The experimental protocol was approved by the Animal Ethics Committee of Jichi Medical University and performed in accordance with the Jichi Medical University Guide for Laboratory Animals and its guidelines concerning the use and care of experimental animals. This protocol uses four 50-week-old SAMR1 male mice and SAMP1 male mice (40-45 g).
1. Serum sample collection
<ol>
	<li>Prepare the following for each mouse: 30 G needles with a 1.0 mL syringe, a 1.0 mL spitz centrifugation tube with heparin, 1.5 mL microcentrifuge tubes, isoflurane anesthetic, a cork sheet, 70% ethanol, two cotton swabs moistened with phosphate-buffered saline (PBS), a Petri dish with PBS, tweezers, and surgical scissors.</li>
	<li>Anesthetize the mouse with 1.5% isoflurane and maintain at 1.5%. Administer an analgesic (Meloxicam 5 mg/kg) subcutaneously, then inject 1.0 mL of 70% ethanol into its abdomen and place it in the supine position on the cork sheet.</li>
	<li>Confirm the depth of anesthesia by the disappearance of the pedal withdrawal reflex. With the tweezers and surgical scissors, incise the skin of the abdomen. Cut the muscles and the peritoneal membrane from the bladder to the lower-left edge of the ribs.</li>
	<li>Use the tweezers to lift the peritoneal membrane, and make a lateral incision in the upper edge of the peritoneal membrane with the surgical scissors. Continue the incision along the lowest edge of the ribs.</li>
	<li>Identify the inferior vena cava by using the two PBS-moistened cotton swabs. Insert one of the 30 G needles with the 1.0 mL syringe into the inferior vena cava and then pull the syringe. Pull the needle out of the inferior vena cava slowly to avoid hemolysis. Transfer the blood to the 1.0 mL spitz tube with heparin and then mix it by inverting the tube several times.&#160; 
	NOTE: The mouse is euthanized by cervical dislocation.</li>
	<li>Spin the spitz tube at room temperature (RT), 3,000 &#215; g for 10 min.</li>
	<li>Aspirate the supernatant slowly, make sure that it does not contain sediment, and then transfer it to an unused 1.5 mL microcentrifuge tube.</li>
	<li>Store the tube at &#8722;80 &#176;C before use. 
	&#8203;NOTE: If proceeding to Step 3 immediately, the tube does not need to be stored at &#8722;80 &#176;C.</li>
</ol>
2. Kidney sample collection
<ol>
	<li>Prepare the following for each mouse: 2.0 mL cryotubes, isoflurane anesthetic, a cork sheet, 70% ethanol, a Petri dish with PBS, tweezers, and surgical scissors.</li>
	<li>Anesthetize the mouse with 1.5% isoflurane and maintain at 1.5%. Administer an analgesic (Meloxicam 5 mg/kg) subcutaneously, then inject 1.0 mL of 70% ethanol into its abdomen, and place it in the supine position on the cork sheet.</li>
	<li>Confirm the depth of anesthesia by the disappearance of the pedal withdrawal reflex. Use the tweezers and the surgical scissors to make an incision in the abdominal skin. Cut the muscles and peritoneal membrane from the bladder to the lower-left edge of the ribs.</li>
	<li>Use the tweezers to lift the peritoneal membrane and make a lateral incision in the peritoneal membrane&#39;s upper edge with the surgical scissors. Continue the incision along the lowest edge of the ribs.</li>
	<li>Identify the left kidney. Reflux it with PBS to flush the blood from the vessels until the kidney turns a yellowish-white color. Excise the entire kidney first using surgical scissors to sever the left renal artery and vein. Place the kidney in a Petri dish and wash it carefully with PBS.&#160; 
	NOTE: The mouse is euthanized by cervical dislocation.</li>
	<li>Use the tweezers and the surgical scissors to cut the kidney into 10 mg samples (10 mg is a suitable sample size for the next step). Place each sample of the kidney in its own 2.0 mL cryotube. Close the tube&#39;s cap.</li>
	<li>For long-term storage, transfer each cryotube to liquid nitrogen and store it at &#8722;80 &#176;C.</li>
</ol>
3. Total RNA extraction from a serum sample
<ol>
	<li>Prepare the following items first: a vortex mixer, phenol/guanidine-based lysis reagent, 80% ethanol, 100% ethanol, 100% chloroform, biopolymer spin columns (in 2.0 mL collection tubes11), membrane-anchored spin columns (in 2.0 mL collection tubes11), wash buffer #1 (i.e., wash buffer containing guanidine and 100% ethanol in a ratio of 1:2), wash buffer #2 (i.e., wash buffer containing guanidine and 100% ethanol in a ratio of 1:4), RNase-free water, 1.5 mL microcentrifuge tubes, and 2.0 mL microcentrifuge tubes.</li>
	<li>First, take a 200 &#181;L serum sample in a 1.5 mL microcentrifuge tube, and then add 1,000 &#181;L of the phenol/guanidine-based lysis reagent. Vortex the mixture for 5 s.</li>
	<li>Incubate the sample at RT for 5 min.</li>
	<li>Add 200 &#181;L of chloroform to the serum sample in the tube and close the tube cap tightly. Invert the tube 15x to mix the chloroform and the serum sample.</li>
	<li>Each sample is incubated at RT for 3 min. Next, spin the sample at 4 &#176;C for 15 min at 12,000 x g.</li>
	<li>Transfer the 300 &#181;L of supernatant to a new 1.5 mL microcentrifuge tube without disturbing the pellet. Add 450 &#181;L of 100% ethanol, and vortex the tube for 5 s. 
	NOTE: In all the following steps, place the membrane-anchored spin column for the separation of RNA and DNA in a 2.0 mL collection tube to centrifuge it.</li>
	<li>Then, remove 700 &#181;L of the sample, load it onto a membrane-anchored spin column, and close the cap. Spin the column at RT for 15 s at 8,000 &#215; g, and leave the supernatant in the column. Discard the pellet remaining in the collection tube.</li>
