RNA FISH in embryos was performed using fluorescence-labeled probes or digoxigenin-labeled probes detected by an anti-dig antibody and a fluorescence-labeled secondary antibody 58 (link). In our current study, probes were prepared from cDNA plasmids or genomic PCR products; for direct fluorescence labeling, the FISH Tag™ RNA kit (Alexa Fluor 488, Life Technology) was used. Embryos used in bcd and nos mRNA FISH were from 0–1-hr collections and those used in hb and eve mRNA FISH from 0–3-hr and 0–4-hr collections, respectively. Imaging was performed under the Zeiss Imager Z1 ApoTome microscope with a Zeiss Plan 10× Aprochromat objective. Imaging acquisition was performed under linear settings and data analysis (MATLAB, Math Works) was based on fluorescent intensities extracted from the cytoplasmic layer of midsagittal images as a function of AP position (for hb and eve) 58 (link), or as epifluorescence intensities (for bcd and nos) 23 (link).
Fish tag rna kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FISH Tag RNA Kit is a tool designed for labeling and detecting RNA molecules within cells. It provides a method for incorporating fluorescent tags into RNA samples, enabling visualization and analysis of RNA localization and expression patterns using fluorescence in situ hybridization (FISH) techniques.
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5 protocols using fish tag rna kit
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RNA FISH Imaging in Drosophila Embryos
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RNA FISH Imaging in Drosophila Embryos
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Visualizing histone H4 mRNA distribution
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We amplified a 351 bp-long genomic DNA of the gene while using a set of a PCR primer, 5′-TACTCGAGCTTTCGTGCTGTGCGTG-3′ and 5′-CGGAATTCTAACCGCCAAATCCGTA-3′, for RNA in situ hybridization to examine a distribution of histoneH4 mRNA in larval LGs. The DNA fragment was inserted into pOT2 plasmid to produce a RNA probe for the FISH using FISH Tag RNA Kit (Catalogue #F32954 Invitrogen, Carlsbad, CA, USA). The LGs were collected from third instar larvae at mature stage and fixed with 4% paraformaldehyde. Subsequently, they were treated with 80% acetone for 10 min. at −30 °C, rehydrated in PBS-10% Triton X-100 (PBST), and then fixed again with 4% paraformaldehyde. RNA hybridization was carried out with fluorescence-labelled RNA probe described above in a hybridization buffer provided at 56 °C, for 16 h following the manufacturer’s instructions. After repeated rinse steps, the LG samples were observed while using a confocal microscope (Fv-10i, Olympus, Tokyo, Japan).
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Subcellular Localization of circLMO1 by FISH
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The subcellular localization of circLMO1 was evaluated using FISH assay. Specific RNA probes against cricMLO1 and miR-4291 was synthesized using FISH Tag™ RNA Kit (F32952; Invitrogen). CaSki and C33A cells (about 1.5×104/well) were mounted on a coverslip and fixed with 4% PFA (Beyotime) at room temperature for 15 min. The cells were digested with protein K at 37°C for 1 h in the presence of glycine and acetic anhydride. Then, cells were treated with pre-hybridization solution for 90 min and treated with the probe (300 μL, 250 ng/mL) against cricMLO1 at 42°C overnight. Finally, cells were stained with DAPI for 5 min at room temperature before sealing. A fluorescence microscope (Keyence, Osaka, Japan) was used to capture the signals of cricMLO1, miR-4291 and the nucleus.
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Fluorescent Probe Labeling and Imaging of linc-MYH
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Fluorescent-labeled antisense linc-MYH probes were synthesized according to manufacturer's instruction (FISH Tag RNA kit, Invitrogen). FISH experiments were performed on isolated EDL myofibres and images acquired on a Leica SP2 confocal microscope.
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