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

Summary

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

Abstract

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3' untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA's translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

Introduction

MicroRNAs (miRNAs) — the short, noncoding RNAs that cause the degradation and transcriptional inhibition of messenger RNA (mRNA)¹ — have been shown to regulate the expression of various mRNAs that have crucial roles in both physiology and disease (e.g., inflammation, fibrosis, metabolic disorders, and cancer). Some of the miRNAs may therefore be candidate novel biomarkers and therapeutic targets for a variety of diseases^2,3,4,5. Although miRNA expression profiles in mouse organs and tissues including brain, heart, lung, liver, and kidney have been described^6,7,8,9,10, there have been no standard methods for the extraction and evaluation of miRNAs in mouse kidney with renal interstitial fibrosis.

We have designed a protocol to reliably purify and detect the expressions of miRNAs in the kidneys of mice with renal interstitial fibrosis. The protocol involves five main steps, as follows. (1) 8-week-old C57BL/6 male mice are divided into groups of mice that undergo a sham-operation (controls) and mice that are subjected to a surgery providing unilateral ureteral obstruction (UUO), which is linked to renal interstitial fibrosis. (2) Kidney samples are extracted from the sham and UUO mice, homogenized separately in a silicon homogenizer, and then transferred to a biopolymer-shredding system on a microcentrifuge spin column^11,12. (3) The total RNA containing miRNA is extracted from the kidney samples by a silica membrane-based spin column^12,13. (4) Using this extracted total RNA, complementary DNA (cDNA) is synthesized from the total RNA with the use of reverse transcriptase, poly(A) polymerase, and oligo-dT primer^14,15. (5) The expressions of miRNAs are evaluated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) using an intercalating dye^14,15.

This protocol is based on investigations that obtained meaningful extractions and evaluations of miRNAs in a variety of tissues^{11,12,13,14,15}, and the biopolymer-shredding system used in the protocol was shown to purify high-quality, total RNA from tissues in 2006¹². In addition, prior studies have confirmed the accuracy and sensitivity of aspects of the protocol (i.e., the cDNA synthesis with reverse transcriptase, poly(A) polymerase, and oligo-dT primers from extracted total RNA) for the determination of miRNA expression by qRT-PCR with an intercalating dye^14,15. Since the new protocol has the advantages of simplicity, time-saving, and the reduction of technical errors, the protocol can be used in research that requires the accurate and sensitive identification of the miRNA profile in mouse kidney. Moreover, the protocol could be applied to investigations of many pathological conditions.

We next describe the determination of the miRNA expression profiles in mice with UUO, which is linked to renal interstitial fibrosis. In humans, renal interstitial fibrosis is a common and important feature of both chronic kidney disease and end-stage renal disease, regardless of their etiology^16,17. This renal interstitial fibrosis is associated with the progression of renal failure, and it is characterized by increased expressions of extracellular matrix components in the interstitial spaces (e.g., collagen, fibronectin, and α-smooth muscle actin)^17,18.

Protocol

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. The sham surgery

1. Prepare the following items: isoflurane, cork sheet, depilatory cream, laboratory wipes, Petri dish with phosphate-buffered saline (PBS), 4-0 nylon, tweezers, surgical scissors, cotton swabs, and 8-week-old C57BL/6 male mice.
2. Anesthetize a mouse with 1.5% isoflurane and maintain at 1.5%. Then, apply depilatory cream to the mouse's abdomen. After a few minutes, wipe the depilatory cream off with a PBS-soaked laboratory wipe.
3. Inject 70% ethanol into the mouse's abdomen and then place the mouse on the cork sheet in the supine position.
4. Using surgical scissors and tweezers, 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.
5. Moisten two cotton swabs with PBS and then pull the intestines carefully to the side. Place the moistened swabs to identify the left kidney and ureter.
6. Close the peritoneal membrane and then close the incision with 4-0 nylon.

