RNA Imaging with Dimeric Broccoli in Live Bacterial and Mammalian Cells

Grigory S. Filonov1, Samie R. Jaffrey1

1 Department of Pharmacology, Weill Medical College, Cornell University, New York, New York
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch150174
Online Posting Date:  March, 2016
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Abstract

RNA spatial dynamics play a crucial role in cell physiology, and thus the ability to monitor RNA localization in live cells can provide insight into important biological problems. This unit focuses on imaging RNAs using an RNA mimic of GFP. This approach relies on an RNA aptamer called dimeric Broccoli, which binds to and switches on the fluorescence of DFHBI, a small molecule mimicking the fluorophore in GFP. Dimeric Broccoli is tagged to heterologously expressed RNAs and, upon DFHBI binding, the fluorescent signal of dimeric Broccoli reports the transcript's localization in cells. This protocol describes the process of validating the fluorescence of dimeric Broccoli−labeled transcripts in vitro and in cells, flow cytometry analysis to determine overall fluorescence levels in cells, and fluorescence imaging in bacterial and mammalian cells. Overall, the protocol should be useful for researchers seeking to image high‐abundance RNAs, such as those transcribed off the T7 promoter in bacteria or off Pol III−dependent promoters in mammalian cells. © 2016 by John Wiley & Sons, Inc.

Keywords: RNA imaging; RNA aptamer; aptamer expression; fluorescence microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In‐Gel Staining of Broccoli‐Tagged RNA in Bacteria to Assess Expression Level and Integrity
  • Alternate Protocol 1: In‐Gel Staining of Broccoli‐Tagged RNA in Mammalian Cells
  • Basic Protocol 2: Flow Cytometry Analysis of Transformed Bacterial Cells
  • Alternate Protocol 2: Flow Cytometry Analysis of Transformed Mammalian Cells
  • Basic Protocol 3: dBroccoli Fluorescence Imaging in Live Bacterial Cells
  • Alternate Protocol 3: dBroccoli Imaging in Live Mammalian Cells
  • Support Protocol 1: In Vitro Transcription and Fluorescence Measurement of RNA‐Broccoli Fusions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In‐Gel Staining of Broccoli‐Tagged RNA in Bacteria to Assess Expression Level and Integrity

  Materials
  • Chemically competent Escherichia coli BL21 Star (DE3) cells (Invitrogen, cat. no. C6010‐03)
  • pET28c‐F30‐2×dBroccoli plasmid (Addgene)
  • pET28c (Novagen)
  • SOC medium (Life Technologies, cat. no. 15544)
  • LB medium and LB agar plates with 50 μg/μl kanamycin (see recipe)
  • PBS (see recipe)
  • 1 M IPTG solution (see recipe)
  • TRIzol LS (Life Technologies, cat. no. 10296028)
  • Chloroform (Fisher Scientific, cat. no. C607‐4)
  • Glycogen, 20 mg/ml (Thermo Scientific, cat. no. R0551)
  • Isopropanol (Thermo Scientific, cat. no. BP26181)
  • 70% (v/v) ethanol (Koptec, cat. no. V1016) in DNase‐free, RNase‐free water
  • TE buffer (see recipe)
  • Precast Novex 8% or 10% TBE‐Urea Gel (Life Technologies, cat. no. EC6875BOX)
  • 1× TBE buffer (see recipe)
  • DNase‐free, RNase‐free water (Gibco, cat. no. 10977)
  • RiboRuler Low Range RNA Ladder (Life Technologies, cat. no. SM1831)
  • Novex TBE‐Urea 2× Sample Buffer (Life Technologies, cat. no. LC6876)
  • DFHBI‐1T staining buffer (see recipe)
  • RNA fluorophore, e.g., SYBR Gold (Life Technologies, S‐11494)
  • 42° and 75°C water baths or heat blocks
  • 37°C incubator
  • Culture tubes (VWR International, cat. no. 60818‐667)
  • NanoDrop (Thermo Scientific) or equivalent
  • 25‐ml Erlenmeyer flasks with baffles (Corning, cat. no. 355115)
  • 1.5‐ml microcentrifuge tubes (USA Scientific, cat. no. 1615‐5500)
  • XCell SureLock Mini‐Cell Electrophoresis System (Life Technologies, cat. no. EI0001)
  • Power supply (Bio‐Rad, cat. no. 1645050)
  • ChemiDoc MP imager (Bio‐Rad)

Alternate Protocol 1: In‐Gel Staining of Broccoli‐Tagged RNA in Mammalian Cells

  Additional Materials (also see protocol 1)
  • HEK293T/17 cells (ATCC, cat. no. CRL‐11268)
  • TrypLE Express Enzyme (Life Technologies, cat. no. 12604021)
  • Mammalian cell culture medium (see recipe)
  • Plasmids:
  • pAVU6+27 (available from the corresponding author of Paul et al., )
  • pAV5S (available from the corresponding author of Paul et al., )
  • pAVU6+27‐F30‐2×dBroccoli (Addgene; Filonov et al., )
  • pAV5S‐F30‐2×dBroccoli (Addgene)
  • FuGENE HD (Promega, cat. no. E2311)
  • Hemacytometer
  • 15‐ml conical tubes (Corning, cat. no. 430791)
  • 12‐well cell culture plate (Costar, cat. no. 3513)
  • Cell culture incubator

