Mircury lnatm microrna ish optimization kit

Manufactured by Qiagen

The MiRCURY LNA microRNA ISH Optimization Kit is a laboratory equipment product designed for the in situ hybridization (ISH) of microRNA (miRNA) molecules. The kit provides reagents and protocols to facilitate the optimization and detection of specific miRNA targets within tissue samples.

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Lab products found in correlation

Hybridization buffer, by Qiagen (3 mentions) Las v 4, by Leica (3 mentions) Thermo block, by Labnet (2 mentions) Lsm 710, by Zeiss (2 mentions) Nf κb p65, by GeneTex (1 mentions) Dylight633 conjugate, by AmyJet Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Dm6000b, by Leica (1 mentions) Ab8878, by Abcam (1 mentions) Ab13248, by Abcam (1 mentions) Ab83364, by Abcam (1 mentions)

Close spelling variants of this product

MiRCURY LNA microRNA ISH Optimization Kits (FFPE) ISH optimization kit LNA miRCuRY

5 protocols using mircury lnatm microrna ish optimization kit

Immunofluorescence and FISH for Circular RNA

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Cultured cells were prepared as described previously (18 (link)). The VSMCs were washed in PBS and fixed in 4% paraformaldehyde for 10 min and permeabilized overnight in 70% ethanol. Then the cells were rehydrated for 10 min in 50% formamide and 2 × SSC. For immunofluorescence, cells were blocked with 10% BSA in PBS for 2 h followed by incubation with NF-κB p65 (1:200, GeneTex) in PBS treated with DEPC at 4°C overnight. After washing three times in PBS, cells were incubated with secondary antibody (DyLight633 Conjugate, AmyJet Scientific).
For FISH, the cells was incubated using specific probes of circ-Sirt1 and miR-132/212 according to user manual of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon). Hybridization was performed using fluorescence-labeled probes in hybridization buffer by incubation at 55°C for 1 h. After stringent washing with SSC buffer, cell nuclei were counterstained with DAPI (Invitrogen). Images were acquired using a Leica microscope (Leica SP5, Switzerland) and digitized with a software program LAS AF Lite.

Kong P., Yu Y., Wang L., Dou Y.Q., Zhang X.H., Cui Y., Wang H.Y., Yong Y.T., Liu Y.B., Hu H.J., Cui W., Sun S.G., Li B.H., Zhang F, & Han M. (2019). circ-Sirt1 controls NF-κB activation via sequence-specific interaction and enhancement of SIRT1 expression by binding to miR-132/212 in vascular smooth muscle cells. Nucleic Acids Research, 47(7), 3580-3593.

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KLF16 mRNA Expression Analysis via In Situ Hybridization

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Cell slides were fixed in 4% paraformaldehyde and performed in situ hybridization according to the user manual of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon) as previous description [33 (link)]. Hybridization was performed using fluorescence-labeled KLF16 mRNA probes in hybridization buffer (Exiqon). After stringent washing with SSC buffer, slides were blocked with 10% normal goat serum. The sections were then incubated with anti-SF3B4 primary antibody (Ptoteintech, 10482-1-AP) for 1 h. After washing, the slides were incubated with a rhodamine-labeled secondary antibody (KPL, USA, 031506). Images were acquired using a Leica microscope (Leica DM6000B, Switzerland) and digitized with software of LAS V.4.4 (Leica).

Yang Z., Wang Y.X., Wen J.K., Gao H.T., Han Z.W., Qi J.C., Gu J.F., Zhao C.M., Zhang H., Shi B., Wang D.D., Wang X.L, & Qu C.B. (2023). SF3B4 promotes Twist1 expression and clear cell renal cell carcinoma progression by facilitating the export of KLF 16 mRNA from the nucleus to the cytoplasm. Cell Death & Disease, 14(1), 26.