2. The UUO surgery

1. Prepare the following items: isoflurane, cork sheet, depilatory cream, laboratory wipes, Petri dish with PBS, 4-0 silk, 4-0 nylon, a 2.5 mL syringe, cotton swabs, tweezers, surgical scissors, and 8-week-old C57BL/6 male mice.
2. Anesthetize a mouse with 1.5% isoflurane and maintain at 1.5%. Then apply depilatory cream to the mouse's abdomen. After a few minutes, wipe the depilatory cream off with a PBS-soaked laboratory wipe.
3. Inject 70% ethanol into the mouse's abdomen mouse and then place the mouse in the supine position on the cork sheet.
4. Using surgical scissors and tweezers, 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.
5. Place the 2.5 mL syringe underneath the mouse. Take two cotton swabs and moisten them with PBS. Pull the intestines carefully to the side with the tweezers, and place the moistened swabs appropriately to identify the left ureter. Using the tweezers, lift the left kidney.
6. Use the 4-0 silk to ligate the left ureter in two places approx. 1 cm 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.

3. Collection of kidney samples

1. Prepare the following: 1.5 mL microcentrifuge tubes, isoflurane, cork sheet, 70% ethanol, Petri dish with PBS, tweezers, and surgical scissors.
2. Anesthetize a mouse with 1.5% isoflurane and maintain at 1.5%. Inject 70% ethanol into its abdomen and put the mouse on the cork sheet in the supine position.
3. Using surgical scissors and tweezers, 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.
4. Lift up the peritoneal membrane with the tweezers. With the surgical scissors, make a sideways incision at the upper edge of the peritoneal membrane, and continue the incision along the lowest edge of the ribs.
5. Next, identify the left kidney, reflux it with PBS until the kidney turns yellow-white to wash out blood from vessels, and remove the kidney by cutting the left renal artery and vein with the surgical scissors. Place the kidney in the Petri dish and wash it carefully with PBS.
6. Cut the kidney into 10 mg samples with the surgical scissors and tweezers (10 mg is an appropriate size for the next step). Put each piece of the kidney in its own 1.5 mL microcentrifuge tube and close the tube's cap.
7. Transfer each microcentrifuge tube into liquid nitrogen, and keep the tubes at −80 °C for long-term storage before use.

4. Extraction of total RNA from the kidney samples

1. Prepare the following items: 1.5 mL microcentrifuge tubes, 2.0 mL microcentrifuge tubes, 100% ethanol, chloroform, silicon homogenizer, ice, a vortex mixer, biopolymer spin columns in 2.0 mL collection tubes^11,12, membrane-anchored spin columns in 2.0 mL collection tubes^12,13, phenol/guanidine-based lysis reagent, wash buffer containing guanidine and ethanol (wash buffer 1), wash buffer containing ethanol (wash buffer 2), and RNase-free water.
2. Put a 10 mg kidney sample in the silicon homogenizer. Add 700 µL of the phenol/guanidine-based lysis reagent in the homogenizer.
3. Prepare the homogenizer and then slowly press/twist the homogenizer's pestle against the kidney sample to homogenize the sample. Repeat the pressing/twisting until the kidney sample is completely dissolved in the phenol/guanidine-based lysis reagent.
4. To further homogenize the sample, transfer the homogenized lysate (in a 2.0 mL collection tube) to the biopolymer spin column.
5. Spin the homogenized lysate at 14,000 x g for 3 min at room temperature (RT), and then transfer the precipitated lysate to an unused 1.5 mL microcentrifuge tube.
6. Combine the lysate in the tube with 140 µL of chloroform and then close the tube cap tightly. To mix the lysate and chloroform, invert the tube 15 times.
   NOTE: The chloroform can be used without a hood.
7. Incubate each sample for 2–3 min at RT and then spin each sample at 12,000 x g for 15 min at 4°C.
8. Without disturbing the precipitate, transfer the supernatant (which is typically ~300 µL) to a new 1.5 mL microcentrifuge tube, and then add 1.5x its volume (typically ~450 µL) of 100% ethanol. Vortex the mixture for 5 s.
9. Load 700 µL of the sample onto a membrane-anchored spin column in a 2.0 mL collection tube. Close the column cap and spin the column at 15,000 x g for 15 s. Throw away the precipitated lysate in the collection tube.
10. Wash the sample thoroughly by adding 700 µL of wash buffer 1 to the membrane-anchored spin column in a 2.0 mL collection tube. Close the column cap, and spin the column at 15,000 x g for 15 s. Throw away the precipitated lysate in the collection tube.
11. Load 500 µL of wash buffer 2 onto the membrane-anchored spin column in a 2.0 mL collection tube to remove trace salts. Close the column cap, and spin the column at 15,000 x g for 15 s. Throw away the precipitated lysate in the collection tube.
    NOTE: A membrane-anchored spin column can separate RNA and DNA.
12. Perform step 4.11 again.
13. Spin the membrane-anchored spin column in a 2.0 mL collection tube again at 15,000 x g for 1 min. Throw away the precipitated lysate in the collection tube.
14. Transfer the membrane-anchored spin column to a new 1.5 mL collection tube. Dissolve total RNA by adding 30 µL of RNase-free water to the column. Close the column cap, and wait 5 min at room temperature. Then, spin the column at 15,000 x g for 1 min.
15. Transfer the total amount of sample containing total RNA to a new microcentrifuge tube. Put each of the tubes on ice, and measure the concentration of total RNA by spectrophotometry.
16. Keep the tubes with samples at −80 °C for long-term storage before use.

