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Rnascope multiplex fluorescent v1 assay

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The RNAscope Multiplex Fluorescent v1 Assay is a proprietary in situ hybridization technology developed by Advanced Cell Diagnostics. It enables the simultaneous detection and visualization of up to 3 different RNA targets within a single cell or tissue sample.

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Lab products found in correlation

Vglut2 slc17a6, by Advanced Cell Diagnostics (2 mentions) Dm5500 widefield microscope, by Leica (2 mentions) Ab49873, by Abcam (2 mentions) Mm per1, by Advanced Cell Diagnostics (2 mentions) Tissue tek, by Sakura Finetek (2 mentions) Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)

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RNAscope assay (49 citations)

7 protocols using rnascope multiplex fluorescent v1 assay

1

Confirming Pdyn-GFP Expression in LJA5 Neurons

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Pdyn‐GFP mice were described previously (Geerling et al., 2016; Krashes et al., 2014). To confirm that in the LJA5 region specifically, GFP expression was limited to prodynorphin‐expressing neurons, we performed in situ hybridization with RNAscope using a probe directed against mouse Pdyn (318771) and the Advanced Cell Diagnostics RNAscope multiplex V1 fluorescent assay.
We additionally labeled Pdyn‐GFP tissue for GABAergic and glutamatergic genetic markers. We used probes directed against mouse Vgat/Slc32a (319191), Gad1 (400951), Vglut2/Slc17a6 (319171) (Advanced Cell Diagnostics).
Agostinelli L.J, & Bassuk A.G. (2020). Novel inhibitory brainstem neurons with selective projections to spinal lamina I reduce both pain and itch. The Journal of Comparative Neurology, 529(8), 2125-2137.
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2

Validating Pdyn-GFP Expression in LJA5 Region

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Pdyn-GFP mice were described previously (Geerling et al., 2016 (link); Krashes et al., 2014 (link)). To confirm that in the LJA5 region specifically, GFP expression was limited to prodynorphin-expressing neurons, we performed in situ hybridization with RNAscope using a probe directed against mouse Pdyn (318771) and the Advanced Cell Diagnostics RNAscope multiplex V1 fluorescent assay.
We additionally labeled Pdyn-GFP tissue for GABAergic and glutamatergic genetic markers. We used probes directed against mouse Vgat/Slc32a (319191), Gad1 (400951), Vglut2/Slc17a6 (319171) (Advanced Cell Diagnostics).
Agostinelli L.J, & Bassuk A.G. (2020). Novel inhibitory brainstem neurons with selective projections to spinal lamina I reduce both pain and itch. The Journal of Comparative Neurology, 529(8), 2125-2137.
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3

Zonal Expression of Clock Genes in Liver

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smFISH of R genes were done on fresh-frozen liver cryosections (8μm) embedded in O.C.T Compound (Tissue-Tek; Sakura-Finetek USA), sampled every three hours (ZT0 to ZT21). RNAscope® probes for Bma1l mRNA (Mm-Arntl, catalog #: 438748-C3) and Per1 mRNA (Mm-Per1, catalog #: 438751) were used, according to the manufacturer’s instructions for the RNAscope Fluorescent Multiplex V1 Assay (Advanced Cell Diagnostics). To detect the central vein, an immunofluorescence of Glutamine Synthetase (ab49873, Abcam, diluted 1:2000 in PBS/BSA 0.5%/Triton-X0.01%) was done together with smFISH. Nuclei were counterstained with DAPI and sections were mounted with ProLong™ Gold Antifade Mountant. Liver sections were imaged with a Leica DM5500 widefield microscope and an oil-immersion x63 objective. Z-stacks were acquired (0.2μm between each Z position) and mRNA transcripts were quantified using ImageJ, as described previously in ref.50 (link). Pericentral (PC) and Periportal (PP) veins were manually detected based on Glutamine Synthetase IF or on bile ducts (DAPI staining). The Euclidean distance between two veins and the distance from the vein of each mRNA transcript were calculated. mRNA transcripts were assigned to a PP or PC zone if the distance from the corresponding vein was smaller than one-third of the distance between the PP and PC veins (ranging from 50 to 130μm).
Droin C., El Kholtei J., Halpern K.B., Hurni C., Rozenberg M., Muvkadi S., Itzkovitz S, & Naef F. (2021). Space-time logic of liver gene expression at sublobular scale. Nature metabolism, 3(1), 43-58.
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4

Zonal Expression of Clock Genes in Liver

Cited 1 time
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smFISH of R genes were done on fresh-frozen liver cryosections (8μm) embedded in O.C.T Compound (Tissue-Tek; Sakura-Finetek USA), sampled every three hours (ZT0 to ZT21). RNAscope® probes for Bma1l mRNA (Mm-Arntl, catalog #: 438748-C3) and Per1 mRNA (Mm-Per1, catalog #: 438751) were used, according to the manufacturer’s instructions for the RNAscope Fluorescent Multiplex V1 Assay (Advanced Cell Diagnostics). To detect the central vein, an immunofluorescence of Glutamine Synthetase (ab49873, Abcam, diluted 1:2000 in PBS/BSA 0.5%/Triton-X0.01%) was done together with smFISH. Nuclei were counterstained with DAPI and sections were mounted with ProLong™ Gold Antifade Mountant. Liver sections were imaged with a Leica DM5500 widefield microscope and an oil-immersion x63 objective. Z-stacks were acquired (0.2μm between each Z position) and mRNA transcripts were quantified using ImageJ, as described previously in ref.50 (link). Pericentral (PC) and Periportal (PP) veins were manually detected based on Glutamine Synthetase IF or on bile ducts (DAPI staining). The Euclidean distance between two veins and the distance from the vein of each mRNA transcript were calculated. mRNA transcripts were assigned to a PP or PC zone if the distance from the corresponding vein was smaller than one-third of the distance between the PP and PC veins (ranging from 50 to 130μm).
Droin C., El Kholtei J., Halpern K.B., Hurni C., Rozenberg M., Muvkadi S., Itzkovitz S, & Naef F. (2021). Space-time logic of liver gene expression at sublobular scale. Nature metabolism, 3(1), 43-58.
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5

