A Functional MicroRNA Screening Method for Organ Morphogenesis

Ivan T. Rebustini1

1 Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.19
DOI:  10.1002/cpcb.15
Online Posting Date:  March, 2017
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The increasing repertoire of microRNAs expressed during organ development and their role in regulating organ morphogenesis provide a compelling need to develop methods to assess microRNA function using various in vitro and in vivo experimental models. Methods to assess microRNA function during organ morphogenesis include transfection of microRNA inhibitors (antagomirs) and activators (mimics) into mouse embryonic explanted organs using liposomes, which can potentially result in low efficiency of transfection and off‐target effects. We devised a method to assess microRNA function in explanted organs by transfecting antagomirs and mimics using peptide‐based nanoparticles, increasing functional microRNA targeting efficiency, and decreasing off‐target effects. Our method can be applied to a variety of embryonic organs that can be explanted and provides an alternative to efficiently and functionally prioritize microRNAs during organ morphogenesis for further in vivo genetic approaches. © 2017 by John Wiley & Sons, Inc.

Keywords: microRNA; functional screening; organ morphogenesis

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Table of Contents

  • Significance Statement
  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Transfection of Antagomirs and Mimics in Explanted Organs Using Peptide‐Based Nanoparticles
  • Support Protocol 1: Embryonic Organs Explanted in Trowell‐Type or Floating Filters
  • Basic Protocol 2: microRNA Pulldown (MIR‐PD)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Transfection of Antagomirs and Mimics in Explanted Organs Using Peptide‐Based Nanoparticles

  • Organ explants (see protocol 2Support Protocol)
  • N‐TER peptide (Nanoparticle‐Forming Solution; Sigma‐Aldrich, cat. no. N2913‐1ML)
  • Antagomirs (QIAGEN)
  • Biotinylated mimics (Dharmacon)
  • Culture medium (see recipe)
  • Off‐target miR‐Cy3 microRNA (QIAGEN)
  • Fetal bovine serum (FBS; Stasis Stem Cells, cat. no. 100‐125 or equivalent)
  • mirVana total RNA isolation kit, with phenol (AMBION, cat. no. AM1560)
  • iScript cDNA synthesis kit (Bio‐Rad, cat. no. 720001180)
  • iQ SYBR Green Supermix (Bio‐Rad, cat. no. 170‐8880)
  • miScript II RT kit (QIAGEN, cat. no. 218160)
  • miScript SYBR Green PCR kit (QIAGEN, cat. no. 218073)
  • qPCR primers:
  • miScript primer assays (for mature miRNA expression; QIAGEN)
  • Invitrogen brand primers (for miRNA targets and other genes)
  • 0.7‐ml sterile plastic tubes (Eppendorf or equivalent)
  • Rainin Pipet Plus (Rainin, P1000, P200, and P10)
  • Sterile filter pipet tips (1000, 200, and 10 μl)
  • Table vortex mixer
  • CO 2 incubator (Thermo Fisher Scientific NAPCO Series 8000 WJ)
  • Bench top centrifuge Eppendorf 5148 (Eppendorf)
  • 12‐well multiwell tissue culture plates (Falcon, cat. no. 353043)
  • Metal mesh (corrosion‐resistant stainless steel, 0.7 mm mesh size)
  • Whatman Nuclepore track‐etched polycarbonate membrane filter, diameter: 1.3 cm, pore size: 0.1 µm (Capitol Scientific, cat. no. 110405)

Support Protocol 1: Embryonic Organs Explanted in Trowell‐Type or Floating Filters

  Additional Materials (also see protocol 1)
  • Pregnant mice
  • DMEM‐F12 (Thermo Fisher Scientific, Gibco brand, cat. no. 11330‐032)
  • Penicillin and streptomycin (PS) antibiotics (Thermo Fisher Scientific, Gibco brand, cat. no. 15140‐122)
  • 70% ethanol (spray bottle, 70:30, v/v, ethanol/distilled water)
  • Ethanol, molecular biology grade
  • PBS (Thermo Fisher Scientific, cat. no. SH30378.02)
  • Parafilm (Sigma‐Aldrich, cat. no. P7793)
  • Laboratory rocker
  • Plastic metric pipets (5.0, 10.0, and 25.0 ml)
  • Petri dishes (100 and 150 mm; VWR, cat. no. 25384‐342 and 25384‐139)
  • Sterilized 60‐mm Corning glass dish
  • Surgical Instruments:
  • mirVana No. 5 CO forceps (Dumont, cat. no. 11295)
  • Dumont no. 7 tweezers, 12 cm, Inox (Dumont, cat. no. 14097)
  • Iris 10 cm, CVD serrated forceps (Dumont, cat. no. 15915)
  • Scalpel handle no. 4, 14 cm (Dumont, cat. no. 500237)
  • Scalpel blade no. 11 (Sterling, cat. no. 500240)
  • Scissors for tenotomy, 10 cm (Dumont, cat. no. 14396)
  • Perforated round spoon, 15 mm (Dumont, cat. no. 10370)
  • Stereomicroscope Leica M80 and camera Leica IC80 HD
  • Stereomicroscope Zeiss Discovery.V8

Basic Protocol 2: microRNA Pulldown (MIR‐PD)

  • Organ explants (see protocol 2Support Protocol)
  • Dynabeads MyOne streptavidin T1 (Thermo Fisher Scientific, Invitrogen brand, cat. no. 65601)
  • Complete miR‐PD lysis buffer (see recipe)
  • miR‐PD blocking solution (see recipe)
  • Nuclease‐free water
  • Biotinylated microRNA mimics (Dharmacon)
  • mirVana total RNA isolation kit, with phenol (AMBION, cat. no. AM1560)
  • Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific)
  • 0.7‐ml sterile plastic tubes (Eppendorf or equivalent)
  • 12‐well multiwell tissue culture plates (Falcon, cat. no. 353043)
  • Bench centrifuge Eppendorf (for PCR strip tubes)
  • Thermal cycler Bio‐Rad CFX96 C100
  • MagnaRack (for microcentrifuge tubes; Thermo Fisher Scientific, Invitrogen brand, cat. no. CS15000)
  • Rainin Pipet Plus (Rainin, P1000 and P200)
  • Table vortex mixer
  • Fine dressing forceps, 12.5 cm (Dumont, cat. no. 503283)
  • Homogenizer
  • 96‐well PCR Plate, non‐skirted, 25 plates, low profile (Thermo Fisher Scientific, cat. no. AB0600)
  • Combitips advanced, 0.5 ml, standard
  • Microseal sealing tape (for 96‐well PCR plate; Bio‐Rad, cat. no. MSB1001)
  • Parafilm (Sigma‐Aldrich, cat. no. P7793)
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