5. Synthesis of cDNA with the reverse transcription of total RNA

NOTE: The MIQE (Minimum Information for the Publication of Quantitative Real-Time PCR Experiments) guidelines were issued to encourage better experimental practices and help obtain reliable and unequivocal results¹⁹. In this protocol, cDNA is synthesized from 1.0 µg of purified total RNA in a two-step procedure using reverse transcriptase, poly(A) polymerase, and oligo-dT primer.

1. Prepare the following: 1.5 mL microcentrifuge tubes, eight-well strip tubes with caps, the cap of each eight-strip tube, distilled water, ice, a reverse transcriptase kit (see the Table of Materials)^14,15 in the melted state, a thermal cycler, and a vortex mixer.
2. Start the thermal cycler.
3. Prepare a master mix solution: Add 2.0 µL of reverse transcriptase mix (included in the kit) and 2.0 µL of 10x nucleic acid mix into 4.0 µL of 5x hi-spec buffer (to obtain a total of 8.0 µL master mix per tube).
4. Put 8.0 µL of the master mix solution into each tube of an eight-well strip tube.
5. Adjust the total RNA density. To isolate 1.0 µg of total RNA from the kidney samples in 12 µL of RNase-free water, transfer the appropriate amount of total RNA into distilled water, using the density data measured as described in step 4.15.
   NOTE: If any DNA contamination is present, the contaminated DNA will be co-amplified in the qRT-PCR.
6. Place a 12 µL aliquot of total RNA into each tube and close the tube's cap. Centrifuge the tube for 15 x.
7. Put the tube in the thermal cycler and incubate the sample for 60 min at 37 °C. Next, immediately incubate the sample for 5 min at 95 °C for the synthesis of cDNA.
8. When the incubation is complete, transfer the cDNA into a new 1.5 mL microcentrifuge tube and dilute the cDNA ten times (1:10) with distilled water. Vortex and centrifuge the tube for 5 s.
9. Temporarily store the diluted cDNA on ice and move the samples to −80 °C for long-term storage before use.

6. qRT-PCR of miRNA

NOTE: We used the intercalator method to perform the qRT-PCR of miRNA. Primers for RNA are U6 small nuclear 2 (RNU6-2), miRNA-3070-3p, miRNA-6401, miRNA-7218-5p, and miRNA-7219-5p were used.

1. Prepare the following: 1.5 mL microcentrifuge tubes, a vortex mixer, a 96-well reaction plate for the qRT-PCR, adhesive film for the 96-well reaction plate, an adhesive film applicator, a 96-well centrifuge rotor, miRNA-specific primers, a real-time PCR instrument, and a green dye-based PCR kit (see the Table of Materials)^14,15 containing 2x PCR master mix and 10x universal primer.
2. After mixing them in 1.5 mL microcentrifuge tubes, vortex the following items: 6.25 µL of distilled water, 1.25 µL of each 5 µM miRNA primer dissolved in nuclease-free water, 12.5 µL of 2× PCR master mix, and 2.5 µL of 10x universal primer.
3. Prepare the cDNA synthesized as described in step 5, and melt it. Vortex and centrifuge the cDNA for 5 s.
4. Put a 22.5 µL aliquot of the reagent (created as described in step 6.2) in each well of the 96-well plate.
5. Put a 2.5 µL aliquot of cDNA in each well of the plate.
6. Using the adhesive film applicator, secure the adhesive film tightly on the 96-well plate. Centrifuge the plate with the 96-well centrifuge rotor at 1,000 x g for 30 s to settle the reactions at the bottom of each well.

