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This article presents a protocol for detecting microRNA expression in the kidneys of an acute kidney injury mouse model using quantitative real-time reverse-transcription polymerase chain reaction. This protocol emphasizes an ischemic kidney injury mouse model and the careful extraction of microRNA samples.
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.
Ischemia-reperfusion injury (IRI) of the kidney represents one of the major risk factors for Acute kidney injury (AKI) development1. AKI plays a significant role in patient prognosis, but specific therapies and early diagnostic biomarkers have not been established.
MicroRNAs (miRNAs) are short, non-coding RNAs with approximately 18–25 bases. miRNAs are stable in body fluids, and their sequences are highly conserved among animals2. MiRNAs regulate the expression of multiple proteins through thousands of targets, thereby influencing diverse signaling pathways2,3,4,5,6,7,8. In recent years, it has been reported that miRNAs are involved in various disease states and are effective biomarkers for the early diagnosis of several diseases.
The overall goal of this protocol is to successfully purify and detect miRNAs in the kidneys of an AKI mouse model induced by IRI. This IRI model is a widely used model of AKI and renal fibrosis. The advantages of the IRI model include being able to visually confirm whether ischemia-reperfusion has been achieved and determine the exact time of AKI onset. However, a clamp duration that is long enough to cause broad tubular damage is associated with a high mortality rate, whereas a short clamp duration does not cause tubular damage, which results in a large variation in tubular damage progression in this experimental model. Compared with the bilateral clamping model, the right nephrectomy tissue harvested in the unilateral renal IRI model acts as a control and ensures renal failure.
The kidney sample is mashed using a glass homogenizer, wherein, proteins and nucleic acids are separated, and DNA and RNA are separated9. Total RNA, including miRNA, is purified from the kidney sample using a silica-membrane-based spin column9. Subsequently, cDNA synthesis (reverse transcription) is performed from total RNA using poly(A) polymerase and oligo-dT primers10. Finally, the miRNA expression is determined by qRT-PCR using an intercalating dye10. qRT-PCR can more accurately quantify gene expression compared with PCR of amplification volumes at endpoints. qRT-PCR measures high concentrations and is characterized by a wide dynamic range, which allows accurate quantification that depends on the number of cycles. Previous studies reported that simple processes could be used to effectively purify and detect miRNAs in tissues8,9,10. The methods described in this protocol for cDNA synthesis (reverse transcription) and the detection of miRNA expression by qRT-PCR using an intercalating dye have been reported to show high accuracy and sensitivity10.
Additionally, this protocol is simple and achieves consistent results between laboratories. Therefore, this protocol is useful in studies that require highly accurate and sensitive miRNA detection in mouse AKI kidneys.
All animal experimental protocols were approved by the animal ethics committee of Jichi Medical University and performed in accordance with the Use and Care of Experimental Animals guidelines from the Jichi Medical University Guide for Laboratory Animals.
1. IRI model
NOTE: Carefully monitor for hypothermia, intestinal moisturization, and depth of anesthesia throughout the procedure.
2. Kidney sample collection
NOTE: This video has a 45 min clamping time, collecting the kidneys 24 h later.
3. Purifying miRNAs from kidney samples
NOTE: Here, kidney samples weighing 30 mg are homogenized using a glass homogenizer and a biopolymer-shredding system in a microcentrifuge spin column. Subsequently, miRNA from the kidney sample is isolated using a silica-membrane-based spin column.
4. Reverse transcription of miRNA
NOTE: Here, 1.0 µg of isolated RNA is reverse-transcribed using reverse transcriptase, poly(A) polymerase, and oligo-dT primers.
5. qRT-PCR of miRNA
NOTE: qRT-PCR of miRNA is performed using an intercalating dye. Here, primers for U6 small nuclear 2 (RNA-2), miRNA-17-5p, miRNA-18a-5p, miRNA-21a-5p, miRNA-132-3p, miRNA-212-3p, miRNA-223-3p, and miRNA-574-5p were used.
Here, the miRNA expression profile in AKI mice was investigated. The miRNA expression profile has been investigated in various organs and tissues in mice. MiRNAs are important post-transcriptional regulators and are now being extensively studied in the characterization of a variety of diseases, including AKI. MiRNAs have the potential to help elucidate pathological conditions and be applied to the treatment of AKI12. The major causes of AKI are IRI, nephrotoxic insult, and sepsis. IRI of the kidne...
Using the protocol presented in this manuscript, miRNAs from the kidney of IRI mice were successfully purified and detected using qRT-PCR. Critical points of the IRI-inducing procedure in the protocol include careful monitoring of body temperature and anesthesia concentrations, which are known to affect AKI20. The strength of this protocol is that it allows visual confirmation of whether ischemia-reperfusion has been achieved. However, there are some limitations to this IRI model. Prolonging the c...
The authors declare that they have no conflicts of interest.
We thank Nam Nguyen, PhD for editing a draft of this manuscript.
Name | Company | Catalog Number | Comments |
Bent Tip Tapered Tweezers without Hook | Natsume Seisakusho | MA-47 | |
Buffer RLT | Qiagen | 79216 | wash buffer 1 |
Buffer RWT | Qiagen | 1067933 | wash buffer 2 |
C57B6 mice | SLC | not assign | |
forcep with Teeth | Natsume Seisakusho | MA-49 | |
forcep without Teeth | Natsume Seisakusho | MA-48-1 | |
Hemostatic clips | Natsume Seisakusho | KN−353 | |
MicroAmp Optical 96 well reaction plate for qRT-PCR | Thermo Fisher Scientific | 4316813 | 96-well reaction plate |
MicroAmp Optical Adhesive Film | Thermo Fisher Scientific | 4311971 | adhesive film for 96-well reaction plate |
miRNA-132-3p primer | Qiagen | MS00003458 | 5'UAACAGUCUACAGCCAUGGUCG |
miRNA-17-5p primer | Qiagen | MS00029274 | 5'CAAAGUGCUUACAGUGCAGGUAG |
miRNA-18a-5p primer | Qiagen | MS00031514 | 5'UAAGGUGCAUCUAGUGCAGAUAG |
miRNA-212-3p primer | Qiagen | MS00003815 | 5'UAACAGUCUCCAGUCACGGCC |
miRNA-21a-5p primer | Qiagen | MS00009079 | 5'UAGCUUAUCAGACUGAUGUUGA |
miRNA-223-3p primer | Qiagen | MS00003871 | 5'UGUCAGUUUGUCAAAUACCCCA |
miRNA-574-5p primer | Qiagen | MS00043617 | 5'UGAGUGUGUGUGUGUGAGUGUGU |
miRNeasy Mini kit | Qiagen | 217004 | silica-membrane based spin column |
miScript II RT kit | Qiagen | 218161 | |
miScript SYBR Green PCR kit | Qiagen | 218073 | |
QIA shredder | Qiagen | 79654 | biopolymer-shredding system in a micro centrifuge spin-column |
QIAzol Lysis Reagent | Qiagen | 79306 | phenol/guanidine-based lysis reagent |
QuantStudio 12K Flex Flex Real-Time PCR system | Thermo Fisher Scientific | 4472380 | real-time PCR instrument |
QuantStudio 12K Flex Software version 1.2.1. | Thermo Fisher Scientific | 4472380 | real-time PCR instrument software |
RNU6-2 primer | Qiagen | MS00033740 | not disclosed |
surgical scissors | Natsume Seisakusho | B-2 | |
Vascular clip applier | VITALITEC | 1621420 |
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