Evaluating Virus Spread Inhibition Through microRNA Mimic-Mediated Genome Silencing

Abstract

Source: Ruiz, A. J. and Russell, S. J. MicroRNA-based Regulation of Picornavirus Tropism. J. Vis. Exp. (2017)

This video demonstrates the use of synthetic microRNA mimics to inhibit viral replication in cultured epithelial-like cells selectively. It highlights how microRNA-directed genome silencing can block virus spread and confirm targeting specificity through downstream infectivity analysis.

Protocol

1. Evaluating microRNA-targeting Specificity: Virus-spreading Assay Using Synthetic microRNA Mimics

NOTE: Use of a synthetic microRNA mimic is performed to show the specificity of the microRNA:microRNA-target interaction.

1. Include mock transfection, negative control microRNA mimic, and experimental microRNA mimic only controls for every assay to evaluate microRNA-mediated toxicity. It is also ideal to include a positive control (i.e., microRNA-targeted gene or genome) as low transfection efficiency of microRNA mimics can result in false negatives.
2. Plate H1-HeLa cells in a 96-well tissue culture plate such that they are 80-90% confluent at the time of transfection (24 h post-seeding). Plate cells in DMEM (Dulbecco’s modified eagle medium) supplemented with 10% fetal bovine serum at 0.1 mL per well.
   NOTE: Perform this assay in cells permissive for viral replication that do not express the cognate microRNAs.
3. Warm the transfection reagents to room temperature. Combine 9 µL serum-free media, 200 nM final concentration per well of microRNA mimic stock, 0.18 µL boost reagent, 0.18 µL transfection reagent, and mix. Incubate the mixture at room temperature for 2-5 min.
   NOTE: Volumes listed are per well. It is recommended that a master mix be assembled for the transfection of all wells to maintain consistency and minimize pipetting error. The optimal concentration of the microRNA mimic will vary. Consult the manufacturer's instructions accompanying the microRNA mimic for a reasonable starting concentration. This will generally range from 5-200 nM final concentrations.
4. Aspirate the media from the wells and add 92 µL of fresh complete medium.
   NOTE: If there are a significant number of samples, complete this step prior to assembling the transfection solution and store the plate at 37 °C until ready.
5. Add the entire transfection mixture to the cells drop-wise.
6. Incubate at 37 °C for 6 h.
7. Infect each well at a low MOI (Multiplicity of infection) to ensure a low percentage of cells get infected to allow analysis of virus spread. This protocol uses an MOI of 0.2.
   1. Dilute virus stocks in serum-free media to a concentration of MOI = 0.2 per 100 µL. Remove media from the wells and add 100 µL of virus dilution per well.
   2. Incubate at 37 °C for 2 h.
8. Aspirate media from each well and add 100 µL of fresh complete media.
9. Incubate at 37 °C for 20-22 h.
10. Determine the virus titration in the supernatants.
    1. Collect the supernatant from each well and replace with 100 µL of fresh complete media.
       NOTE: If using suspension cells, resuspend the cell pellet from the following step in 100 µL of fresh complete media and return to the sample well for the viability assay.
    2. Remove cellular debris from the collected supernatants by centrifuging at 300 x g for 5 min at 4 °C.
    3. Transfer the cleared supernatant to a fresh tube and titrate infectious virus on permissive cells that do not express the cognate microRNAs. 
       NOTE: Keep all samples on ice to prevent loss of infectivity.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nuclease-free water	Fisher Scientific	AM9938
TransIT-2020 Transfection reagent	Mirus	MIR 5404
TransIT-mRNA Transfection reagent	Mirus	MIR 2225
2 mL Screw-cap tubes	Sarstedt	72.694.005
MicroRNA mimics	Dharmacon	Varied
MTT Cell proliferation assay	ATCC	30-1010K