The overall goal of this procedure is to successfully purify and detect miRNAs in the rat peritoneal membrane. To begin this procedure, after inhalational anesthesia with 4%isoflurane. Spray the abdominal skin of the rat with 70%ethanol and mount it on the cork sheet, on its'back.
Next make a longitudinal incision in the animal's abdominal skin, muscle, and peritoneal membrane using scissors and forceps. Pick up the peritoneal membrane using the forceps and make horizontal incisions on its'upper portion along the lowest rib bone under the diaphragm. And on its'lower side on the lateral region using the surgical scissors.
Next, make lateral longitudinal incisions to remove the peritoneal membrane from the body, using the surgical scissors. Then wash it with PPS in a petri dish. Cut out and trim 20 milligrams of peritoneal membrane sample.
In this procedure, place a 20 milligram peritoneal membrane sample into a glass homogenizer. And add 700 microliters phenol guanidine based lysis reagent. After that, homogenize the peritoneal membrane sample on ice, by slowing pressing a pestle onto the sample with twisting.
Repeat this process several dozen times, until the peritoneal membrane sample has completely dissolved into the phenol guanidine based lysis reagent. for further homogenization, transfer the homogenized lysate to a bio-polymer shredding system in the micro centrifuge spin column in a two milliliter collection tube. Then centrifuge it at 14, 000 times G for three minutes at four degrees Celsius.
Next, transfer the homogenized lysate to a new micro centrifuge tube. Add 140 microliters of chloroform to the homogenized lysate and cap the tube securely. Subsequently, mix the tube by inversion for 15 seconds.
Afterward, incubate the sample for two to three minutes at room temperature. Following that, centrifuge it at 12, 000 times G for 15 minutes at four degrees Celsius. Transfer 300 microliters of the supernatant to a new micro centrifuge tube, without disturbing the precipitant.
And add 450 micro liters of 100%ethanol. Then mix the sample by vortexing for five seconds. Next pipette up to 700 micro liters of the sample into a silicone membrane based spin column, placed in a two milliliter collection tube, and close the cap.
Then centrifuge it at 15, 000 times G for 15 seconds. After centrifugation, discard the flow through in the collection tube. Then add 700 microliters of wash buffer 1, to the silicon membrane based spin column to stringently wash the sample and close the cap.
Centrifuge it at 15, 000 times G for 15 seconds. After centrifugation, discard the flow through in the collection tube. Add 500 microliters of wash buffer 2, to the silica membrane based spin column, to remove any trace of salt, and close the cap.
Then centrifuge it at 15, 000 times G for 15 seconds. After centrifugation, discard the flow through and the collection tube and repeat this procedure again. Next, centrifuge the silica membrane based spin column again at 15, 000 times G for one minute.
After centrifugation, discard the collection tube and any flow through. Place the silica membrane based spin column in a new, one point five milliliter collection tube. Then transfer 25 microliters of RNase free water onto the column and close the cap.
Leave the sample at room temperature for five minutes. Centrifuge it at 15, 000 times G for one minute. Following that, transfer the 25 microliter alouette containing the total RNA to a new micro centrifuge tube and store it at minus 80 degrees Celsius before use.
In this procedure, prepare a master mix solution that contains two microliters of 10x nucleic acid mix, two microliters of reverse transcriptase mix, and four microliters of 5x Hispec buffer, for a total of 8 microliters per tube. Then dispense 8 microliters of the master mix solution into each tube. Measure the quantity of the total RNAs using a Spectrophotometer.
Afterward, add one microgram of the isolated total RNAs, purified from the peritoneal membrane sample, to each tube. Following that, add RNase free water to each tube to bring it to a total of 20 microliters. Then mix it thoroughly by pipetting and centrifuge it for 15 seconds.
Following that, incubate the sample for 60 minutes at 37 degrees Celsius. Then incubate it for five minutes at 95 degrees Celsius. Transfer the newly generated cDNA to a new micro centrifuge tube and dilute it 10 times with RNase free water.
In this procedure, prepare a master mix containing 12 point five microliters of 2x PCR master mix, two point five microliters of five micromolar of each miRNA primer dissolved in nuclease free water, and one point two five microliters of 10x universal primer, for each well. Dispense 22 point five microliters of this master mix into each well of a 96 well plate. Then add two point five microliters of template cDNA to each well.
Seal the 96 well reaction plate with a piece of film. Then centrifuge it at 1, 000 times G for 30 seconds. To run the PCR cycling program, set the plate in the real time PCR instrument.
In the real time PCR software, define the experimental properties. Next, assign names to the samples and target miRNAs in each well. Then in the real time PCR software input a reaction volume of 20 micro liters and the following PCR cycling conditions in accordance with the instructions.
Pre incubation at 95 degrees Celsius for 15 minutes. And then 40 cycles of denaturation at 94 degrees Celsius for 15 seconds, and kneeling at 55 degrees Celsius for 30 seconds, And extension at 70 degrees Celsius for 30 seconds. Based on miRNA, micro array screening, the levels of six miRNAs have been found to be significantly increased in the peritoneal membrane of peritoneal fibrosis rats, compared with those in mock rats and control rats, using QRTCPR, following the protocol presented in this manuscript.
