (2 mentions) Microspin g 25 column, by GE Healthcare (2 mentions) Mmessage mmachine sp6 transcription kit, by Thermo Fisher Scientific (2 mentions) Nanodrop, by Thermo Fisher Scientific (1 mentions) Pgem t easy vector system, by Promega (1 mentions) Genelute pcr clean up, by Merck Group (1 mentions) Gelred, by Biotium (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Rneasy minielute cleanup kit, by Qiagen (1 mentions) T7 rna polymerase, by Promega (1 mentions) Dnase, by Promega (1 mentions) Pgex 6p 3, by GE Healthcare (1 mentions) Proteiniso gst resin, by Transgene (1 mentions) Biomax ms, by Kodak (1 mentions) Microspin g 50 column, by GE Healthcare (1 mentions)

Spelling variants of this product

Riboprobe system (31 citations) Riboprobe (14 citations) In vitro transcription (9 citations) SP6/T7 Riboprobe® in vitro Transcription Systems (2 citations)

29 protocols using riboprobe in vitro transcription system

Plasmid Cloning and RNA Probe Synthesis

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The GenElute™ PCR Clean‐Up (Sigma‐Aldrich) kit and protocol was used for PCR clean‐up of genes of interest (GOI) fragments, obtained by RT‐PCR. After the products had been purified, their concentration was measured with NanoDrop (Thermo Scientific). Ligation of the GOI fragments and transformation was done using the pGEM®‐T Easy Vector Systems (Promega) kit and protocol.
For the transformation, DH5α competent cells (in‐house) were used. Colonies with the right insert were midiprepped using the GenElute® Plasmid Midiprep (Sigma‐Aldrich) kit and protocol. The plasmid, in the eluate, had its concentration measured using NanoDrop (Thermo Scientific). Linearised plasmid was used as a template to synthesise digoxigenin (DIG)‐labelled RNA probes using in vitro transcription with SP6 or T7 RNA polymerases. The reaction was set up using Riboprobe® in vitro Transcription Systems (Promega) reagents and protocol. Riboprobes were purified using the ProbeQuant G‐50 micro columns (Illustra) kit and protocol.

Perdios C., Parnall M., Pang K.L, & Loughna S. (2019). Altered haemodynamics causes aberrations in the epicardium. Journal of Anatomy, 234(6), 800-814.

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Northern Blotting for RNA Detection

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TRIzol (Invitrogen) reagent was used to isolate total RNA from cells as per the manufacturer’s instructions. About 4 μg of total RNA was resolved in 1.2% agarose gels with 0.7% formaldehyde and GelRed® (Biotium) in 1× MOPS buffer. The RNA was transferred to a nylon membrane overnight by capillary-based transfer and used for probing rRNAs and mRNAs. For tRNA northern blots, RNA was resolved in TBE-Urea Gel (15%) in 1× TBE buffer followed by wet transfer at 30 V for 1 h in 0.5× TBE buffer. Membranes were then UV crosslinked and hybridized with probes corresponding to the mitochondrially encoded mRNAs or tRNAs. Washing the membranes after probing was performed with 1× SSC buffer (150 mM NaCl, 15 mM tri-sodium citrate (pH 7.0)) supplemented with 0.1% SDS. Probes for mRNA and tRNA were prepared by Prime-It II Random Primer Labeling Kit (Agilent technologies) with 50 μCi of CTP and Riboprobe® in vitro Transcription Systems (Promega) with 50 μCi of UTP respectively, according to manufacturer’s instructions. Oligonucleotides and primers used for the synthesis of tRNA and mRNA probes are listed in the Supplementary Tables S3 and S4, respectively. Membranes were then exposed on a phosphoimager screen and visualized using Typhoon FLA 7000 phosphoimager.

