Riboprobe system

Manufactured by Promega

Sourced in United States, Germany

The Riboprobe system is a laboratory tool used for the synthesis of radioactively or non-radioactively labeled ribonucleic acid (RNA) probes. It allows for the in vitro transcription of single-stranded RNA molecules from DNA templates using specific RNA polymerases.

Automatically generated - may contain errors

Lab products found in correlation

Cm1950, by Leica (7 mentions) Micro bio spin 30 column, by Bio-Rad (5 mentions) Biomax mr, by Kodak (4 mentions) 35s utp, by Cytiva (3 mentions) Rneasy mini kit, by Qiagen (2 mentions) Amersham hyperfilm, by GE Healthcare (2 mentions) Illustra microspin g 50 column, by GE Healthcare (2 mentions) Tat and recombinant smyd5, by Active Motif (2 mentions) Dnase 1, by Promega (2 mentions) Ribotrap kit, by Medical & Biological Laboratories (2 mentions) Protein g plus agarose, by Thermo Fisher Scientific (2 mentions) Ntb photographic emulsion, by Carestream (2 mentions) Dektol developer, by Kodak (2 mentions) 35s utp, by PerkinElmer (2 mentions) T7 polymerase, by Promega (2 mentions) Anti digoxigenin ap antibody, by Roche (1 mentions) Smz800n, by Nikon (1 mentions) Ltq orbitrap discovery hybrid ftms, by Thermo Fisher Scientific (1 mentions) Proteome discoverer 1.4 interface, by Thermo Fisher Scientific (1 mentions) Nanoacquity uplc, by Waters Corporation (1 mentions)

31 protocols using riboprobe system

Cloning and In Vitro Processing of pri-miR-21

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The DNA segment containing the pri-miR-21 sequence (274 nucleotides, comprising the full pre-miR-21 sequence plus 109 nucleotides at the 5′-end and 107 at the 3′-end) was amplified from human genomic DNA by PCR, and cloned into a pUC19 vector along with a T7 promoter. The coding sequence was verified by sequencing. The DNA plasmid was linearized downstream of the pri-miRNA sequences using BamH1 and gel purified. The internally labeled pri-miR-21 was transcribed in vitro using T7 RNA polymerase (Riboprobe system, Promega) in the presence of [α-³²P]-CTP, and purified with QIAGEN RNeasy Mini kit. Cell nuclear extracts (293 or HeLa) from ProteinOne were used for pri-miRNA processing assays, which were carried out (typically 10⁴ ~ 10⁵ cpm of labeled pri-miRNA, 25 μg cell nuclear extract per 20 μl reaction) at 30°C for 1 hour in buffer containing 20 mM Tris (pH7.5), 6.4 mM MgCl₂, 100 mM KCl, 0.5 mM DTT and 0.05 μg/μl tRNA. Following incubation, samples were phenol/chloroform extracted, precipitated and re-suspended in RNA loading dye prior to denaturing polyacrylamide gel electrophoresis (PAGE) separation using 8% acrylamide-8M urea gel.

Chen Y., Yang F., Zubovic L., Pavelitz T., Yang W., Godin K., Walker M., Zheng S., Macchi P, & Varani G. (2016). Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins. Nature chemical biology, 12(9), 717-723.

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Spatiotemporal Expression of Clmp in Mouse Brain

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Mouse brain sections (14 μm thick) at embryonic day (E18) and post-natal days (P1, P7, P14, P21, and P56) were prepared using a cryostat (Leica CM 1950). Hybridization probes specific for mouse Clmp mRNAs were prepared using the following regions: nt 1351–1650 (C-term) of Clmp (NM_133733.4). Antisense riboprobes were generated using 35^S-uridine triphosphate (UTP) and the Riboprobe system (Promega).

Jang S., Yang E., Kim D., Kim H, & Kim E. (2020). Clmp Regulates AMPA and Kainate Receptor Responses in the Neonatal Hippocampal CA3 and Kainate Seizure Susceptibility in Mice. Frontiers in Synaptic Neuroscience, 12, 567075.

