Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

All animal experimental protocols were approved by the animal ethics committee of Jichi Medical University and were performed in accordance with the Use and Care of Experimental Animals guidelines from the Jichi Medical University Guide for Laboratory Animals.

1. Peritoneum Sample Collection

1. Collect the following items: 50 mL centrifuge tube with cotton drenched in isoflurane, cork sheet, Petri dish with phosphate-buffered saline (PBS), surgical scissors, and forceps.
2. Euthanize a rat with an overdose of isofluorane, then spray the abdominal skin of the rat with 70% ethanol, and mount the rat on the cork sheet on its back.
3. Make a longitudinal incision in the abdominal skin, muscle, and peritoneal membrane using the scissors and forceps.
4. 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 PBS in a petri dish.
5. Cut out and trim 20 mg (3-5 mm²) of peritoneal membrane samples, which is a suitable size for the following steps, using the surgical scissors and forceps.

2. Purifying total RNAs from Peritoneal Membrane Samples

NOTE: Here, peritoneal membrane samples weighing 20 mg are homogenized using a glass homogenizer and a biopolymer-shredding system in a microcentrifuge spin column. Then, total RNA from the peritoneal membrane sample is isolated using a silica-membrane-based spin column.

1. Collect the following items: 1.5 or 2.0 mL microcentrifuge tubes, 100% ethanol, chloroform, glass homogenizer, ice, biopolymer-shredding system in a microcentrifuge spin column¹⁰, silica-membrane-based spin column¹⁰, phenol/guanidine-based lysis reagent, wash buffer including guanidine and ethanol (wash buffer 1), and wash buffer including ethanol (wash buffer 2).
2. Put a 20 mg peritoneal membrane sample into a glass homogenizer and add 700 µL of the phenol/guanidine-based lysis reagent.
3. Homogenize the peritoneal membrane sample by slowly pressing the pestle on to the sample with twisting. Repeat this process several dozen times on ice until the peritoneal membrane sample has completely dissolved into the phenol/guanidine-based lysis reagent.
4. For further homogenization, transfer homogenized lysate to the biopolymer-shredding system in a microcentrifuge spin column in a 2.0 mL collection tube. Then, centrifuge it at 14,000 x g for 3 min at 4 °C.
5. Transfer the homogenized lysate to a new microcentrifuge tube.
6. Add 140 µL of chloroform to the homogenized lysate and cap the tube securely. Then, mix the tube by inversion for 15 s.
7. Incubate the samples for 2-3 min at room temperature. Then, centrifuge them at 12,000 x g for 15 min at 4 °C.
8. Transfer the supernatant (usually 300 µL) to a new microcentrifuge tube without disturbing the precipitate, and add 1.5x its volume (usually 450 µL) of 100% ethanol. Then, mix the sample by vortexing for 5 s.
9. Pipette up to 700 µL of the sample into a silica-membrane-based spin column placed in a 2.0 mL collection tube. Close the cap of the column. Then, centrifuge it at 15,000 x g for 15 s. After centrifugation, discard the flow-through in the collection tube.
10. Add 700 µL of wash buffer 1 to the silica-membrane-based spin column to stringently wash the sample. Close the cap of the column. Then, centrifuge it at 15,000 x g for 15 s. After centrifugation, discard the flow-through in the collection tube.
11. Add 500 µL of wash buffer 2 to the silica-membrane-based spin column to remove any trace of salt. Close the cap of the column. Then, centrifuge it at 15,000 x g for 15 s. After centrifugation, discard the flow-through in the collection tube.
12. Repeat 2.11.
13. Centrifuge the silica-membrane-based spin column again at 15,000 x g for 1 min. After centrifugation, discard the flow-through in the collection tube.
14. Place the silica-membrane-based spin column in a new 1.5 mL collection tube. Then, transfer 25 µL of RNase-free water into the column. Close the cap of the column. Leave the sample at room temperature for 5 min. Then, centrifuge it at 15,000 x g for 1 min.
15. Transfer the 25 µL eluate containing total RNA to a new microcentrifuge tube. Then, store it at −80 °C before use.