	<li>Add 700 &#181;L of wash buffer #1 which is part of the serum/plasma kit (see the Table of Materials) to the membrane-anchored spin column to clean the sample thoroughly. Close the column cap and spin the column at RT for 15 s at 8,000 &#215; g, and leave the supernatant in the column. Discard the pellet remaining in the collection tube.</li>
	<li>For the removal of trace salts, take 500 &#181;L of wash buffer #2 and load it onto a membrane-anchored spin column. After closing the column cap, spin the column at RT for 15 s at 8,000 &#215; g, and leave the supernatant in the column. Discard the pellet in the collection tube.</li>
	<li>For the removal of trace salts, take 500 &#181;L of 80% ethanol and load it onto a membrane-anchored spin column. After closing the column cap, spin the column at RT for 15 s at 8,000 &#215; g, and leave the supernatant in the column. Discard the pellet in the collection tube.</li>
	<li>Spin the membrane-anchored spin column again at RT for 5 min at 15,000 &#215; g.</li>
	<li>After transferring the membrane-anchored spin column to a new 1.5 mL collection tube, add 14 &#181;L of RNase-free water to the column to dissolve the total RNA. After closing the column cap, wait 5 min with the tube left at RT. Spin the column again for 1 min at 15,000 &#215; g at RT.</li>
	<li>Store the tubes with samples at &#8722;80 &#176;C before use.</li>
</ol>
4. Extraction of total RNA from a kidney sample
<ol>
	<li>Prepare the following items first: a silicon homogenizer, ice, a vortex mixer, 100% ethanol, 100% chloroform, biopolymer spin columns (in 2.0 mL collection tubes11), membrane-anchored spin columns (in 2.0 mL collection tubes11), phenol/guanidine-based lysis reagent, wash buffer #1 (the same buffer as Step 3.1.), wash buffer #2 (the same buffer as Step 3.1.), RNase-free water, 1.5 mL microcentrifuge tubes, and 2.0 mL microcentrifuge tubes.</li>
	<li>Place a 10 mg kidney sample in the silicon homogenizer, and add 700 &#181;L of the phenol/guanidine-based lysis reagent.</li>
	<li>Set up the homogenizer. Gently press and twist the homogenizer&#39;s pestle against the kidney sample to homogenize the sample. Continue to press and twist the pestle until the kidney sample is completely homogenized in the phenol/guanidine-based lysis reagent.</li>
	<li>For further homogenization, take the homogenized lysate (in a 2.0 mL collection tube) and transfer it to a biopolymer spin column.</li>
	<li>Centrifuge the homogenized lysate at RT for 3 min at 14,000 &#215; g and then transfer the entire precipitated pellet to an unused 1.5 mL microcentrifuge tube to invert the precipitated pellet</li>
	<li>Mix the pellet with 140 &#181;L of chloroform in the tube; then, close the tube cap tightly. Invert the tube 15x to mix the lysate and chloroform. 
	NOTE: The chloroform can be used safely without a hood.</li>
	<li>Incubate the sample at RT for 2-3 min. Centrifuge the sample at 4 &#176;C for 15 min at 12,000 &#215; g.</li>
	<li>Transfer the supernatant (which is usually ~300 &#181;L) to a new 1.5 mL microcentrifuge tube, without disturbing the precipitate. Add 1.5 times its volume (which is usually ~450 &#181;L) of 100% ethanol. Vortex the mixture for 5 s. 
	NOTE: In all the following steps, place the membrane-anchored spin column for separating RNA and DNA in a 2.0 mL collection tube to centrifuge it.</li>
	<li>Load 700 &#181;L of sample onto one of the membrane-anchored spin columns. Close the cap and centrifuge the column at 15,000 &#215; g for 15 s. Discard the precipitated lysate remaining in the collection tube.</li>
	<li>Add 700 &#181;L of wash buffer #1 to the spin column to thoroughly wash it. Close the cap and centrifuge the column at 15,000 &#215; g for 15 s. Discard the precipitated lysate remaining in the collection tube.</li>
	<li>For the removal of trace amounts of salt, load 500 &#181;L of wash buffer #2 onto the membrane-anchored spin column. After closing the column cap, centrifuge the column at 15,000 &#215; g for 15 s. Discard the precipitated lysate in the collection tube.</li>
	<li>Repeat Step 4.11.</li>
	<li>Centrifuge the membrane-anchored spin column again for 1 min at 15,000 &#215; g. Discard the precipitated lysate in the collection tube.</li>
	<li>Take the membrane-anchored spin column and transfer it to a new 1.5 mL collection tube. Add 30 &#181;L of RNase-free water to the column to dissolve the total RNA. After closing the column&#39;s cap, wait for 5 min with the tube at RT, and then centrifuge the column for 1 min at 15,000 &#215; g.</li>
	<li>Transfer the total volume of the sample containing total RNA to a new microcentrifuge tube. Place the tube on ice, and measure the total RNA concentration by spectrophotometry. Make sure that the total RNA concentration is ~300-1,500 ng/&#181;L.</li>
	<li>Store tubes containing samples at &#8722;80 &#176;C before use.</li>
</ol>
5. Synthesis of cDNA with the reverse transcription of total RNA in serum
NOTE: The minimum information for the publication of quantitative real-time PCR experiments (MIQE) guidelines recommend using better experimental practices to obtain reliable, unequivocal results17. In this protocol, cDNA is synthesized from the total RNA purified in a two-step procedure using reverse transcriptase, poly(A) polymerase, and oligo-dT primers.
<ol>
	<li>Prepare the following first: a vortex mixer, a thermal cycler, eight-well strip tubes, the cap of each eight-strip tube, distilled water, ice, the reverse transcriptase kit (see the Table of Materials)13,14 in the melted state, and 1.5 mL microcentrifuge tubes.</li>
	<li>Start the thermal cycler.</li>
