This method can be used to easily verify the change of microRNAs in IGA nephropathy using a small animal model, making it possible to study IGA nephropathy without a human kidney biopsy. The protocol can provide new insights into the genetics of IGA nephropathy, which can potentially aid in discovery of new diagnostic and new therapeutic methods for the kidney disease. After ensuring that the mouse is properly anesthetized, use surgical scissors and forceps to make a three-to four-centimeter midline incision in the abdominal wall of the mouse and identify both sides of the kidney.
Incise the ribs and diaphragm to expose the heart. After incising the right atrium, inject PBS into the left ventricle until the kidney color changes to pale yellow, indicating that the whole body of the mouse is perfused with PBS. Cut the renal artery, renal vein, and ureter, then remove the kidney.
Homogenize the 30-milligram kidney samples using a silicon homogenizer and 700 microliters of phenol guanidine-based lysis reagent at room temperature. Transfer the lysate to the biopolymer shredding spin column and centrifuge it at 15, 000 G for two minutes at room temperature. Continue with RBNA purification following manuscript directions.
After the final wash, add 30 microliters of nuclease-free water to the silica membrane-based spin column and centrifuge it at 8, 000 G for one minute to elute the RNA. For cDNA synthesis, prepare a master mix with two microliters of nucleic acid mix, two microliters of reverse transcriptase mix, and four microliters of buffer in a total of eight microliters per sample. Add eight microliters of the master mix to each well of an eight-well tube strip.
Dilute total RNA in nuclease-free water. Then, add 12 microliters of the total RNA solution to each well of the strip. Place the samples in the thermocycler and incubate them at 37 degrees Celsius for 60 minutes, then at 95 degrees Celsius for five minutes.
To perform quantitative real-time PCR, prepare the master mix according to manuscript directions, then add 22.5 microliters of the master mix to each well of a 96-well reaction plate. Add 2.5 microliters of the prepared cDNA to each well. Place the reaction plate in the real-time PCR instrument.
Then, program it for an initial activation at 95 degrees Celsius for 15 minutes, followed by 40 cycles of denaturation at 94 degrees Celsius for 15 seconds, annealing at 55 degrees Celsius for 30 seconds, and extension at 70 degrees Celsius for 30 seconds. Quantify gene expression using the delta-delta CT method. This protocol was used to investigate the expression levels of microRNAs in the kidneys of HIGA mice using the kidneys of Balb/c mice as control.
The expression levels of three IGA nephropathy-associated microRNAs were measured. Relative microRNA expression levels of the two groups were compared using a T-test with a P of less than 0.05 being statistically significant. miR-155-5p was expressed at 3.3-fold higher levels in the HIGA group compared with the control group, and miR-21-5p was expressed at 1.58-fold higher levels.
miR-1461-5p expression did not differ significantly between the two groups. When attempting this protocol, it is important to wear a mask, gloves, and cap and to keep the laboratory table and tools clean in order to prevent nuclease in the environment from affecting the experiment. This experimental method is currently in practical use in our laboratory.
It's goal, to establish new diagnostic and therapeutic methods for IGA nephropathy.
