Test MicroRNA Target with in vitro Cell Culture Luciferase Assay Protocol

Experiment Summary

MicroRNAs (miRNAs) are small non-coding RNAs of 21-24 nucleotides in length that modulate gene expression by targeting the untranslated region (UTR) of mRNA. This protocol is to be used to test the binding and activity of miRNA on putative UTR target sequences. It is based on the expression of Luciferase as a reporter gene fused to the UTR sequence in the presence of plasmids containing pre-miRNA of interest or synthetic miRNA to test in an in vitro cell culture assay.

Materials and Reagents

HEK293T cells
DMEM (High Glucose) (GutaMAXTM)
Fetal bovine serum (FBS) heat inactivated
Penicillin-Streptomycin
pmirGLO Dual-Luciferase miRNA Target Expression Vector
miScript miRNA Mimic
Lipofectamine®2000 Transfection Reagent
Dual-Glo Luciferase assay system
Cuiture medium

Equipment

Luminometer
White 96-well plates

Procedure

Clone the putative UTR sequence to test into the pmirGLO Dual-Luciferase miRNA Target Expression Vector.
Plate 1 x 10⁴ cells per well in 50 μl culture medium from a sub-confluent HEK293T cell suspension (cell density < 1.3 x 10⁵ cells/cm²). Plate 3 wells for each condition to produce triplicates (conditions should include negative controls for miRNA and UTR sequence and positive control when existing such as known miRNA for the target or known target for the miRNA).
Transfect cells after 5-16 h in culture. The following is transfection condition for 1 well.

1) Dilute 300 ng of reporter plasmid (an example of vector is pmirGLO Dual-Luciferase miRNA Target Expression Vector) in combination with miRNA Mimic (such as miScript miRNAMimic) at a final concentration of 5 to 50 nM in 25 μl of pure DMEM.

2) Dilute 0.5 μl Lipofectamine in 25 μl of pure DMEM.

3) Incubate both diluted solutions 5 min at room temperature.

4) Combine diluted nucleic acids with diluted Lipofectamine (50 μl final), mix gently and incubate complexes 20 min at room temperature.

5) Add the transfection complexes (50 μl) to the well containing plated cells in 50 μl medium.

4. Incubate transfected cells at 37 °C in a 5% CO₂ incubator for 48 h without medium change.

5. Measure luciferase using the Dual-Glo Luciferase assay system as recommended by the manufacturer:

1) Prepare the stop solution, according to the number of wells, by diluting Dual-Glo® Stop &Glo®Substrate (1:100) in Dual-Glo® Stop &Glo® Buffer.

2) Due to evaporation of the medium with time, the final volume per well is less than 100 μl after 48 hin culture. Measure the volume of the medium left in one well by pipetting and discard the appropriate amount of medium to reduce the volume to 50 μl from the well by pipetting out.

3) Add 50 μl of Dual-Glo® Luciferase Reagent to each well (equal volumes). Avoid depositing Reagent solution along the walls of the well. Mix gently by tapping the plate. Because the plate is not shaken, when a drop of reagent is on the wall of the well, the volume of reagent in contact with the culture medium is reduced and this has an impact on the measurement of the luciferase.

4) Incubate 10 min in the dark at room temperature without shaking.

5) Measure firefly luciferase activity using the luminometer.

6) Remove the plate from the luminometer and add 50 μl of Dual-Glo®Stop&Glo® Reagent (stop solution prepared in a.) per well. Mix gently.

7) Incubate 10 min in the dark at room temperature.

8) Measure Renilla luciferase activity using the luminometer.

6. Calculate the ratio of luminescence from the experimental reporter (firefly) to luminescence from the control reporter (Renilla). Calculate the mean ratio for each triplicate and normalize this ratio to the ratio of control wells. Experimental conditions will include a miRNA negative control (which does not target the UTR), a reporter plasmid devoid of UTR or containing an irrelevant UTR sequence.

* For research use only. Not intended for any clinical use.

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Test MicroRNA Target with in vitro Cell Culture Luciferase Assay Protocol

Experiment Summary

Materials and Reagents

Equipment

Procedure

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