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A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model
Published on: September 11, 2020
A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model
Akinori Aomatsu1, Keiji Hirai1, Hiroki Ishii1
1Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University.
Abstract:
MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, standard protocols for the purification of miRNAs and detection of their expression in the kidneys of acute kidney injury (AKI) mice have not been well established. This study developed an effective and simple protocol to purify and quantify miRNAs in the kidneys of an AKI mouse model induced by renal ischemia-reperfusion using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). This protocol comprises five steps: 1) induction of AKI by renal ischemia-reperfusion, 2) harvesting of kidneys, 3) purification of total RNA, including miRNAs, from kidneys, 4) cDNA synthesis by reverse transcription of miRNA, and 5) qRT-PCR to detect miRNA expression. Using this protocol, the renal ischemia-reperfusion injury model can be generated with mild to severe forms of AKI. Additionally, if the procedure is followed properly, a consistent AKI model with minimal individual differences can be obtained. This qRT-PCR assay shows a very wide dynamic range and enables the discrimination of mature miRNAs, which can be accurately quantified with high specificity. This protocol can be used to study the miRNA expression profile in AKI kidneys.
Insights
Researchers developed a simple protocol to purify and quantify microRNAs (miRNAs) in mouse kidneys with acute kidney injury (AKI). This method enables accurate measurement of miRNA expression for studying AKI pathogenesis.
Area of Science:
- Molecular Biology
- Renal Physiology
- Biomarker Discovery
Background:
- MicroRNAs (miRNAs) are crucial in disease and serve as potential biomarkers.
- Established protocols for miRNA purification and expression analysis in acute kidney injury (AKI) mouse models are lacking.
Purpose of the Study:
- To develop and validate a simple, effective protocol for purifying and quantifying miRNAs in the kidneys of AKI mouse models.
- To establish a reliable method for studying miRNA expression profiles in AKI kidneys.
Main Methods:
- Induction of AKI in mice via renal ischemia-reperfusion.
- Isolation of total RNA, including miRNAs, from kidney tissues.
- Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) for miRNA expression analysis.
Main Results:
- A five-step protocol was successfully established for AKI model generation and miRNA quantification.
- The protocol allows for the creation of consistent AKI models with varying severity.
- The qRT-PCR assay demonstrated a wide dynamic range, high specificity, and accurate quantification of mature miRNAs.
Conclusions:
- The developed protocol provides a standardized and effective method for miRNA purification and expression analysis in AKI mouse kidneys.
- This method facilitates the study of miRNA roles in AKI pathogenesis and their potential as diagnostic or therapeutic targets.