	<li>Prepare the master mix solution; to obtain a total of 8.0 &#181;L of master mix per eight-well strip tube, add 2.0 &#181;L of reverse transcriptase mix (included in the kit) and 2.0 &#181;L of 10x nucleic acid mix to 4.0 &#181;L of reverse-transcription buffer in a 1.5 mL microcentrifuge tube.</li>
	<li>Place 8.0 &#181;L of the master mix solution in each tube of an eight-well strip tube.</li>
	<li>Place a 12 &#181;L aliquot of total RNA in each tube of the eight-well strip tube, and close the tube&#39;s cap. Centrifuge the tube for 15 s at RT and 2,000 &#215; g.</li>
	<li>Place the tube in the thermal cycler and incubate for 60 min at 37 &#176;C. Incubate the sample for an additional 5 min at 95 &#176;C min to synthesize the cDNA.</li>
	<li>Following the incubation, transfer the cDNA to a new 1.5 mL microcentrifuge tube. Dilute the cDNA tenfold (1:10) with distilled water. Vortex and centrifuge the tube for 5 s at RT and 2,000 &#215; g.</li>
	<li>Store the diluted cDNA temporarily on ice. Store the diluted samples at &#8722;80 &#176;C before use.</li>
</ol>
6. Synthesis of cDNA with the reverse transcription of total RNA in kidney
NOTE: The MIQE guidelines encourage better experimental practices to ensure reliable and unequivocal results17. This protocol uses reverse transcriptase, poly(A) polymerase, and oligo dT primers to synthesize cDNA from 1.0 &#181;g of purified total RNA in a two-step procedure.
<ol>
	<li>First, prepare the following: a vortex mixer, a thermal cycler, 1.5 mL microcentrifuge tubes, eight-well strip tubes, the cap of each eight-strip tube, distilled water, ice, and a reverse transcriptase kit (see the Table of Materials)13,14 in the melted state.</li>
	<li>Start the thermal cycler.</li>
	<li>Prepare the master mix solution; to obtain a total of 8.0 &#181;L of master mix solution per tube, add 2.0 &#181;L of the reverse transcriptase mix that is included in the kit and 2.0 &#181;L of 10x nucleic acid mix to 4.0 &#181;L of the reverse-transcription buffer.</li>
	<li>Add 8.0 &#181;L of the master mix solution to each tube of an eight-well strip tube.</li>
	<li>Adjust the total RNA density as follows. For the separation of 1.0 &#181;g of total RNA from a kidney sample with 12 &#181;L of RNase-free water, take a suitable amount of total RNA and transfer it to distilled water by using the concentration measured as described above (in Step 4.15.). 
	&#8203;NOTE: In the presence of DNA contamination, the contaminated DNA is co-amplified by qRT-PCR.</li>
	<li>Perform the same process as that described above in Steps 5.5.-5.8.</li>
</ol>
7. The qRT-PCR of miRNA
NOTE: An intercalator method is used for the qRT-PCR of the miRNAs. Primers are used for RNA: U6 small nuclear 2 (RNU6-2), miRNA-223-3p, miRNA-423-5p, miRNA-7218-5p, and miRNA-7219-5p.
<ol>
	<li>Prepare the following: a vortex mixer, a real-time PCR system, a 96-well reaction plate for qRT-PCR, adhesive film for the 96-well reaction plate, adhesive film applicator, a 96-well centrifuge rotor, miRNA-specific primers, a green dye-based PCR kit containing 2x PCR master mix and 10x universal primer (see the Table of Materials)13,14, and a 1.5 mL microcentrifuge tube.</li>
	<li>Vortex the following after mixing them in a 1.5 mL microcentrifuge tube: 6.25 &#181;L of distilled water, 1.25 &#181;L each of 5 &#181;M miRNA primer dissolved in nuclease-free water, 12.5 &#181;L of 2x PCR master mix, and 2.5 &#181;L of 10x universal primer.</li>
	<li>Prepare and melt the cDNA synthesized as described in Step 5 (serum) or Step 6 (kidney). Vortex and centrifuge the cDNA for 5 s.</li>
	<li>Take 22.5 &#181;L aliquots of the reagent (as described in Step 7.2. above) and place them separately in each well of the 96-well plate.</li>
	<li>Add a 2.5 &#181;L aliquot of cDNA to each well of the plate.</li>
	<li>To secure the adhesive film to the plate, use the adhesive film applicator. Centrifuge the plate for 30 s at 1,000 x g in the 96-well centrifuge rotor. Stabilize the reaction at the bottom of each well.</li>
</ol>
8. Using the real-time PCR system and software to run the PCR cycling program
<ol>
	<li>Begin the real-time PCR system. Place the plate created as described in Step 7.6. in the real-time PCR system. Modify the settings; provide a name for the experiment, and then select 96-well (0.2 mL) as the system&#39;s experiment type, Comparative CT (&#916;&#916;CT) as the quantitation method, standard as the system run mode, and SYBR Green Reagents as the reagents for detecting the target sequence.</li>
	<li>Provide names for the sample and the target miRNAs, and name the sample and target miRNA in each well. Assign duplicate samples to obtain data suitable for confirming the results, and choose a reference sample and the endogenous control. For the dye to use as the passive reference, select none. To eliminate reagent cross-contamination, set up negative reverse transcriptase and a non-template control for miRNA expression.</li>
	<li>Next, make sure that the reaction volume is set to 20 &#181;L and the PCR cycling conditions are set as follows: 95 &#176;C for 15 min, then 40 cycles of denaturation at 94 &#176;C for 15 s, annealing at 55 &#176;C for 30 s, and lastly extension at 70 &#176;C for 30 s.</li>
	<li>Click on analyze in the system&#39;s software program to analyze the qRT-PCR data after the process is complete. Confirm that the threshold line automatically selected by the program is appropriate for each well.</li>
	<li>Check the threshold cycle (CT) 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. 
	NOTE: In the present study, RNU6-2 and miRNA-423-5p were used as the endogenous controls for target miRNA expression levels, and the &#916;&#916;CT method was used to determine the relative expression levels of each target miRNA18.</li>
</ol>

