nRCMs were seeded into 1% gelatin coated 96-well black, clear bottom, culture plates (Corning) accompanied with the transfection complex mix. This complex mix contained Lipofectamine 2000 transfection reagent (Invitrogen) in combination with mirVana mimics (Life-Technologies; f.c. 10 nM.), prepared according to manufacturer’s protocol. After 24 h of starvation, cells were stimulated for 72 hours with 5μM phenylephrine (PE)(Sigma #P6126) or treated with PBS as control.
Mirvana mimic
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Ireland
The MirVana mimics are small, chemically synthesized RNA molecules designed to mimic endogenous microRNA (miRNA) molecules. They are used as positive controls or to study the function of specific miRNAs in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Plvx puro, by Takara Bio (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Pmir report vector, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Lna gapmer, by Qiagen (1 mentions)
Mircury lna microrna inhibitor, by Qiagen (1 mentions)
Accell sirna, by Thermo Fisher Scientific (1 mentions)
Mirvana isolation kit, by Thermo Fisher Scientific (1 mentions)
Mircury lna microrna target site blockers, by Qiagen (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Hek293t cells, by Thermo Fisher Scientific (1 mentions)
Invivofectamine 2, by Thermo Fisher Scientific (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Mirvana paris kit, by Thermo Fisher Scientific (1 mentions)
Hrp secondaryantibodies, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse crosslinker, by Merck Group (1 mentions)
Mir nc oligonucleotides, by Thermo Fisher Scientific (1 mentions)
16 protocols using mirvana mimic
1
Stimulating nRCM Hypertrophy with PE
2
Modulating miR-103 and KLF4 in HAECs
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Lipofectamine RNAiMAX (Life Technologies) was used to transfect HAECs with a LNA-miR-103 inhibitor (50 nM, miRCURY LNA microRNA Inhibitors; Exiqon), a miR-103 mimic (15 nM, mirVana mimics; Life Technologies), Dicer GapmeRs (10 nM, LNA GapmeRs; Exiqon), miR-103-KLF4 target site blockers (50 nM miRCURY LNA microRNA Target Site Blockers; Exiqon), premade KLF4 mRNA (2 μg, mRNAExpress Human KLF4 Transcript; BioCat GmbH) or scrambled controls. Total RNA was isolated after 24 or 48 h using the RNeasy Mini Kit (Qiagen) or mirVana Isolation Kit (Life Technologies). HAECs were transfected with a small interfering RNA (siRNA) against KLF4 or a non-targeting siRNA (1 μM Accell siRNA in Accell Delivery Cell Culture Medium; Thermo Scientific) for 72 h. Additional treatment with the LNA-miR-103 inhibitor was performed as described above.
3
Transient miRNA Overexpression in HEK 293T
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HEK 293T cells were purchased and maintained according to the manufacturer’s guidelines (ATCC). Transient expression of miR-16, -103, -107 or a scrambled control was achieved by using miRVana mimics according to the manufacturer’s instruction (Life Technologies). Lipofectamine 2000 was used according to the manufacturer’s protocol for transient transfection of HEK 293T cells (Invitrogen). Endpoints were analyzed 48 h post transfection.
4
Targeted miR-22 Modulation in Mice
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Coordinates for i.c.v. injections were bregma: AP = −0.3 mm, L = −1.0 mm, V = −2.0 mm33 . Antagomirs were from Exiqon (locked nucleic acid (LNA)- and 3′-cholesterol modified oligonucleotides). The miR-22 antagomir sequence was CTTCAACTGGCAGCT and scrambled was ACGTCTATACGCCCA. Mice received 2 μL infusion of 0.5 nmol antagomir/scrambled in artificial cerebrospinal fluid (aCSF) (Harvard Apparatus UK, Kent, UK). To overexpress miR-22 we used chemically-modified double-stranded RNAs (mirVana™ mimics; Life technologies). Nanoparticles of the mimics or non-targeting controls (range 0.3 pmol–1 nmol) were generated using Invivofectamine®2.0 (Life technologies) following manufacturer instructions. Two microliters of the complex was injected i.c.v. into mice. For systemic injections during long-term studies, JNJ-47965567 or minocycline (both 30 mg/kg) or vehicle (DMSO or PBS, respectively) were injected intraperitoneal twice daily (with first injection 1 h after intra-amygdala kainic acid injection).
5
Evaluating miRNA Transfection Efficacy
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Cells were grown until reach a 60-70% confluence. Then, cells were transfected separately with the miRNAs miR-100, miR-21 and miR-139. The miRNA let7c was included as a positive control of transfection. Cells were transfected according to the manufacturer's protocol. Briefly, cells were incubated with a mix of Opti-MEM medium (Lifetechnologies), Lipofectamine RNAi max (Lifetechnologies) and miRNA mimics (mirVana mimics, Life Technologies) to a final concentration of 20 nM. After 48 hours, total RNA and proteins from transfected and control cultures were extracted with the mirVana PARIS kit (Ambion) according to manufacturer's protocol. All transfections were performed in triplicate.
6
Firefly Luciferase Assay for miRNA Targets
7
Overexpressing Cap1 using miRNA mimics
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Primers for qRT-PCR and sgRNA are listed in Supplementary Table S1 . The cDNA clone for Cap1 was purchased from Origene (CAT#: MR207594) and subsequently cloned into lentiviral vector pLVX-puro (Clontech, Mountain View, United States). For overexpression of the candidate miRNAs, mirVana mimics (Thermo Fisher) were used together with RNAi-max (Thermo Fisher) following the manufacturer’s instructions.
8
Evaluating miRNA Effects on Cap1 Expression
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Both the C2C12 and LHCN-M2 cells were transiently transfected with miRNA mimics (mirVana mimics, Thermo Fisher). Briefly, the cells were seeded at sub-confluent density in the six well plate format and were transfected with 50 nmoles of miR-Ctl, miR-1a-3p, miR-133a-3p, miR-206-3p, miR-378-3p, or miR-486-5p mimics using 10 µl of RNAimax following the manufacturer’s instruction. After 72 h, cells were lysed and analyzed for the effect of the miRNA transfection on Cap1 mRNA and protein level by reverse transcription quantitative PCR and immunoblotting respectively.
9
Molecular Immunology Techniques
10
Molecular Techniques for Studying MRTF-A
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Primers for cloning, mutagenesis and qRT-PCR are listed in Supplementary Table S2 . The SRF reporter plasmid p3D.A-Luc was described previously (12 (link),20 (link),21 (link)). Murine MRTF-A 3′UTR region was obtained by PCR from cDNA and cloned into pMIR-REPORT vector (Thermo Fisher, Waltham, MA, USA). Potential miRNA binding sites were deleted using SOMA mutagenesis (22 (link)) For miTRAP analysis, the murine MRTF-A 3′UTR was subcloned into pCDNA3.1-MS2 as described previously (23 (link)). For overexpression of the candidate miRNAs mirVana mimics (Thermo Fisher) were used. MRTF-A and Dicer1 knockdown was performed with ON-TARGETplus pool siRNA (Dharmacon, Lafayette, CO, USA) and RNAiMax (Thermo Fisher) following the manufacturers’ instructions. Hemagglutinin-tagged (HA) murine full length MRTF-A and a constitutively active variant (lacking the G-actin binding region derived from exons 3–5) were cloned from cDNA via PCR into pLVX-Puro (Clontech, Mountain View, USA). For transient transfection Lipofectamine 3000 was used following the manufacturer's instructions. TGF-β treatment (10 ng/ml, 6 h pretreatment) was performed using recombinant TGF-β1 (Promokine, Heidelberg, Germany).
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