Multiplex Fluorescent In Situ Hybridization Protocol

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ISH experiments were performed using the RNAscope® technology (Advanced Cell Diagnostics, Inc) at the Histology Core Facility of the EPFL. RNAscope Multiplex Fluorescent V1 assay (Advanced Cell Diagnostics, Cat. No. 320850) was performed according to manufacturer's protocol on 10 μm fixed frozen cryosections, hybridized with the probes Mm-Alk1-C1 (ACD, Cat. No. 501131), Mm-Ascl1-CDS-C3 (ADC, Cat. No. 476321-C3), Mm-Insm1-C1 (ADC, Cat. No. 430621), 3Plex positive control Mm-Ppib (ACD, Cat. No. 313911), and negative control DapB (ACD, Cat. No. 310043) at 40°C for 2 h and revealed with Atto550 for C1 and Atto647 for C3.
Vivancos Stalin L., Gualandi M., Schulte J.H., Renella R., Shakhova O, & Mühlethaler-Mottet A. (2019). Expression of the Neuroblastoma-Associated ALK-F1174L Activating Mutation During Embryogenesis Impairs the Differentiation of Neural Crest Progenitors in Sympathetic Ganglia. Frontiers in Oncology, 9, 275.
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6

Fluorescence In Situ Hybridization of DRG

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Mice were anesthetized with isoflurane and rapidly decapitated. The ipsilateral L2 and L3 DRG of mice were removed within 7 min, placed into OCT, and flash frozen using 2-methylbutane chilled on dry ice. Tissue was kept on dry ice until cryosectioning. DRG samples were cryosectioned at 14 μm, mounted directly onto Super Frost Plus slides, and fluorescence in situ hybridization (FISH) studies were performed according to the protocol for fresh frozen samples using the RNAscope Multiplex Fluorescent v1 Assay (Advanced Cell Diagnostics, 320851) with minor modifications. Briefly, DRG sections were fixed for 15 min in ice-cold 4% PFA and dehydrated with ethanol. Sections were then treated with Protease IV for 15 min at room temperature. Probes for Eyfp (Advanced Cell Diagnostics, Eyfp, 312131), Mrgprd (Mm-Mrgprd, 417921), Mrgpra3 (Mm-Mrgpra3, 548161), and Sst (Mm-Sst, 404631) were hybridized for 2 hr at 40°C in a humidified oven, and then a series of incubations was performed to amplify and label target probes with the assigned fluorescence detection channel (C1–C3). Sections were counterstained with DAPI using ProLong Gold Antifade Mountant (Invitrogen, P36931).
Warwick C., Cassidy C., Hachisuka J., Wright M.C., Baumbauer K.M., Adelman P.C., Lee K.H., Smith K.M., Sheahan T.D., Ross S.E, & Koerber H.R. (2021). MrgprdCre lineage neurons mediate optogenetic allodynia through an emergent polysynaptic circuit. Pain, 162(7), 2120-2131.
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7

Characterization of Sensory Neuron Subsets via FISH

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Mice were anesthetized with isoflurane and rapidly decapitated. The ipsilateral L2 and L3 DRG of mice were removed within 7 minutes, placed into optimal cutting temperature compound (OCT), and flash frozen using 2-methylbutane chilled on dry ice. Tissue was kept on dry ice until cryosectioning. Dorsal root ganglia samples were cryosectioned at 14 μm and mounted directly onto Superfrost Plus slides, and fluorescence in situ hybridization (FISH) studies were performed according to the protocol for fresh frozen samples using the RNAscope Multiplex Fluorescent v1 Assay (Advanced Cell Diagnostics, Newark, CA; 320851) with minor modifications. In brief, DRG sections were fixed for 15 minutes in ice-cold 4% paraformaldehyde and dehydrated with ethanol. Sections were then treated with protease IV for 15 minutes at room temperature. Probes for enhanced yellow fluorescent protein (eYFP) (Advanced Cell Diagnostics, eYFP, 312131), Mrgprd (Mm-Mrgprd, 417921), Mrgpra3 (Mm-Mrgpra3, 548161), and Sst (Mm-Sst, 404631) were hybridized for 2 hours at 40°C in a humidified oven, and then, a series of incubations was performed to amplify and label target probes with the assigned fluorescence detection channel (C1-C3). Sections were counterstained with DAPI using ProLong Gold Antifade Mountant (Invitrogen, P36931).
Warwick C., Cassidy C., Hachisuka J., Wright M.C., Baumbauer K.M., Adelman P.C., Lee K.H., Smith K.M., Sheahan T.D., Ross S.E, & Koerber H.R. (2021). MrgprdCre lineage neurons mediate optogenetic allodynia through an emergent polysynaptic circuit. Pain, 162(7), 2120-2131.
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