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The authors declare that they have no conflicts of interest.

The expression levels of the target miRNAs were successfully determined by the above-described protocol using qRT-PCR. The evaluation of the extracted miRNAs is an important step in obtaining meaningful qRT-PCR data. To confirm the adequate quality of miRNAs before performing the qRT-PCR, spectrophotometry should be used to determine the ratio of absorbance at 260 nm to that at 280 nm. DNA contamination may occur, and/or primer dimers in each well of the reaction plate may be present if the qRT-PCR does not provide a single PCR amplification of the expected length and melting temperature, or if it provides a monomodal melting curve.
MiRNA expression levels can be evaluated by several methods other than qRT-PCR, including northern blotting, a microarray, and ribonuclease protection assays. However, the qRT-PCR method is a sensitive, accurate, simple, and reproducible procedure that requires a smaller sample volume than those required for northern blotting and ribonuclease protection assays19. Since microarrays can measure the expression of tens of thousands of miRNAs simultaneously, they can be used to identify candidate miRNA markers. Microarray data also show a high overall correlation with data obtained by qRT-PCR20. However, no consensus has been reached regarding the optimal methodology for comparing microarray data obtained in different studies21.
The evaluation of serum miRNAs has the following features. First, it is easy to collect serum, and as serum miRNAs are stable against freezing and thawing, temperature, and acid, the miRNAs can be good biomarkers. Second, there is high homology of miRNAs among species, and the results of animal experiments are easily extrapolated to humans. Third, serum miRNAs have shown potential for use as therapeutic drugs3. Several studies have also demonstrated that the level of expression of miRNA in organs is correlated with miRNA in serum22,23,24. In the present study, miRNA-223-3p, the kidney levels of which did not show a significant difference between the SAMR1 and SAMP1 mice, also showed no significant between-strain difference in the serum. In contrast, miRNA-7218-5p and miRNA-7219-5p, the kidney levels of which showed a significant difference between the SAMR1 and SAMP1 mice, showed considerable between-strain differences in the serum.
This protocol has the following limitations. First, its usefulness has not been verified in other organs such as the liver and lung, and second, it has not been tested on other laboratory animals such as rats, dogs, and pigs. Several research groups have used this protocol for the purification and detection of miRNAs by qRT-PCR and reported that this protocol enabled the purification of high-quality RNA from tissues and serum13,14,22,23,24. This method has been demonstrated to have high accuracy and sensitivity for detecting the expression of miRNAs13,14,22,23,24. The present study&#39;s results demonstrate that this protocol can successfully detect miRNA expression in the serum and kidney of mice. Therefore, the protocol can be used to determine the serum and kidney miRNA expression profiles in mice with a variety of pathologies. Due to the protocol&#39;s simplicity, a large number of samples can be processed simultaneously. Analyses of the expression of many miRNAs in various pathological conditions of the kidney can, thus, use the protocol described herein.
There are certain aspects of the protocol to keep in mind. To avoid the degradation of the purified miRNAs that would occur at room temperature, the miRNAs must be kept on ice. The kidney samples must be homogenized until they are completely dissolved in the lysis reagent. Mouse kidney contains a substantial amount of connective tissue that is insoluble in lysis reagent, and thus a column shredder is required for further homogenization. In addition, the appropriate endogenous control miRNA (with stable expression among the samples) should be validated throughout the setup of a qRT-PCR experiment. This is because the interference of various substances during the performance of this protocol can alter the expression levels of endogenous control miRNAs, possibly compromising the results. In conclusion, this paper describes a qRT-PCR protocol for the detection, purification, and evaluation of miRNA expression in the serum and kidney of mice with age-dependent renal impairment.

