An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

Summary

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3’ untranslated regions (3’ UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

Abstract

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3’ untranslated regions (3’ UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3’ UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

Introduction

MicroRNAs (miRNAs) are the small regulatory RNAs that mainly modulate the process of translation and degradation of messenger RNAs (mRNAs) by reacting to the 3’ untranslated regions (3’ UTR) in bona fide target genes¹. Expression of miRNAs can be regulated by transcriptional and post-transcriptional mechanisms. The imbalance of such regulatory mechanisms brings uncontrolled and distinctive miRNAs expression levels in numerous diseases including cancers². A single miRNA can have multiple interactions with diverse mRNAs. Correspondingly, an individual mRNA can be controlled by various miRNAs. Therefore, intrace....

Protocol

1. Mature MicroRNA (miRNA) Expression Analysis

1. Mature miRNA complementary DNA (cDNA) synthesis
   1. Add 254 ng of total RNA and 4.5 µL of deoxyribonuclease I (DNase I) mixtures, and then add ultrapure water into PCR strip-tubes to make up to 18 µL (Figure 2A). Prepare the reaction for each total RNA sample purified from several cell lines using enough amount of DNase I mixtures based on the total number of reactions.
      NOTE: DNase I mixtures are composed of DNase I (1.8 µL), ribonuclease inhibitor (0.3 µL), and 25 mM MgCl₂ (2.4 µL). To reproduci....

Results

Successful and accurate confirmation of miRNA levels is important for the interpretation of data by which the classification of miRNAs is possible based on the anticipated roles of miRNAs in the development and progression of a disease. The levels of miRNA-107 and miRNA-301 were measured in three pancreas cell lines using the probe-based quantitative PCR. The synthesis of cDNAs of both a specific miRNA and a reference gene in the same reaction can increase the reproducibility of data. PANC-1 and CAPAN-1 are human pancrea.......

Discussion

Strategies for the determination of bona fide miRNA targets with the functions of a miRNA of interest are indispensable for the understanding of multiple roles of miRNAs. Identification of miRNA target genes can be a guideline for interpreting the cell signaling events modulated by miRNAs in a cell. An unveiling of functionally important target genes of miRNAs can provide the fundamental knowledge to develop a miRNA-based therapy in cancer.

Several methods such as microarrays, small RNA librar.......

Materials

Name	Company	Catalog Number	Comments
15 mL conical tube	SPL Life Sciences	50015
24-well plate	Thermo Scientific	142475
50 mL conical tube	SPL Life Sciences	50050
6-well plate	Falcon	353046
6X DNA loading dye	Real Biotech Corporation	RD006	1 mL
8-cap strip	Applied Biosystems	N8010535	For cDNA synthesis
8-tube strip	Applied Biosystems	N8010580	For cDNA synthesis
96-well plate	Falcon	353072
Acetic acid	Sigma	A6283-1L	1 L
Agarose A	Bio Basic	D0012	500 g
Alkaline phosphatase	New England Biolabs	M0290S	10,000 U/mL
Ampicillin	Bio basic Canada Inc	AB0028	25 g
AriaMx 96 tube strips	Agilent Technologies	401493	For real time PCR
AriaMx real-time PCR system	Agilent Technologies	G8830A	qPCR amplification, detection, and data analysis
AsiSI	New England Biolabs	R0630	10,000 units/mL
CAPAN-1 cells	ATCC	HTB-79
Cell culture hood	Labtech		Model: LCB-1203B-A2
Counting chambers with V-slash	Paul Marienfeld	650010	Cells counter
CutSmart buffer	New England Biolabs	B7204S	10X concentration
DMEM	Gibco	11965-092	500 mL
DNA gel extraction kit	Bionics	DN30200	200 prep
DNA ladder	NIPPON Genetics EUROPE	MWD1	1 Kb ladder
DNase I	Invitrogen	18068015	100 units
Dual-luciferase reporter assay system	Promega	E1910	100 assays
Fetal bovine serum	Gibco	26140-079	500 mL
HIT competent cells	Real Biotech Corporation(RBC)	RH617	Competent cells
HPNE cells	ATCC	CRL-4023
LB agar broth	Bio Basic	SD7003	250 g
Lipofectamine 2000	Invitrogen	11668-027	0.75 mL
Lipofectamine RNAiMax	Invitrogen	13778-075	0.75 mL
Luminometer	Promega		Model: E5311
Microcentrifuge tube	Eppendorf	22431021
Microplate reader	TECAN		Infinite F50
miRNA control mimic	Ambion	4464058	5 nmole
miRNA-107 mimic	Ambion	4464066	5 nmole
miRNeasy Mini Kit	Qiagen	217004	50 prep
Mupid-2plus (electrophoresis system)	TaKaRa		Model: AD110
NotI	New England Biolabs	R3189	20,000 units/mL
Oligo explorer program	GeneLink		For primer design
Optical tube strip caps (8X Strip)	Agilent Technologies	401425	For real time PCR
Opti-MEM	Gibco	31985-070	500 Ml
PANC-1 cells	ATCC	CRL-1469
Penicillin/streptomycin	Gibco	15140-122	100 mL
Phosphate buffer saline	Gibco	14040117	1000 mL
Plasmid DNA miniprep S& V kit	Bionics	DN10200	200 prep
PrimeSTAR GXL DNA polymerase	TaKaRa	R050A	250 units
Shaker	TECAN		Shaking platform
Shaking incubator	Labtech		Model: LSI-3016A
Sigmaplot 14 software	Systat Software Inc		For dose-response curve generation
Sulforhodamine B powder	Sigma	S1402-5G	5 g
SYBR green master mix	Smobio	TQ12001805401-3	Binding fluorescent dye for dsDNA
T4 DNA ligase	TaKaRa	2011A	25,000 U
TaqMan master mix	Applied Biosystems	4324018	200 reactions, no AmpErase UNG
TaqMan microRNA assay (hsa-miR-107)	Applied Biosystems	4427975	Assay ID: 000443 (50RT, 150 PCR rxns)
TaqMan microRNA assay (hsa-miR-301)	Applied Biosystems	4427975	Assay ID: 000528 (50RT, 150 PCR rxns)
TaqMan miR RT kit	Applied Biosystems	4366597	1000 reactions
Thermo CO2 incubator (BB15)	ThermoFisher Scientific		37 °C and 5% CO2 incubation
Trichloroacetic acid	Sigma	91228-100G	100 g
Trizma base	Sigma	T4661-100G	100 g
Ultrapure water	Invitrogen	10977-015	500 mL
Veriti 96 well thermal cycler	Applied Biosystems		For amplification of DNA (or cDNA)
XhoI	New England Biolabs	R0146	20,000 units/mL