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Pre mir s

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Pre-miR™s are synthetic RNA oligonucleotides that mimic endogenous microRNA (miRNA) precursors. They are designed to be processed into mature miRNAs by the cellular machinery.

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Lab products found in correlation

Opti mem reduced serum medium, by Thermo Fisher Scientific (2 mentions) Silencer, by Thermo Fisher Scientific (1 mentions) Anti mir, by Thermo Fisher Scientific (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Sirna, by Horizon Discovery (1 mentions) Hiperfect transfection reagent, by Qiagen (1 mentions) Siimporter, by Merck Group (1 mentions) Anti mir, by Qiagen (1 mentions) Rnaimax, by Thermo Fisher Scientific (1 mentions) Fugene hd, by Promega (1 mentions) Amaxa 96 well shuttle system, by Lonza (1 mentions) Facscalibur system, by BD (1 mentions)

6 protocols using pre mir s

1

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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2

Transfection and Overexpression Assay

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Cells were plated and allowed to adhere and grow overnight at 37°C with 5% CO2. The following day, cells were transfected with 50 nM of pre‐miRs (Ambion) or 30 nM of siRNAs (Dharmacon, Appendix Table S1) using the HiPerFect transfection reagent (Qiagen) according to the manufacturer's instructions. Twenty‐four hours later, cells were transfected with control (pCDNA 3.0) or HA‐MARK4 plasmid (kind gift from Dr. Drewes 6) using Attractene and according to the manufacturer instructions.
Pardo O.E., Castellano L., Munro C.E., Hu Y., Mauri F., Krell J., Lara R., Pinho F.G., Choudhury T., Frampton A.E., Pellegrino L., Pshezhetskiy D., Wang Y., Waxman J., Seckl M.J, & Stebbing J. (2016). miR‐515‐5p controls cancer cell migration through MARK4 regulation. EMBO Reports, 17(4), 570-584.
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3

In Vitro miRNA Inhibition Protocol

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PremiRs (Ambion, Applied Biosystems) or antagomiR oligonucleotides (Eurogentec) were transfected (100 μM) using siIMPORTER (Millipore) according to the manufacturer’s procedures. Negative control premiR (Ambion) or scrambled antagomiR were transfected as controls. Chemically modified antisense oligonucleotides (antagomiR) have been used to inhibit miR expression in vitro. The sequences of antagomiR-145 used is as follows: 5′-AGGGATTCCTGGGAAAACTGGAC-3′. The scrambled antimiR sequence was 5′-CAGCTGAAGTAAATACCGACCAG-3′.
Zhao J., Zhou K., Ma L, & Zhang H. (2020). MicroRNA-145 overexpression inhibits neuroblastoma tumorigenesis in vitro and in vivo. Bioengineered, 11(1), 219-228.
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4

Transfection of pre-miRs, anti-miRs, and siRNAs

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Pre-miRs (25 nM; Ambion) and anti-miRs (25 nM; Exiqon) were transfected into HUVECs and MDA-MB-231 cells using Dharmafect-4 (Dharmacon Research Inc.) according to the manufacturer's instructions. CCND2, CCND3 and control siRNAs (20 nM) were transfected based on the calcium phosphate transfection method. Transfected HUVECs and MDA-MB-231 cells were plated in EGM2 or complete DMEM, respectively. After a 24-hour transfection, the cells were washed and kept in their media for an additional 48 or 72 hours. Functional assays were performed as described above.
Bovy N., Blomme B., Frères P., Dederen S., Nivelles O., Lion M., Carnet O., Martial J.A., Noël A., Thiry M., Jérusalem G., Josse C., Bours V., Tabruyn S.P, & Struman I. (2015). Endothelial exosomes contribute to the antitumor response during breast cancer neoadjuvant chemotherapy via microRNA transfer. Oncotarget, 6(12), 10253-10266.
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5

Modulating miR-29 Expression in Leiomyoma and Myometrial Cells

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To modify the miR-29 expression levels in leiomyoma and myometrial cells, we used the Life Technologies PremiRs to overexpress the target miRNAs in leiomyoma cells, and AntimiRs to down-regulate the targeted miRNAs in myometrial cells (Life Technologies, Austin, TX). Pre-miR and Anti-miR transfection controls were utilized for the respective experiments. For the Pre-miR transfections of leiomyoma cells, the passage one primary cells were plated in 6cm dishes and grown to 50% confluency in standard media (DMEM/F12 with 10% FBS and 1% antimycotic/antibiotic solution). The cells were then incubated in OptiMEM Reduced Serum Medium (Life Technologies) and transfected with Pre-miRs using FuGENE HD (Promega, Madison, WI) as the transfection agent, or Anti-miRs using RNAiMAX (Life Technologies) as the transfection agent.
MARSH E.E., STEINBERG M.L., PARKER J.B., WU J., CHAKRAVARTI D, & BULUN S.E. (2016). Decreased expression of MicroRNA-29 family in leiomyoma contributes to increased major fibrillar collagen production. Fertility and sterility, 106(3), 766-772.
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6

Nucleofection of miR-9 in L2.3 Cells

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PremiRs (Life Technologies) for miR-9 were nucleofected into L2.3 cells using Rat Neuron 96-well Nucleofector Kit (VHPG-1003) in conjunction with an amaxa 96-well shuttle system (Lonza, Cologne, Germany). Observed transfection efficiencies using the 96-well shuttle system were consistently >95% for both the L2.2 and L2.3 NSCs. At four hours after transfection, L2.3 cells were differentiated by removing FGF2 from the growth medium and allowed to differentiate for 72 hours. At 3 days, cells were fixed with 2% paraformaldehyde and analyzed for anti-βIII-tubulin (TuJ1, 1∶500, Covance) expression by flow cytometry on the FACSCalibur System (BD Biosciences, San Jose CA). Data was analyzed using BD's CellQuest Pro v. software.
Davila J.L., Goff L.A., Ricupero C.L., Camarillo C., Oni E.N., Swerdel M.R., Toro-Ramos A.J., Li J, & Hart R.P. (2014). A Positive Feedback Mechanism That Regulates Expression of miR-9 during Neurogenesis. PLoS ONE, 9(4), e94348.
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