Anti mirs

Manufactured by Thermo Fisher Scientific

Sourced in Belgium, United States

Anti-miRs are synthetic oligonucleotides designed to bind and inhibit the function of specific microRNA (miRNA) molecules. They are used as research tools to study the biological roles of miRNAs in various cellular processes.

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Close spelling variants of this product

(anti-miR-9) Anti-miR-23a Anti-miR-146a Anti-miR-681 oligonucleotide Anti-miR-21-5p Anti-miR-218 Anti-miR-199a-5p Anti-miR-con Cy3-PreCy3 dye-labeled anti-miR-negative inhibitor control Anti-miR-372

5 protocols using anti mirs

Investigating miRNA Regulation in NIH3T3 Cells

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Mouse NIH3T3 cells in DMEM, 10% FBS, 1% pen/strep were transfected with miR-206, miR-1 or miR-133 (50 nM; Sigma) with and without antimiRs (100 nM; Ambion) using Lipofectamine 2000. Mock-transfected cells served as controls. Protein was extracted after 48 h and 20 μg was run on 8-12% polyacrylamide gels and blotted onto PVDF membrane. Primary antibodies (Abcam) to BAF60a (1:500; ab83208), BAF60b (1:1000; ab81622), BAF60c (1:500; ab50556), Brg1 (1:500; ab110641) and actin (1:1000; ab3280) were applied at 4°C overnight; secondary antibodies (Dako, P0447; and Jackson ImmunoResearch, 111-035-003) were applied for 1 h at room temperature.
RNA was isolated with TRIzol (Invitrogen). cDNA synthesis used SuperScript II reverse transcriptase (Invitrogen) with 1 μg RNA (cells) or 400 ng RNA (somites). For miRNA qPCR, the MystiCq miRNA cDNA Synthesis Kit (Sigma) was used. Primers (supplementary material Table S1), miRNA primers (designed by Sigma) and SYBR Green MasterMix (Applied Biosystems, Sigma) were used with the Applied Biosystems 7500 Fast Real-Time PCR System following the manufacturer's protocols. All qPCR was normalized to beta-actin (mRNA) or RNU-6 (miRNA).

Goljanek-Whysall K., Mok G.F., Fahad Alrefaei A., Kennerley N., Wheeler G.N, & Münsterberg A. (2014). myomiR-dependent switching of BAF60 variant incorporation into Brg1 chromatin remodeling complexes during embryo myogenesis. Development (Cambridge, England), 141(17), 3378-3387.

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Functional Assays with Transfected Cells

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For functional assays performed with cells transfected with pre-miRs and anti-miRs (Figure 1b and Figure 2), pre-miRs (25 nM; Ambion,Thermo Fisher Scientific, Merelbeke, Belgium) and anti-miRs (25 nM; Ambion) were transfected into ECs and SMCs using Dharmafect-4 (Dharmacon) according to manufacturer’s instructions. Transfected ECs and SMCs were plated in EGM-2 or SMCM, respectively. After a 24-h transfection, cells were washed and kept in their media for an additional period, depending on the assay. For functional assays using EVs enriched in miR-539 and miR-582, ECs and SMCs were transfected with pre-miR-539 and pre-miR-582, respectively, as described above. After a 24 h transfection, cells were washed and kept for 72 h; EVs were isolated as described below and are referred to here as miR-539 EVs and miR-582 EVs.

Fontaine M., Herkenne S., Ek O., Paquot A., Boeckx A., Paques C., Nivelles O., Thiry M, & Struman I. (2021). Extracellular Vesicles Mediate Communication between Endothelial and Vascular Smooth Muscle Cells. International Journal of Molecular Sciences, 23(1), 331.

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Transfection of Breast Cancer Cell Lines

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MCF-7, LCC9, and LY2 cells were transiently transfected for 48 h with miR-29b-1/a inhibitor (Anti-miR™s, Ambion, Austin, TX, USA), siERα (Silencer^®, Ambion), pre-miR-29b-1/a precursor (Pre-miR™s, Ambion), using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) and Opti-MEM® Reduced Serum Medium (Invitrogen, Carlsbad, CA). Negative controls were Anti-miR ™ negative control #1 (Ambion), Pre-miR™ negative control #1 (Ambion), or Negative Control #1 (Silencer^®, Ambion). Treatments were performed following transfections.

Muluhngwi P., Krishna A., Vittitow S.L., Napier J.T., Richardson K.M., Ellis M., Mott J.L, & Klinge C.M. (2016). Tamoxifen differentially regulates miR-29b-1 and miR-29a expression depending on endocrine-sensitivity in breast cancer cells. Cancer letters, 388, 230-238.

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Investigating miR-99a/100 Regulation of FOXA1

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To examine functional implications of miR expression, we manipulated expression using specific anti-miRs and a scrambled RNA sequence controls (Ambion, TX). All experiments were performed in triplicate using non-immortalized NHU cells at 70% confluence, as detailed [44 ]. Briefly, each well of a six-well tissue culture plate was transfected with 100 pmol anti-miR using 5 μl siPORT (Life Technologies) in 200 μl Optimem (Life Technologies). Knockdown of relevant miR was confirmed by miR-specific taqman PCR (Applied Biosystems) after 48 hours.
To investigate direct targeting of the FOXA1 3'UTR we synthesized a Luciferase reporter construct (methods detailed [44 ]). We cloned 800 bases around the miR-99a/100 seed sequence in the FOXA1 3' UTR (chr14: 37,129,203 - 37,129,210) into EJ cells and ligated into pMIR REPORT (Invitrogen, UK). Dual luciferase assays were conducted in a 6 well plate format at 70% confluence. Forty-eight hours post transfection, firefly and renilla luciferase were quantified sequentially using the Dual Luciferase Assay kit (Promega, UK) and luminescence was measured using the manufacturers recommended luminometer (Promega Glomax). Firefly luciferase expression was quantified and normalized to Renilla luciferase expression.

Drayton R.M., Peter S., Myers K., Miah S., Dudziec E., Bryant H.E, & Catto J.W. (2014). MicroRNA-99a and 100 mediated upregulation of FOXA1 in bladder cancer. Oncotarget, 5(15), 6375-6386.

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Validated miRNA Transfection Protocol

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Validated microRNAs were purchased as pre-miRNAs from Ambion (Grand Island, New York), and used with the recommended Ambion pre-miRNA control. Anti-miRs and a scrambled Anti-miR control for validated oncogenic miRNAs were also purchased from Ambion. miRNA transfections were carried out using Oligofectamine (Invitrogen, Grand Island, New York) reagent as previously described [36] (link).

Floyd D.H., Zhang Y., Dey B.K., Kefas B., Breit H., Marks K., Dutta A., Herold-Mende C., Synowitz M., Glass R., Abounader R, & Purow B.W. (2014). Novel Anti-Apoptotic MicroRNAs 582-5p and 363 Promote Human Glioblastoma Stem Cell Survival via Direct Inhibition of Caspase 3, Caspase 9, and Bim. PLoS ONE, 9(5), e96239.

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