Gopalakrishna S., Pearce S.F., Dinan A.M., Rosenberger F.A., Cipullo M., Spåhr H., Khawaja A., Maffezzini C., Freyer C., Wredenberg A., Atanassov I., Firth A.E, & Rorbach J. (2019). C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis. Nucleic Acids Research, 47(17), 9386-9399.

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Quantification of Viral RNA Transcripts

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The NP gene of AIVs and the HA genes of clade 2.3.4.4 H5, H6 and H7 viruses were amplified using one-step RT-PCR (Qiagen, Germany) with each of the designed RPA primer pairs. The RT-PCR products were purified using the PCR cleanup kit (GeneMark, Taiwan) and cloned into pGEM-T Easy Vector (Promega, USA). The recombinant plasmid was linearized and the 3’ overhang was conversed with the DNA polymerase Klenow (Promega). In vitro transcription was performed using Riboprobe in vitro Transcription Systems (Promega) with T7 RNA Polymerase according to the manufacturer's recommendations. DNase (Promega) was added to remove the remaining template DNA. The produced RNA was purified using RNeasy MiniElute Cleanup Kit (Qiagen) and verified by electrophoresis gel. The RNA was quantified using a spectrophotometer (Thermo Fisher Scientific, USA) and the copy number was calculated.

Tsai S.K., Chen C.C., Lin H.J., Lin H.Y., Chen T.T, & Wang L.C. (2020). Combination of multiplex reverse transcription recombinase polymerase amplification assay and capillary electrophoresis provides high sensitive and high-throughput simultaneous detection of avian influenza virus subtypes. Journal of Veterinary Science, 21(2), e24.

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Purification and Characterization of OsTRM13

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OsTRM13 full length cDNA was amplified and cloned into pGEX-6P-3 (GE healthcare Life Sciences, Shanghai, China) using BamHI and NotI sites, resulting in a fusion protein with glutathione S-transferase (GST) at the N-terminus. The recombinant vector was transformed into BL21 cells; expression of fusion protein was induced with 0.5 µM isopropyl β-D-1-thiogalactopyranoside (IPTG) and purified with ProteinIso GST Resin (Transgen Biotech). The N-terminal GST tag was cleaved off by ProScission Protease (Genscript Biotechnology Co. Ltd, Nanjing, China). Tag-free protein was eluted in the presence of 50 mM Tris–HCl (pH 7.0), 150 mM NaCl, 1 mM EDTA, and 1 mM DTT.
Yeast tRNA-Gly-GCC was synthesized in pGEM-T easy vector (Promega, Beijing, China). tRNA-Gly-GCC was in vitro transcribed with Riboprobe in vitro Transcription Systems (Promega). Buffers for tRNA methylation were described previously (Wilkinson et al., 2007 (link)), with 0.5 mM AdoMet as methyl donor. The substrate tRNA was provided in a final concentration of 1 or 2 µM (designated as + or ++), and the final concentration of OsTRM13 protein was 2 or 10 µM (designated as + or ++).

Wang Y., Li D., Gao J., Li X., Zhang R., Jin X., Hu Z., Zheng B., Persson S, & Chen P. (2017). The 2′-O-methyladenosine nucleoside modification gene OsTRM13 positively regulates salt stress tolerance in rice. Journal of Experimental Botany, 68(7), 1479-1491.

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Radioactive RNA Probe Binding Assay