7. PCR cycling

1. Start the real-time PCR system, and place the plate created as described in step 6.6. in the real-time PCR system. Set the experiment properties next; identify the experiment name. Select the following: "96-well (0.2 mL)" as the system's experiment type, "Comparative CT (ΔΔCT)" as the quantitation method, "SYBR Green Reagents" as the reagents to detect the target sequence, and "standard" as the system's run.
2. Assign a name to the sample and the target miRNA, and then assign a name to the sample and target miRNA in each well. Samples should be assigned in duplicate in order to obtain appropriate data for confirmation of the results. Select a reference sample and an endogenous control, and select "none" for the dye to be used as the passive reference. Make sure to set the negative reverse transcriptase and non-template control for the miRNA expression in order to eliminate the cross-contamination of reagents.
3. Make sure the reaction volume setting is "20 µL" and the PCR cycling conditions are set at 95 °C for 15 min, followed by 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.
4. After the qRT-PCR process is complete, use the system's software program to analyze the qRT-PCR data. Ensure that the threshold line that is automatically selected by the software program is appropriate for each well.
5. Check the threshold cycle (CT) value of the endogenous control and target miRNA analyzed in each sample. The CT value is determined by the intersection of the amplification curve and the threshold line. In the present study, we used RNU6-2 as an endogenous control for the target miRNA expression level, and we used the ΔΔCT method to determine the relative expression level of each target miRNA²⁰.

Results

A UUO mouse model was created by left ureteral ligation as described²¹ in 8-week-old male mice weighing 20–25 g. Ureters were completely obstructed by double ligation with 4-0 silk sutures. An analgesic (meloxicam 5 mg/kg, subcutaneous injection) was administered before surgery and also daily on the 2 days post-surgery. At 8 days post-surgery, kidneys were collected, rinsed with PBS, dissected, and stored in liquid nitrogen for further analysis. Sham-operated mice served as controls. The double li...

Discussion

The above-described protocol with qRT-PCR successfully determined the expression levels of the targeted miRNAs. The assessment of extracted miRNAs is important when seeking to obtain meaningful qRT-PCR data, and in order to confirm the quality of the miRNAs before performing the qRT-PCR, the ratio of absorbance at 260 nm to that at 280 nm should be checked with a spectrophotometer. If a single PCR amplification of the expected length and melting temperature or a monomodal melting curve cannot be obtained by qRT-PCR, ther...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Qiagen	79216	Wash buffer 2
Qiagen	1067933	Wash buffer 1
Tokyo Laboratory Animals Science	Not assigned
Thermo Fisher Scientific	4316813	96-well reaction plate
Thermo Fisher Scientific	4311971	Adhesive film for 96-well reaction plate
Qiagen	MS00001701	5'-UUAAUGCUAAUUGUGAUAGGGGU-3'
Qiagen	MS00065141	5'-UUACACUCCAGUGGUGUCGGGU-3'
Qiagen	MS00068067	5'-UGCAGGGUUUAGUGUAGAGGG-3'
Qiagen	MS00068081	5'-UGUGUUAGAGCUCAGGGUUGAGA-3'
Qiagen	217004	Membrane anchored spin column in a 2.0 mL collection tube
Qiagen	218161	Reverse transcriptase kit
Qiagen	218073	Green dye-based PCR kit
Qiagen	79654	Biopolymer spin columns in a 2.0 mL collection tube
Qiagen	79306	Phenol/guanidine-based lysis reagent
Thermo Fisher Scientific	4472380	Real-time PCR instrument
Thermo Fisher Scientific	4472380	Real-time PCR instrument software
Qiagen	129112
Qiagen	MS00033740	Not disclosed
Takara Bio	9790B	Silicon homogenizer
ASKUL	GA04SW
AS ONE	ER1004NA45-KF2,62 -9968-32