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Visualization of miR-145 in Metastatic Lung

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miRNA in situ hybridization was performed using the miRCURY LNA^TM microRNA ISH Optimization kit contained double-DIG-labeled LNA^TM detection probe for miR-145-5p and scramble-miR negative control (EXIQON). Formalin-fixed tissue sections of metastatic lungs from tumor-bearing mice were incubated with 15 μg/ml Proteinase-K for 10 min at 37 °C. The miR-145 (20 nM) or control probe (40 nM) were hybridized at 60 °C for 16 h. After washing in SSC and blocking by 5% normal donkey serum for 1 h, hybridized sections were incubated overnight at 4 °C with primary antibodies and anti-DIG conjugated with Alexa 488 (IC7520G, Novus 1:100). anti-E-cadherin (24E10, CST 1:100) and anti-CD11b (ab8878, Abcam 1:100) were used to detect tumor cells and myeloid cells. The distribution of miR-145 was examined by confocal laser scanning microscope (LSM710, Zeiss).

Ishii H., Vodnala S.K., Achyut B.R., So J.Y., Hollander M.C., Greten T.F., Lal A, & Yang L. (2018). miR-130a and miR-145 reprogram Gr-1+CD11b+ myeloid cells and inhibit tumor metastasis through improved host immunity. Nature Communications, 9, 2611.

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In Situ Hybridization of miR-193a-5p in Prostate Cancer

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In situ hybridization was performed as described previously [28 ]. In brief, according to user manual of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon), paraffin cross-sections (5-μm thick) from clinical PC tissues were deparaffinized and rehydrated for fluorescence in situ hybridization. Hybridization was performed using fluorescence-labeled miR-193a-5p probes with hybridization buffer (Exiqon) by incubation at 56 °C for 1 h in a thermo-block (Labnet, USA). After stringent washing with SSC buffer, nonspecific binding sites were blocked with 10% normal goat serum (710,027, KPL, USA). According to need, the sections were then incubated for 1 h at 37 °C with anti-HO-1 primary antibody (ab13248, Abcam) or anti-Bach2 (ab83364, Abcam) diluted 1:50 in PBS or incubated with secondary antibody directly. After washing with PBS, the sections were incubated with a rhodamine-labeled secondary antibody (031506, KPL, USA) at 37 °C for 30 min. Images were acquired by using a Leica microscope (Leica DM6000B, Switzerland) and digitized with a software of LAS V.4.4 (Leica).

Yang Z., Chen J.S., Wen J.K., Gao H.T., Zheng B., Qu C.B., Liu K.L., Zhang M.L., Gu J.F., Li J.D., Zhang Y.P., Li W., Wang X.L, & Zhang Y. (2017). Silencing of miR-193a-5p increases the chemosensitivity of prostate cancer cells to docetaxel. Journal of Experimental & Clinical Cancer Research : CR, 36, 178.

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miR-140-5p Expression by FISH

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In situ hybridization was performed as described previously [18 (link)]. In brief, according to user manual of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon), cultured cells smears were deparaffinized and rehydrated for fluorescence in situ hybridization (FISH). Hybridization was performed using fluorescence-labeled miR-140-5p probes with hybridization buffer (Exiqon) by incubation at 56°C for 1 h in a thermo-block (Labnet, U.S.A.). After stringent washing with SSC buffer and PBS, images were acquired by using a Leica microscope (Leica DM6000B, Switzerland) and digitized with a software of LAS V.4.4 (Leica). miR-140-5p probe was purchased from GenePharma Co., Ltd (Shanghai, China). The sequence of miR-140-5p-cy3-probe was as follows: TACCATAGGGTAAAACCACTG. The in situ hybridization image analysis was done according to the protocol of FISH image analysis in clinical diagnosis. Briefly, 200 cell nuclei were (stained with DAPI) counted under fluorescence microscopy. The FISH-positive cells were stained with red probes and emitted red fluorescence. Positive-cell numbers (per 200) cells were used to statistical analysis. Each experiment was repeated three times.

Nie Z.Y., Liu X.J., Zhan Y., Liu M.H., Zhang X.Y., Li Z.Y., Lu Y.Q., Luo J.M, & Yang L. (2019). miR-140-5p induces cell apoptosis and decreases Warburg effect in chronic myeloid leukemia by targeting SIX1. Bioscience Reports, 39(4), BSR20190150.

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