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Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction
Published on: August 27, 2020
Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction
Katsunori Yanai1, Shohei Kaneko1, Hiroki Ishii1
1Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University.
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.
Insights
Researchers developed a new method to measure microRNA (miRNA) levels in mouse kidneys with fibrosis. This technique identified specific miRNA changes, offering insights into kidney disease progression.
Area of Science:
- Molecular Biology
- Genetics
- Nephrology
Background:
- MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression.
- Standardized methods for miRNA extraction and quantification in mouse kidneys, especially with fibrosis, were lacking.
- Understanding miRNA profiles is crucial for studying kidney diseases.
Purpose of the Study:
- To establish a reliable protocol for miRNA extraction and expression analysis in mouse kidneys.
- To investigate miRNA expression changes in a mouse model of renal interstitial fibrosis.
- To identify specific miRNAs associated with kidney fibrosis.
Main Methods:
- Development of a five-step protocol for miRNA analysis in mouse kidney samples.
- Utilizing quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for miRNA quantification.
- Employing a unilateral ureteral obstruction (UUO) mouse model to induce renal interstitial fibrosis.
Main Results:
- Successfully extracted and quantified miRNA from mouse kidney samples.
- Identified significant upregulation of miRNA-3070-3p in fibrotic kidneys compared to controls.
- Observed significant downregulation of miRNA-7218-5p and miRNA-7219-5p in the UUO mouse model.
Conclusions:
- The established protocol is effective for determining miRNA expression in mouse kidneys with renal interstitial fibrosis.
- Specific miRNAs (miRNA-3070-3p, miRNA-7218-5p, miRNA-7219-5p) show altered expression patterns in kidney fibrosis.
- This method provides a valuable tool for future research into the role of miRNAs in kidney disease pathogenesis.
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