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³²P-labeled and unlabeled mRNA probes were made in vitro using Riboprobe In Vitro Transcription Systems (Promega, Southampton, UK) in the presence or absence of 40 μCi of [α-³²P]rUTP (GE Healthcare) respectively. The BCL2 ARE probe was generated using the plasmidPCRII/bcl-2 ARE [30] (link). The resulting probe was separated from unincorporated nucleotides using ProbeQuant G-50 Micro Columns (GE Healthcare). E.coli-expressed purified ZFP36L1 protein (10–100 ng) or cell protein cell lysates (30 µg) were incubated with 50 000 to 100 000 cpm of α-³²P-labeled RNA probes, corresponding to approximately 30–100 fmol RNA. The proteins were incubated with RNA probes for 20 min on ice in RNA-binding buffer containing 20 mM HEPES (pH 7.6), 3 mM MgCl2, 40 mM KCl, 2 mM DTT, and 5% Glycerol in a total volume of 20 µl. RNase T1 and heparan sulfate (Sigma) were added to final concentrations of 50 U/ml and 5 mg/ml, respectively, and incubated on ice for another 20 min. RNA-protein complexes were resolved by electrophoresis (150 V for 3 hours at 4°C) on a 4% nondenaturing polyacrylamide gel using a Hoefer SE600 electrophoresis unit (Hoefer Scientific, Holliston, MA, USA). The gel was dried on a gel dryer (Hoefer SE1160 Gel Dryer), exposed to X-ray film (Kodak BioMax MS) and developed.

Zekavati A., Nasir A., Alcaraz A., Aldrovandi M., Marsh P., Norton J.D, & Murphy J.J. (2014). Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells. PLoS ONE, 9(7), e102625.

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In Vitro Transcription and Embryonic Injections

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Sat1 RNA and control RNAs were in vitro transcribed using Riboprobe in vitro transcription systems (Promega). Oligos to amplify the DNA template for in vitro transcription are included in Supplementary file 3. Sense and anti-sense transcripts were transcribed in vitro using the T3 and T7 RNA polymerases respectively. RNA was purified illustra MicroSpin G-50 Columns (GE Healthcare) and 50 ng of sense and antisense RNA was co-injected into zebrafish embryos at the 1 cell stage.

Rajshekar S., Yao J., Arnold P.K., Payne S.G., Zhang Y., Bowman T.V., Schmitz R.J., Edwards J.R, & Goll M. (2018). Pericentromeric hypomethylation elicits an interferon response in an animal model of ICF syndrome. eLife, 7, e39658.

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Biotinylation of Synthesized tRNA

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The pGEM-T Easy plasmids containing the tRNA^Leu_CAG sequences were used as templates and amplified by PCR for the in vitro transcription of tRNA sequences using corresponding primer pairs (Table S2) and Pfu polymerase. After gel extraction, in vitro transcription was performed using the Riboprobe® in vitro Transcription Systems (Promega). The residual DNA was digested using DNase I, and the tRNAs were purified by phenol/chloroform precipitation. After rehydration with RNase-free destilled water (DW), tRNA was eluted using Illustra NAP columns (GE Healthcare, USA).
For the in vitro biotinylation of the synthesized tRNA, we generated eFx flexizyme, according to the protocol described previously,⁴⁰ and thus charged the synthesized tRNA with biotin-Phe-DBE.

Kwon N.H., Lee M.R., Kong J., Park S.K., Hwang B.J., Kim B.G., Lee E.S., Moon H.G, & Kim S. (2018). Transfer-RNA-mediated enhancement of ribosomal proteins S6 kinases signaling for cell proliferation. RNA Biology, 15(4-5), 635-648.

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In vitro Transcribed RNA Analysis for Reverse Transcription

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In vitro transcribed RNA for primer extension and reverse transcription in circle (cRT) was obtained with Riboprobe in vitro Transcription Systems (Promega) using linear pGemT-3′UTR_At3g18145. The [³²P]CTP-labeled RNA substrates were transcribed from linear pBSIIk+ plasmids containing rcr1 sequences (GeneCust). Primer extension (20 (link)) analysis was performed using in vitro transcribed RNA treated or untreated with His-RTL1 and specific 5′ end labeled primers p1, p2 and p7. For Reverse Transcription in circle (cRT) reactions, His-RTL1 treated RNA was incubated with T4 RNA ligase (Promega) for 1 h at 37°C. The reaction was then used to perform RT with UTRrt primers, followed by 42 cycles PCR with p1/p8 primers. PCR products were purified on 2% agarose gel, cloned and sequenced. Total RNA from Wt and RTL1-Flag plants was prepared using TriZol reagent (GE Healthcare, Littler Chalfont, Buckinghamshire, UK). All RNA samples were then treated with Turbo DNase (Ambion) to eliminate contaminant DNA (21 (link)).