3. Reverse Transcription of Total RNA

NOTE: Reference and adherence to The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines encourage better practice and help in obtaining reliable and unequivocal results¹².
NOTE: Here, a total of 1.0 µg of isolated RNA is reverse-transcribed using the reverse transcriptase, poly(A) polymerase, and oligo-dT primer.

1. Collect the following items: 1.5 mL microcentrifuge tubes; eight-well strip tubes; RNase-free water; ice; reverse transcriptase kit (see materials table)¹¹.
2. Prepare a master mix solution that contains 2.0 µL of 10x nucleic acid mix, 2.0 µL of reverse transcriptase mix (from the kit), and 4.0 µL of 5x hi-spec buffer for a total of 8.0 µL per tube.
3. Dispense 8.0 µL of master mix solution (from the kit) into each tube.
4. Measure the quantity of total RNAs using a spectrophotometer. Add 1.0 µg of isolated total RNAs purified from the peritoneal membrane sample to each tube.
   NOTE: qRT-PCR may be performed using total RNAs as a template without reverse transcription for the quality control of purified total RNAs. If any DNA is contaminated, unexpected amplification products are observed by qRT-PCR.
5. Add RNase-free water to each tube to a total of 20 µL. Then, mix thoroughly by pipetting and centrifuge it for 15 s.
6. Incubate the samples for 60 min at 37 °C. Immediately incubate for 5 min at 95 °C.
   NOTE: This step can be performed in a thermal cycler.
7. Transfer cDNA to a new microcentrifuge tube and dilute ten times (1:10) with RNase-free water.
8. Store the diluted cDNA on ice temporarily, and at −80 °C for long term before use.

4. qRT-PCR of miRNA

NOTE: qRT-PCR of miRNA is performed using intercalating dye. Here, primers for RNA, U6 small nuclear 2 (RNU6-2), miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p were used.

1. Collect the following items: 1.5 mL microcentrifuge tubes, 96-well reaction plate for qRT-PCR, adhesive film for the 96-well reaction plate, miRNA-specific primers, green dye-based PCR kit (see materials table)¹¹, and a real-time PCR Instrument.
2. Prepare a master mix containing 12.5 µL of 2x PCR master mix, 2.5 µL of 5 µM each miRNA primer dissolved in nuclease-free water, and 1.25 µL of 10x universal primer for each well.
3. Dispense 22.5 µL of master mix into each well of the 96-well plate.
4. Add 2.5 µL of template cDNA to each well.
5. Seal the plate with adhesive film for the 96-well reaction plate. Then, centrifuge the plate at 1,000 x g for 30 s.
6. Run the PCR cycling program with the real-time PCR Instrument and software as follows.
   1. Set the plate in the real-time PCR instrument. In the real-time PCR software, define experimental properties (input experimental name, choose "96-wells" for the experimental type of instrument, choose "2^−ΔΔCT method" for the quantitation method, choose "SYBR green reagents" for the reagent to detect the target sequence, choose "standard ramp speed" for the instrument run).
   2. Then, assign names to the samples and target miRNAs in each well. Samples are always tested in duplicate or triplicate to obtain enough data for validation of the results. Select reference sample and endogenous control. Select "none" for the dye to use as the passive reference.
   3. Input a reaction volume of "20 µL" and the following PCR cycling conditions in the real-time PCR software in accordance with the instructions: preincubation at 95 °C for 15 min, and then 40 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s, and extension at 70 °C for 30 s.
7. Analyze qRT-PCR data using the software of the real-time PCR instrument. Confirm that the threshold and baseline that are automatically determined by software are appropriate in each well.
8. Check the threshold cycle of target miRNAs in each sample. The threshold cycle is the intersection between an amplification curve and a threshold line. Then, normalize the expression level of target miRNAs against RNU6-2 as an endogenous control, and calculate the relative expression level of target miRNAs using the 2^−ΔΔCT method¹³.
   NOTE: It should be noted that the stability of the expression level of endogenous control miRNA should be verified. In this experiment, RNU6-2 was confirmed to be expressed at high level and relatively invariant across each group.