The expression of various mRNAs that play important roles in both physiology and disease (e.g., inflammation, fibrosis, metabolic disorders, and cancer) is known to be regulated by miRNAs, which are short, noncoding RNAs that cause the degradation and inhibit the transcription of mRNA1. It is, therefore, possible that certain miRNAs could serve as new candidatebiomarkers and/or therapeutic targets for various diseases2,3,4,5. Research has been conducted on miRNA expression profiles in a variety of mouse organs and tissues (including brain6, heart7, lung8, liver9, and kidney10). However, there are no standard or established methods for extracting and evaluating miRNAs in the kidneys or serum of mice with age-dependent renal impairment.
Therefore, we established a protocol that can be used to reliably purify and detect miRNA expression in the serum and kidney of mice with age-dependent renal impairment. There are six main steps in the protocol: (1) preparation of both 50-week-old SAMR1 male mice and SAMP1 male mice; (2) extraction of blood samples from the inferior vena cava of both strains of mice, with the subsequent use of a spitz tube with heparin, followed by centrifugation, to obtain a serum sample; (3) kidney sample extraction from the mice-a silicon homogenizer is used to homogenize the kidney sample separately, and the sample is then transferred to a biopolymer-shredding system on a microcentrifuge spin column11; (4) total RNA (containing miRNA) extraction from the serum samples with the use of a silica membrane-based spin column12 and total RNA containing miRNA extraction from the kidney samples with the use of a silica membrane-based spin column11; (5) synthesis of complementary DNA (cDNA) from the total RNA using reverse transcriptase, poly(A) polymerase, and oligo-dT primer13,14; and (6) finally, determination of miRNA expression using qRT-PCR and an intercalating dye13,14.
This new protocol was based on studies that succeeded in extracting and evaluating miRNAs in various types of tissue11,12,13. The protocol&#39;s biopolymer-shredding system was demonstrated to be able to purify high-quality total RNA from tissues11. The accuracy and sensitivity of aspects of this protocol used for the evaluation of miRNA expression by qRT-PCR with an intercalating dye have been established13,14, for example, the cDNA synthesis with reverse transcriptase, poly(A) polymerase, and oligo-dT primers from the extracted total RNA. The new protocol has several advantages: simplicity, time savings, and reduced technical errors. It can, thus, be used for investigations that require accurate and sensitive identification of kidney and serum miRNA profiles. Studies of many pathological conditions can also utilize the new protocol.
The miRNA expression profiles in SAMP1 mice, which are a model of age-dependent renal impairment, can be determined as shown below. In humans, age-dependent renal impairment is associated with the progression of renal failure and is characterized by both an increase in the area of renal interstitial fibrosis and the progression of glomerulosclerosis15,16. Age-dependent renal impairment is also an important and frequent feature of chronic kidney disease and end-stage renal disease15,16.

