Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

Summary

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

Abstract

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3' untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA's translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

Introduction

MicroRNAs (miRNAs) — the short, noncoding RNAs that cause the degradation and transcriptional inhibition of messenger RNA (mRNA)¹ — have been shown to regulate the expression of various mRNAs that have crucial roles in both physiology and disease (e.g., inflammation, fibrosis, metabolic disorders, and cancer). Some of the miRNAs may therefore be candidate novel biomarkers and therapeutic targets for a variety of diseases^2,3,4,5. Although miRNA expression profiles in mouse organs and....

Protocol

All animal experimental protocols were approved by the Animal Ethics Committee of Jichi Medical University and were performed in accordance with the Use and Care of Experimental Animals guidelines from the Jichi Medical University Guide for Laboratory Animals.

1. The sham surgery

1. Prepare the following items: isoflurane, cork sheet, depilatory cream, laboratory wipes, Petri dish with phosphate-buffered saline (PBS), 4-0 nylon, tweezers, surgical scissors, cotton swabs, and 8-week-old C57BL/6 male mice.
2. Anesthetize a mouse with 1.5% isoflurane and maintain at 1.5%. Then, apply depilatory cream to the mouse's abdomen.....

Results

A UUO mouse model was created by left ureteral ligation as described²¹ in 8-week-old male mice weighing 20–25 g. Ureters were completely obstructed by double ligation with 4-0 silk sutures. An analgesic (meloxicam 5 mg/kg, subcutaneous injection) was administered before surgery and also daily on the 2 days post-surgery. At 8 days post-surgery, kidneys were collected, rinsed with PBS, dissected, and stored in liquid nitrogen for further analysis. Sham-operated mice served as controls. The double li.......

Discussion

The above-described protocol with qRT-PCR successfully determined the expression levels of the targeted miRNAs. The assessment of extracted miRNAs is important when seeking to obtain meaningful qRT-PCR data, and in order to confirm the quality of the miRNAs before performing the qRT-PCR, the ratio of absorbance at 260 nm to that at 280 nm should be checked with a spectrophotometer. If a single PCR amplification of the expected length and melting temperature or a monomodal melting curve cannot be obtained by qRT-PCR, ther.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Qiagen	79216	Wash buffer 2
Qiagen	1067933	Wash buffer 1
Tokyo Laboratory Animals Science	Not assigned
Thermo Fisher Scientific	4316813	96-well reaction plate
Thermo Fisher Scientific	4311971	Adhesive film for 96-well reaction plate
Qiagen	MS00001701	5'-UUAAUGCUAAUUGUGAUAGGGGU-3'
Qiagen	MS00065141	5'-UUACACUCCAGUGGUGUCGGGU-3'
Qiagen	MS00068067	5'-UGCAGGGUUUAGUGUAGAGGG-3'
Qiagen	MS00068081	5'-UGUGUUAGAGCUCAGGGUUGAGA-3'
Qiagen	217004	Membrane anchored spin column in a 2.0 mL collection tube
Qiagen	218161	Reverse transcriptase kit
Qiagen	218073	Green dye-based PCR kit
Qiagen	79654	Biopolymer spin columns in a 2.0 mL collection tube
Qiagen	79306	Phenol/guanidine-based lysis reagent
Thermo Fisher Scientific	4472380	Real-time PCR instrument
Thermo Fisher Scientific	4472380	Real-time PCR instrument software
Qiagen	129112
Qiagen	MS00033740	Not disclosed
Takara Bio	9790B	Silicon homogenizer
ASKUL	GA04SW
AS ONE	ER1004NA45-KF2,62 -9968-32