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Spatiotemporal Dlg2 mRNA Expression

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Mouse brain sections (14 μm thick) at embryonic day (E18) and postnatal days (P0, P7, P14, P21, and P56) were prepared using a cryostat (Leica CM 1950). Hybridization probe specific for mouse Dlg2 mRNA was prepared using the following regions: nt 1116–1369 of Dlg2 (NM_011807.3). Antisense riboprobe was generated using 35S-uridine triphosphate (UTP) and the Riboprobe System (Promega). For quantification, 15 of sampling areas (233,000 μm²) were randomly selected within a region of interest including the cortex, striatum, and cerebellum. The mean gray intensity of each region was measured using the ImageJ Fiji software [21 (link)]. Data were pooled from both sagittal and horizontal brain sections of two mice per each time point.

Yoo T., Kim S.G., Yang S.H., Kim H., Kim E, & Kim S.Y. (2020). A DLG2 deficiency in mice leads to reduced sociability and increased repetitive behavior accompanied by aberrant synaptic transmission in the dorsal striatum. Molecular Autism, 11, 19.

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In Situ Hybridization of Mouse Nephrin Genes

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In situ hybridization was performed essentially as previously described (Kim et al., 2002 (link)). Hybridization probes specific for mouse Neph1/2/3 mRNAs were prepared using the following regions: nt 1861-2370 of Neph1 (NCBI accession #: AF480411), nt 2161-2694 of Neph2 (AY169782) and nt 2222-2770 of Neph3 (AK049284). Antisense riboprobes were generated using α-³⁵S-UTP and the Riboprobe system (Promega).

Roh J.D., Choi S.Y., Cho Y.S., Choi T.Y., Park J.S., Cutforth T., Chung W., Park H., Lee D., Kim M.H., Lee Y., Mo S., Rhee J.S., Kim H., Ko J., Choi S.Y., Bae Y.C., Shen K., Kim E, & Han K. (2017). Increased Excitatory Synaptic Transmission of Dentate Granule Neurons in Mice Lacking PSD-95-Interacting Adhesion Molecule Neph2/Kirrel3 during the Early Postnatal Period. Frontiers in Molecular Neuroscience, 10, 81.

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ZENK Expression Detection in Brain

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The expression of ZENK in brain sections was detected with antisense RNA probes labelled with 35S-CTP. Labelling of the probes with 35S-CTP (1250 Ci/mmol; Perkin Elmer, Rodgau, Germany) was performed using the Riboprobe System (Promega). Our in situ hybridisation procedure followed a previously published protocol (Whitfield et al., 1990 (link)) with modifications as previously described in detail (Gahr and Metzdorf, 1997 (link)). For signal detection, sections were exposed to autoradiographic film (Kodak Biomax MR, Rochester, USA). Brains from both groups of males were run through the entire procedure at the same time and sections from both were placed on each autoradiographic film to avoid any possible effect of small differences in procedures on the observed differences in expression level. No labelling was observed with the sense probe.

Voigt C., Hirschenhauser K, & Leitner S. (2018). Neural activation following offensive aggression in Japanese quail. Biology Open, 7(12), bio038026.

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In situ hybridization of SALM4 in mouse brain

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Mouse brain sections (12 μm thick) at embryonic day (E16 and E18) and postnatal days (P7, P14, P21 and P56) were prepared using a cryostat (Leica CM 1950). Mouse brain sections from WT and Salm4^−/− mice (P56) were also generated to visualize the lack of SALM4 mRNAs in Salm4^−/− mice. Hybridization probe specific for mouse SALM4 mRNA was prepared using the following regions: nt 1,429–1,881 of SALM4 (GenBank DQ078787). Antisense riboprobe was generated using 35S-uridine triphosphate (UTP) and the Riboprobe system (Promega).

Lie E., Ko J.S., Choi S.Y., Roh J.D., Cho Y.S., Noh R., Kim D., Li Y., Kang H., Choi T.Y., Nam J., Mah W., Lee D., Lee S.G., Kim H.M., Kim H., Choi S.Y., Um J.W., Kang M.G., Bae Y.C., Ko J, & Kim E. (2016). SALM4 suppresses excitatory synapse development by cis-inhibiting trans-synaptic SALM3–LAR adhesion. Nature Communications, 7, 12328.