<table><tbody><tr><td>1.0 mL spitz with heparin</td><td>Greiner-bio-one</td><td>450534</td><td></td></tr><tr><td>Buffer RPE (wash buffer #2 containing guanidine and ethanol in ratio of 1:4)</td><td>Qiagen</td><td>79216</td><td>Wash buffer 2</td></tr><tr><td>Buffer RWT (wash buffer #1 containing guanidine and ethanol in ratio of 1:2)</td><td>Qiagen</td><td>1067933</td><td>Wash buffer 1</td></tr><tr><td>MicroAmp Optical 96-well reaction plate for qRT-PCR</td><td>Thermo Fisher Scientific</td><td>4316813</td><td>96-well reaction plate</td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Thermo Fisher Scientific</td><td>4311971</td><td>Adhesive film for 96-well reaction plate</td></tr><tr><td>miRNA-223-3p primer</td><td>Qiagen</td><td>MS00003871</td><td>5&#039;-CGUGUAUUUGACAAGCUGAGUU&lt;br/&gt; G-3&#039;</td></tr><tr><td>miRNA-423-5p primer</td><td>Qiagen</td><td>MS00012005</td><td>5&#039;-UGAGGGGCAGAGAGCGAGACU&lt;br/&gt; UU-3&#039;</td></tr><tr><td>miRNA-7218-5p primer</td><td>Qiagen</td><td>MS00068067</td><td>5&#039;-UGCAGGGUUUAGUGUAGAGGG&lt;br/&gt; -3&#039;</td></tr><tr><td>miRNA-7219-5p primer</td><td>Qiagen</td><td>MS00068081</td><td>5&#039;-UGUGUUAGAGCUCAGGGUUGA&lt;br/&gt; GA-3&#039;</td></tr><tr><td>miRNeasy Mini kit</td><td>Qiagen</td><td>217004</td><td>Membrane anchored spin column in a 2.0 mL collection tube</td></tr><tr><td>miRNeasy Serum/Plasma kit</td><td>Qiagen</td><td>217184</td><td>Membrane anchored spin column in a 2.0 mL collection tube</td></tr><tr><td>miScript II RT kit (reverse transcription buffer)</td><td>Qiagen</td><td>218161</td><td>Reverse transcriptase kit</td></tr><tr><td>miScript SYBR Green PCR kit</td><td>Qiagen</td><td>218073</td><td>Green dye-based PCR kit</td></tr><tr><td>QIA shredder</td><td>Qiagen</td><td>79654</td><td>Biopolymer spin columns in a 2.0 mL collection tube</td></tr><tr><td>QIAzol Lysis Reagent (phenol/guanidine-based lysis reagent)</td><td>Qiagen</td><td>79306</td><td>Phenol/guanidine-based lysis reagent</td></tr><tr><td>QuantStudio 12K Flex Flex Real-Time PCR system</td><td>Thermo Fisher Scientific</td><td>4472380</td><td>Real-time PCR instrument</td></tr><tr><td>QuantStudio 12K Flex Software version 1.2.1.</td><td>Thermo Fisher Scientific</td><td>4472380</td><td>Real-time PCR instrument software</td></tr><tr><td>RNase-free water</td><td>Qiagen</td><td>129112</td><td></td></tr><tr><td>RNU6-2 primer</td><td>Qiagen</td><td>MS00033740</td><td>Not disclosed due to confidentiality</td></tr><tr><td>SAMP1 male mice</td><td>Nippon SLC Corporation</td><td>Not assigned</td><td></td></tr><tr><td>SAMR1 male mice</td><td>Nippon SLC Corporation</td><td>Not assigned</td><td></td></tr><tr><td>Takara biomasher standard</td><td>Takara Bio</td><td>9790B</td><td>Silicon homogenizer</td></tr></tbody></table>

