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microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.
Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.
Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA in the renal glomerular mesangial region1,2. It is the most common of the primary glomerulonephritis and leads to the end-stage renal failure in 20%–40% of patients2. The cause is still unknown but persistent mucosal infection has been implicated1,3. Corticosteroids, immunosuppressants, and renin−angiotensin system inhibitors have been proposed as therapeutic methods1....
Animal experiments were approved by the Animal Ethics Committee of Jichi Medical University and comply with the Use and Care of Experimental Animals guidelines from the Jichi Medical University Guide for Laboratory Animals.
1. Obtaining kidney samples from HIGA mice
NOTE: HIGA mice show a stable phenotype of IgA nephropathy after 25 weeks of age8,9,10,
We investigated the expression levels of miRNAs in the kidneys of HIGA mice (n=10). This result was obtained completely based on the described protocol. The kidneys of Balb/c mice were selected as the control (n=10). In both groups, aged 25 weeks were selected. Only female HIGA mice were available from the supplier. The expression levels of three miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p; Figure 1) were detected, which were previously reported to be associated with IgA nephropathy
We were able to measure the expression levels of miRNAs in the kidneys of an IgA nephropathy mouse model (HIGA mice) using this new method. IgA nephropathy is an unexplained disease that needs further research to clarify its etiology and therapeutic targets1,3. However, obtaining human kidney samples is highly invasive. This new technique is advantageous in that it allows the study of IgA nephropathy using small animals and should facilitate future research into .......
We thank Sarah Williams, PhD, from Edanz Group (www.edanzediting.com) for editing a draft of this manuscript.
....Name | Company | Catalog Number | Comments |
BALB/cCrSlc (25-week-old, female) | Japan SLC, Inc. | none | Mouse for control |
HIGA/NscSlc (25-week-old, female) | Japan SLC, Inc. | none | IgA nephropathy mouse model |
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 |
miScript II RT kit | Qiagen | 218161 | Experimental kit for synthesis of cDNA |
miRNeasy Mini kit | Qiagen | 217004 | Experimental kit fot extraction of total RNA |
miScript Primer Assay (RNU6-2) | Qiagen | MS00033740 | miRNA-specific primer |
miScript Primer Assay (miR-155-5p) | Qiagen | MS00001701 | miRNA-specific primer |
miScript Primer Assay (miR-146a-5p) | Qiagen | MS00001638 | miRNA-specific primer |
miScript Primer Assay (miR-21-5p) | Qiagen | MS00009079 | miRNA-specific primer |
miScript SYBR Green PCR kit | Qiagen | 218073 | Experimental kit for real-time PCR |
QIA shredder | Qiagen | 79654 | biopolymer-shredding spin column |
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 |
takara biomasher standard | Takara Bio | 9790B | silicon homogenizer |
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