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In situ Hybridization of Rat Brain

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In situ hybridization was performed as described previously 58 (link). Adult (6 weeks) rat brain sections (12 μm thick) were prepared with a cryostat (Leica CM 1950). The hybridization probes were prepared from pGEM7zf plasmids containing nucleotides 279-1444 of rat IgSF11 (XM_221443) using a Riboprobe System (Promega) and α-[³⁵S]UTP.

Jang S., Oh D., Lee Y., Hosy E., Shin H., van Riesen C., Whitcomb D., Warburton J.M., Jo J., Kim D., Kim S.G., Um S.M., Kwon S.K., Kim M.H., Roh J.D., Woo J., Jun H., Lee D., Mah W., Kim H., Kaang B.K., Cho K., Rhee J.S., Choquet D, & Kim E. (2015). Synaptic adhesion molecule IgSF11 regulates synaptic transmission and plasticity. Nature neuroscience, 19(1), 84-93.

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Developmental Expression of Ank2 mRNA

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Mouse brain sections (14 μm thick) at embryonic day (E18) and postnatal days (P0, P7, P14, P21, and P56) were prepared using a cryostat (Leica CM 1950). A hybridization probe specific for mouse Ank2 mRNA was prepared using the following regions: nucleotide 315–565 of Ank2 (NM_178655.3). Antisense riboprobe was generated using ³⁵S-uridine triphosphate (UTP) and the Riboprobe system (Promega).

Oh H., Lee S., Oh Y., Kim S., Kim Y.S., Yang Y., Choi W., Yoo Y.E., Cho H., Lee S., Yang E., Koh W., Won W., Kim R., Lee C.J., Kim H., Kang H., Kim J.Y., Ku T., Paik S.B, & Kim E. (2023). Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice. Nature Communications, 14, 3547.

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Spatiotemporal Expression of Scn2a in Mice

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Mouse brain sections (14 μm thick) at embryonic day (E18) and postnatal days (P0, P7, P14, P21, and P56) were prepared using a cryostat (Leica CM 1950). Hybridization probes specific for mouse Scn2a mRNAs were prepared using the following regions: nt 181–480 (N-term) and nt 6060–6359 (C-term) of Scn2a (NM_001099298.2). Antisense riboprobes were generated using ³⁵S-uridine triphosphate (UTP) and the Riboprobe system (Promega).

Shin W., Kweon H., Kang R., Kim D., Kim K., Kang M., Kim S.Y., Hwang S.N., Kim J.Y., Yang E., Kim H, & Kim E. (2019). Scn2a Haploinsufficiency in Mice Suppresses Hippocampal Neuronal Excitability, Excitatory Synaptic Drive, and Long-Term Potentiation, and Spatial Learning and Memory. Frontiers in Molecular Neuroscience, 12, 145.

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Synthesis and Characterization of TAR RNA

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TAR RNAs (WT, Δbulge and Δloop) were synthesized in in-vitro transcription reactions with the Riboprobe system (Promega).^{35 (link)} Transcripts were treated with 2 U of DNase I (Promega), extracted with a phenol:chloroform mixture and purified over an Illustra MicroSpin G50 column (GE Healthcare). Gel-mobility reactions (12 μl final volume) were carried out in binding buffer (50 mM Tris, pH 7.4, 0.5 mM EGTA, 150 mM NaCl, 2% glycerol, 0.2% Tween 20, 0.5 mM DTT, 90 mM ZnSO4, 0.005% BSA and 100 μM ATP) and contained 2×10⁴ cpm TAR probes/reaction and the indicated concentrations of Tat and recombinant SMYD5 (Active Motif). Reactions were incubated for 30 min at 30 °C and separated on a pre-run 4% Tris-glycine gel. The gels were dried and exposed to Amersham Hyperfilm (GE Healthcare) overnight.

Boehm D., Lam V., Schnolzer M, & Ott M. (2023). The lysine methyltransferase SMYD5 amplifies HIV-1 transcription and is post-transcriptionally upregulated by Tat and USP11. Cell reports, 42(3), 112234.

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