Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Summary

Luminescent identification of functional elements in 3’ untranslated regions (3’UTRs) (3’LIFE) is a technique to identify functional regulation in 3’UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Abstract

Luminescent Identification of Functional Elements in 3’UTRs (3’LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3’UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3’LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3’LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3’UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3'LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Introduction

The overall goal of this method is to detect and precisely map microRNA (miRNA) targets in high-throughput. MiRNAs are endogenous non-coding RNAs ~22 nucleotides in length. Following transcription and processing, mature miRNAs are incorporated in a protein complex called the RNA induced silencing complex (RISC). Each miRNA guides the RISC to target elements located primarily in the 3’untranslated regions (3’UTRs) of messenger RNAs (mRNAs), resulting in either translation repression or mRNA cleavage ¹. MiRNA recognize target sites based on standard Watson-Crick and G:U wobble base pairing, and are degenerate in nature, containing multiple mismatc....

Protocol

1. Cell Culture (24-48 hr prior to transfection)

1. 24-48 hr prior to transfection seed a sufficient quantity of HEK293T cells based on the number of 96-well plates being transfected.
   NOTE: For consistent transfections, plate cells at a sufficient density to favor rapid division, yet not be at more than 70-90% confluency at the time of transfection.
2. Each 96-well plate requires 9 x 10⁶ cells (75,000 cells per well, and 120 wells per plate to account for use of reservoir and multichannel pipette). Calculate the doubling time of HEK293 cells (typically ~20 hr), and seed the appropriate number of cells to obtain at least 9 x 10

Results

The luminometer output file contains raw measurements for both firefly and Renilla luciferase proteins. This raw format is compatible with the “3’LIFE – single plate analysis” and “3’LIFE – multiplate analysis” spreadsheets available from the Mangone lab website (www.mangonelab.com). The single plate analysis spreadsheet automatically calculates firefly/Renilla ratio, normalizes each miRNA to the appropriate negative control, and normalizes repression values acro.......

Discussion

The 3’LIFE assay identifies functional miRNA targets in 3’UTRs in high-throughput. This assay is useful for researchers who wish to experimentally identify a large number of putative targets for their miRNA of interest. The 3’LIFE assay is a powerful approach to query for 3’UTR driven regulation, in that the assay provides a functional measure of miRNA targeting, and the binary testing of a single reporter::3’UTR against a single miRNA can confidently address the targeting status of individu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
glycylglycine	Sigma	G1127-25G
Kx PO4	Sigma	P2222
EGTA	Sigma	E3889
ATP	Sigma	FLAAS
DTT	Sigma	D0632
MgSO4	Sigma	M7506
CoA	Sigma	C4282
luciferin	Sigma	L9504
NaCl	Sigma	S7653
Na2EDTA	Sigma	E0399
K H2 P O4	Sigma	1551139
BSA	Sigma	A2153
NaN3	Sigma	S2002
Coelenterazine	Sigma	C3230
PBS/HEPES	Corning	21-040-CV
DMEM	Sigma	D5546
FBS	Sigma	F2442
Pennicilin/Streptomicin	Sigma	P4333
Trypsin		T2600000
Consumables
MaxiPrep Kit	Promega	A2392
96-well miniprep plate	Pall	8032
96-well shuttle plates	Lonza	V4SP-2096
5x Lysis Buffer	Promega	E1941
Instruments
96-well GloMax Plate Reader	Promega	E9032
Biomech FX Liquid Handler Robot	Beckmann	A31842
4D-Nucleofector Core Unit	Lonza	AAF-1001
96-well Shuttle System	Lonza	AAM-1001
Cell Counter Countess	Invitrogen	C10227