Basic Protocol 2: Flow Cytometry Analysis of Transformed Bacterial Cells

  Materials
  • PBS (see recipe)
  • 40 mM DFHBI‐1T solution (see recipe)
  • Round‐bottom tubes with cell strainer cap (VWR, cat. no. 21008‐948)
  • FACSAria II (BD biosciences) or equivalent, with filter (488 nm excitation, 530/30 or 525/50 emission)
  • FlowJo software (TreeStar) or equivalent

Alternate Protocol 2: Flow Cytometry Analysis of Transformed Mammalian Cells

  Materials
  • TrypLE Express Enzyme (Life Technologies, cat. no. 12604021)
  • PBS (see recipe)
  • PBS with 4% FBS (see recipe)
  • 40 mM DFHBI‐1T solution (see recipe)
  • Round‐bottom tubes with cell strainer cap (VWR, cat. no. 21008‐948)
  • FACSAria II (BD biosciences) or equivalent, with filter (488 nm excitation, 530/30 or 525/50 emission)
  • FlowJo software (TreeStar) or equivalent

Basic Protocol 3: dBroccoli Fluorescence Imaging in Live Bacterial Cells

  Materials
  • PBS (see recipe)
  • 40 mM DFHBI‐1T solution (see recipe)
  • Microcentrifuge tubes
  • Poly‐D‐lysine‐coated 3.5‐cm glass‐bottom plates (Mattek, cat. no. P35GC‐1.5‐14‐C)
  • Imaging apparatus, including:
  • Inverted wide‐field fluorescence microscope (e.g., Nikon TE2000)
  • Temperature‐controlled environmental chamber (35°‐37°C, 5% CO 2)
  • Motorized stage (e.g., Ludl BioPrecision Motorized Flat Top Stage)
  • 60× high‐NA objective (e.g., Nikon Plan Apo, 1.4 NA, oil immersion)
  • CCD camera (e.g., Photometrics CoolSnap HQ2)
  • Filter cube for green fluorescence, such as FITC or GFP filter sets (e.g., sputter‐coated excitation filter 470 ± 20 nm, 495 nm [long pass] dichroic mirror, and emission filter 525 ± 25 nm; Chroma Technology)
  • NIS Elements (Nikon) or similar image acquisition software
  • Fiji (http://fiji.sc/Fiji), ImageJ, or similar image analysis software
NOTE: If an environmental chamber is not available, a heated stage is also an option, especially for bacteria, which do not require a controlled CO 2 level. Conceivably, imaging can be done at room temperature if temperature is not a critical parameter for the intended experiment.

Alternate Protocol 3: dBroccoli Imaging in Live Mammalian Cells

  Materials
  • Mouse laminin (Fisher Scientific, cat. no. 50948048)
  • PBS (see recipe)
  • TrypLE Express Enzyme (Life Technologies, cat. no. 12604021)
  • Clear mammalian cell culture medium (see recipe)
  • DNase, RNase‐free water (Gibco, cat. no. 10977)
  • 40 mM DFHBI‐1T solution (see recipe)
  • Hoechst 33342 (Life Technologies, cat. no. H3570)
  • Poly‐D‐lysine‐coated 3.5‐cm glass‐bottom plates (Mattek, cat. no. P35GC‐1.5‐14‐C)
  • Cell culture incubator
  • Imaging apparatus, including:
  • Inverted wide‐field fluorescence microscope (e.g., Nikon TE2000)
  • Temperature‐controlled environmental chamber (35°‐37°C, 5% CO 2)
  • Motorized stage (e.g., Ludl BioPrecision Motorized Flat Top Stage)
  • 60× high‐NA objective (e.g., Nikon Plan Apo, 1.4 NA, oil immersion)
  • CCD camera (e.g., Photometrics CoolSnap HQ2)
  • Filter cube for green fluorescence, such as FITC or GFP filter sets (e.g., sputter‐coated excitation filter 470 ± 20 nm, 495 nm [long pass] dichroic mirror, and emission filter 525 ± 25 nm; Chroma Technology)
  • Filter cubes for auxiliary fluorescent signals: e.g., Texas Red filter cube for mCherry (560 ± 20 nm excitation filter, 585 nm [long pass] dichroic mirror, and 630 ± 37.5 nm emission filter; Chroma Technology) and DAPI filter cube for Hoechst 33342 (350 ± 25 nm excitation filter, 400 nm [long pass] dichroic mirror, 460 ± 25 nm emission filter; Chroma technology)
  • NIS Elements (Nikon) or similar image acquisition software
  • Fiji (http://fiji.sc/Fiji), ImageJ, or similar image analysis software
NOTE: If an environmental chamber is not available, a heated stage is also an option. In this case, the medium on the cells must be buffered with 25 mM HEPES, pH 7.4, rather than a bicarbonate buffer.

Support Protocol 1: In Vitro Transcription and Fluorescence Measurement of RNA‐Broccoli Fusions

  Materials
  • AmpliScribe T7‐Flash Transcription Kit (Epicenter, cat. no. ASF3257) or equivalent
  • DFHBI‐1T (Lucerna, cat. no. 410)
  • 5× fluorescence measurement buffer (see recipe)
  • 1.5‐ml microcentrifuge tubes (USA Scientific, cat. no. 1615‐5500)
  • QIAquick PCR Purification Kit (Qiagen, cat. no. 28104)
  • Micro Bio‐Spin P30 gel columns (Bio‐Rad, cat. no. 7326223)
  • NanoDrop (Thermo Scientific) or equivalent
  • FluoroMax‐4 fluorometer (Horiba Scientific) or equivalent
  • Quartz SUPRASIL Ultra‐micro cuvette with PTFE stopper (PerkinElmer, cat. no. B0631124) or equivalent
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Figures

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