quantitative real-time polymerase chain reaction evaluation of microrna expression in kidney and serum of mice with age-dependent renal impairment

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of their target messenger RNAs (mRNAs) by destabilizing them and disrupting their translation. Although the miRNA expression profiles in various mouse organs and tissues have been investigated, there have been no standard methods for purifying and quantifying mouse kidney and serum miRNAs. We have established an effective and reliable method for extracting and evaluating the miRNA expression in the serum and kidney of mice with age-dependent renal impairment. 
The method uses quantitative reverse-transcription-polymerase chain reaction (qRT-PCR), and the protocol requires six steps: (1) preparing senescence-accelerated mouse resistance 1 (SAMR1) mice and senescence-accelerated mouse prone (SAMP1) mice; (2) extracting serum samples from these mice; (3) extracting a kidney sample from each mouse; (4) extracting total RNA (including miRNA) from kidney and serum samples from each mouse; (5) the synthesis of complementary DNA (cDNA) with reverse transcription from the miRNA; (6) conducting a qRT-PCR using the cDNA obtained. 
This protocol was used to confirm that, compared to the controls, the expression of miRNA-7218-5p and miRNA-7219-5p was significantly changed in the kidney and serum of a mouse model of age-dependent renal impairment. This protocol also clarified the relationship between the kidney and serum of the mouse model of age-dependent renal impairment. This protocol can be used to determine miRNA expression in the kidney and serum of mice with age-dependent renal impairment.

