Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

This method makes it possible to provide microRNA expression in kidneys of mice with a wide range of pathological conditions, such as renal fibrosis. The main advantage of this technique is that it enables microRNA expression profiling with high accuracy and sensitivity by using a simple process that saves time and prevents technical error. To perform the sham surgery, use surgical scissors and tweezers to make an incision in the skin at the abdomen and cut the muscle and peritoneal membrane from the bladder to the left lower edge of the ribs.

Moisten two cotton swabs with PBS and carefully pull the intestines to the side. Place the moistened swabs to identify the left kidney and ureter. Close the peritoneal membrane and then the incision with 4-0 nylon.

To perform the unilateral urethral obstruction, or UUO, make an incision in the skin at the abdomen and cut the muscle and peritoneal membrane as demonstrated for the sham surgery. Place a 2.5-milliliter syringe underneath the mouse. Moisten two cotton swabs with PBS, then pull the intestines carefully to the side with the tweezers and place the swabs to identify the left ureter.

Use the tweezers to lift the left kidney. Use 4-0 silk to ligate the left ureter in two places approximately one centimeter apart. Cut the ureter at the center point of the two ligations and then use 4-0 nylon sutures to close the peritoneal membrane and incision.

After cutting the peritoneal membrane, as previously demonstrated, lift it with tweezers and use surgical scissors to make a sideways incision at the upper edge, then continue the incision along the lowest edge of the ribs. Identify the left kidney and reflux it with PBS until the kidney turns yellow-white. Remove the kidney by cutting the left renal artery and vein with the surgical scissors and place it in a Petri dish, then wash it carefully with PBS.

Cut the kidney into approximately 10-milligram samples with the surgical scissors and tweezers, put each piece of the kidney in its own 1.5-milliliter microcentrifuge tube and close the tube's cap. Put a kidney sample in the silicone homogenizer and add 700 microliters of a phenyl/guanidine-based lysis reagent. Slowly press and twist the homogenizer's pestle against the kidney sample to homogenize it.

Repeat the pressing and twisting until the sample is completely dissolved in the lysis reagent. To further homogenize the sample, transfer the homogenized lysate to the biopolymer spin column and spin it at 14, 000 times G for three minutes, then transfer the precipitated lysate to an unused 1.5-milliliter microcentrifuge tube. Combine the lysate in the tube with 140 microliters of chloroform and cap the tube tightly.

To mix the lysate and chloroform, invert the tube 15 times. Incubate the samples for two to three minutes at room temperature, then spin them at 12, 000 times G for 15 minutes at four degrees Celsius. Without disturbing the precipitate, transfer the supernatant to a new 1.5-milliliter microcentrifuge tube, and add 1.5 times its volume of 100%ethanol.

Vortex the mixture for five seconds. Load 700 microliters of the sample onto a membrane-anchored spin column in a two-milliliter collection tube. Close the column cap and spin the column at 15, 000 times G for 15 seconds, then throw away the precipitated lysate in the collection tube.

Wash the sample thoroughly by adding 700 microliters of wash buffer one to the membrane anchored spin column in a two-milliliter collection tube. Then repeat the centrifugation and throw away the collection tube. Repeat the wash twice with 500 microliters of wash buffer two per wash.

After the last wash, spin the membrane-anchored spin column in a two-milliliter collection tube at 15, 000 times G for one minute and throw away the precipitated lysate. Transfer the membrane-anchored spin column to a new 1.5-milliliter tube. Dissolve the total RNA by adding 30 microliters of RNase-free water to the column, close the column cap, and leave it for five minutes at room temperature.

Then spin the column at 15, 000 times G for one minute. Transfer the sample containing total RNA to a new microcentrifuge tube on ice, and measure the concentration of total RNA by spectrophotometry. Prepare a master mix solution according to the manuscript directions and add eight microliters to each tube of an eight-well tube strip.

After adjusting the total RNA density, place a 12-microliter aliquot of total RNA into each tube and close the cap. Centrifuge the tubes for 15 seconds. Put the tubes in the thermocycler and incubate them for 60 minutes at 37 degrees Celsius.

When finished, immediately incubate them for five minutes at 95 degrees Celsius for the synthesis of cDNA. Transfer the cDNA into a new 1.5-milliliter microcentrifuge tube and dilute it 10 times with distilled water. Vortex and centrifuge the tube for five seconds, then perform quantitative real-time PCR, as described in the text manuscript.

This protocol was used to create a unilateral urethral obstruction mouse model via left ureteral ligation in eight week old male mice weighing 20 to 25 grams. Ureters were completely obstructed by double ligation with 4-0 silk sutures. Kidneys were collected at eight days post surgery.

The microRNA qRT-PCR data indicated that the level of microRNA 3070-3p was significantly increased and the levels of microRNA, 7218-5p and microRNA 7219-5p were decreased in the kidneys of the UUO mice compared to the controls. When attempting this protocol, remember to repeat twisting the kidney sample until it is completely dissolved in phenyl/guanidine-based lysis reagent to achieve a good RNA extraction.