Charbonnel C., Niazi A.K., Elvira-Matelot E., Nowak E., Zytnicki M., de Bures A., Jobet E., Opsomer A., Shamandi N., Nowotny M., Carapito C., Reichheld J.P., Vaucheret H, & Sáez-Vásquez J. (2017). The siRNA suppressor RTL1 is redox-regulated through glutathionylation of a conserved cysteine in the double-stranded-RNA-binding domain. Nucleic Acids Research, 45(20), 11891-11907.

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In Vitro Transcription and FISH Analysis of AAV and Tau Transcripts

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In vitro transcription of complementary RNA (cRNA) probes against the AAV 3′UTR transgene WPRE was constructed in the pBluescript II KS(−) vector, and cRNA probe against human P301L tau under the T7 promoter was synthesized as double-stranded DNA fragment as previously described (12 (link)). The 645–base pair reverse complement of the 3′UTR posttranslational regulatory element WPRE sequence in the AAV vector was used for the cRNA probe of AAV transgenes. Digoxigenin (DIG)–labeled cRNA riboprobe was synthesized using DIG labeling mix (catalog no. 1 277 073, Roche Diagnostics GmbH, Mannheim, Germany) and Riboprobe In Vitro Transcription Systems (catalog no. P1420, Promega, Madison, WI) and purified using ProbeQuant G-50 microcolumns (catalog no. GE28–9034, GE Healthcare, Boston, MA). Enhanced fluorescence in situ hybridization was conducted for the detection of human tau and WPRE mRNA on brain tissue sections using anti-digoxigenin antibody conjugated with horse-radish peroxidase (anti–DIG-POD) Fab fragments (catalog no. 11 207 733 910, Roche, Branford, CT), with the TSA Plus Cy5 fluorescence system (catalog no. NEL745, PerkinElmer, Waltham, MA). After in situ hybridization, the tissue sections were processed for immunofluorescence as described above to detect WFS1 or HT7.

Delpech J.C., Pathak D., Varghese M., Kalavai S.V., Hays E.C., Hof P.R., Johnson W.E., Ikezu S., Medalla M., Luebke J.I, & Ikezu T. (2021). Wolframin-1–expressing neurons in the entorhinal cortex propagate tau to CA1 neurons and impair hippocampal memory in mice. Science translational medicine, 13(611), eabe8455.

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Biotin-labeled RNA Pull-down Assay

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Linearized pCDNA3-hTR was used as template to transcribe biotinylated RNA using Riboprobe In Vitro Transcription Systems (Promega) according to the manufacturer’s instructions. One-half microgram of the biotinylated RNA was incubated at room temperature for 1 hour with 5 μg of indicated recombinant proteins in biotin pull-down assay buffer (50 mM tris-HCl at pH 7.9, 10% glycerol, 100 mM KCl, 5 mM MgCl₂, 10 mM β-mercaptoethanol, and 0.1% NP-40). Twenty microliters of washed streptavidin agarose beads (Invitrogen) was added to each binding reaction and further incubated at room temperature for 30 min. Beads were washed briefly five times and boiled in SDS loading buffer, the retrieved proteins were detected by standard Western blot analysis, and the recovered biotin-hTR was examined by the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific).

Cheng L., Yuan B., Ying S., Niu C., Mai H., Guan X., Yang X., Teng Y., Lin J., Huang J., Jin R., Wu J., Liu B., Chang S., Wang E., Zhang C., Hou N., Cheng X., Xu D., Yang X., Gao S, & Ye Q. (2019). PES1 is a critical component of telomerase assembly and regulates cellular senescence. Science Advances, 5(5), eaav1090.

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