This qRT-PCR method can address questions related to microRNA expression profiling in serum of mice in conditions such as age-dependent renal impairment. The technique enables the detection of microRNA expression with high accuracy and sensitivity by a simple process that saves time and prevents technical error. To begin, incise the skin of the abdomen using tweezers and surgical scissors.
Cut the muscles in the peritoneal membrane from the bladder to the lower left edge of the ribs. Lift the peritoneal membrane using tweezers, and make a lateral incision in the upper edge with surgical scissors. Continue the incision along the lowest edge of the ribs.
Identify the inferior vena cava by using two PBS-moistened cotton swabs. Insert a 30 G needle attached to a 1-milliliter syringe into the inferior vena cava, and pull the syringe. Slowly pull the needle out to avoid hemolysis.
Transfer the blood to a 1-milliliter spitz tube with heparin, and mix by inverting. Centrifuge the spitz tube for 10 minutes at 3000 times G at room temperature. Transfer the supernatant to a fresh 1.5-milliliter microcentrifuge tube.
Take 200 microliters of serum sample, and add 1, 000 microliters of phenol/guanidine-based lysis reagent. Vortex the mixture for 5 seconds, and incubate at room temperature for 5 minutes. Add 200 microliters of chloroform, and mix by inverting the tube 15 times.
Incubate the sample at room temperature for 3 minutes, followed by centrifugation. Without disturbing the pellet, transfer the supernatant to a fresh 1.5-milliliter microcentrifuge tube. Add 450 microliters of 100%ethanol, and vortex the tube for 5 seconds.
Then load 700 microliters of the sample onto a membrane-anchored spin column, and centrifuge it. Discard the flow-through, and add 700 microliters of Wash Buffer 1 provided in the kit. Centrifuge again, and discard the flow-through.
Repeat wash with 500 microliters of Wash Buffer 2 to remove trace salts. Add 500 microliters of 80%ethanol, centrifuge, and discard the flow-through from the collection tube. Centrifuge the spin column again, and transfer the column to a fresh 1.5-milliliter collection tube.
Then add 14 microliters of RNase-free water, and incubate for 5 minutes at room temperature. Spin the column again for 1 minute at 15000 times G at room temperature. Prepare a master mix solution, and add 8 microliters per well in an 8-well strip tube.
Add 12 microliters of the extracted total RNA in each tube, and centrifuge the tube for 15 seconds at 2000 times G at room temperature. Place the tube in a thermal cycler, and start the incubation to synthesize the cDNA. After incubation, transfer the cDNA to a fresh microcentrifuge tube.
Dilute the cDNA tenfold with distilled water. Then vortex, followed by centrifugation for 5 seconds at 2000 times G at room temperature. In a microcentrifuge tube, prepare the master mix solution as described in the text protocol, and vortex the solution.
Add 22.5 microliter aliquots in each well of a 96-well plate, followed by 2.5 microliters of cDNA. Seal the plate using an adhesive film, and centrifuge the plate for 30 seconds at 1000 times G.Place the plate in the real-time PCR system. Modify the settings by providing a name for the experiment.
Then select 96-well 0.2 milliliters as the experiment type, comparative CT as the quantitation method, standard as the system run mode, and SYBR Green reagents as the reagents for detecting the target sequence. Provide names for the sample and the target miRNA in each well. Assign duplicate samples, choose a reference sample and endogenous control, and select none for the dye to use as the passive reference.
To eliminate reagent cross-contamination, set up negative reverse transcriptase and a non-template control for miRNA expression. Next ensure that the reaction volume is set to 20 microliters, and the PCR cycling conditions are set at 95 degrees Celsius for 15 minutes, then 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. After the process is complete, click on analyze to analyze the qRT-PCR data.
Confirm that the threshold line automatically selected by the program is appropriate for each well. Check the threshold cycle value of the endogenous control and target miRNAs analyzed in each sample. Determine the CT values by the intersection of the amplification curve and threshold line.
miRNA qRT-PCR data for the age-dependent renal impairment model showed that, as compared to SAMR1 control mice, the kidney level of miRNA-7219-5p significantly increased in SAMP1 experimental mice, while the kidney level of miRNA-7218-5p decreased. The expression levels of miRNA-223-3p did not change in either strain. The serum levels of both miRNA-7219-5p and miRNA-7218-5p were considerably increased in the SAMP1 mice, while the levels of miRNA-223-3p were unchanged.
You should insert the needle into the inferior vena cava and extract blood without penetrating the vessel. This method can be used to answer key questions in the microRNA expression profiling in mice serum for a wide range of pathological conditions.