For the age-dependent renal impairment mouse model, we used 50-week-old SAMP1 male mice weighing 40-45 g. Approximately 0.8 mL of blood was collected per mouse and transferred into a 1.0 mL spitz tube with heparin, inverted, and centrifuged. Each kidney was rinsed with PBS, dissected, and stored in liquid nitrogen for further analysis. Fifty-week-old SAMR1 mice served as controls. Based on the miRNA qRT-PCR data obtained using this age-dependent renal impairment model, we observed that the kidney level of miRNA-7219-5p was significantly increased and the kidney level of miRNA-7218-5p was considerably decreased in the SAMP1 mice compared to the controls (Figure 1). The serum levels of both miRNA-7219-5p and miRNA-7218-5p were considerably increased in the SAMP1 mice compared to the controls (Figure 2). The expression levels of miRNA-223-3p did not change in either strain and between kidney and serum (Figure 1 and Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63258/63258fig01.jpg" /> 
Figure 1: Differentially expressed microRNAs in the kidneys of SAMP1 mice. qRT-PCR analysis of the expression of miRNA-223-3p, miRNA-7218-5p, and miRNA-7219-5p in SAMR1 mice (control, n = 4) and SAMP1 mice (n = 4). The data are mean &#177; standard error (error bars); t-tests were used to analyze between-group differences; p &#60; 0.05 was considered significant (*p &#60; 0.05), n.s.: not significant. Abbreviations: miRNA = microRNA; SAMP1 = senescence-accelerated mouse prone; SAMR1 = senescence-accelerated mouse resistance 1; qRT-PCR = quantitative reverse-transcription-polymerase chain reaction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63258/63258fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63258/63258fig02.jpg" /> 
Figure 2: Differentially expressed miRNAs in the serum of SAMP1 mice. qRT-PCR analysis of the expression of miRNA-223-3p, miRNA-7218-5p, and miRNA-7219-5p in SAMR1 mice (control, n = 4) and SAMP1 mice (n = 4). The data are mean &#177; SE (error bars); t-tests were used to investigate significant differences between groups. *p &#60; 0.05 by t-test. Abbreviations: miRNA = microRNA; SAMP1 = senescence-accelerated mouse prone; SAMR1 = senescence-accelerated mouse resistance 1; qRT-PCR = quantitative reverse-transcription-polymerase chain reaction <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63258/63258fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment at JoVE.com

Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment

We present a method for evaluating microRNA expression in the kidney and serum of mice with age-dependent renal impairment by quantitative reverse-transcription polymerase chain reaction.

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of ...

quantitative-real-time-polymerase-chain-reaction-evaluation-microrna

Nanyang Technological University

Research

JoVE Journal

Genetics

2.2K Views.  Jichi Medical University. Deze qRT-PCR-methode kan vragen beantwoorden met betrekking tot microRNA-expressieprofilering in serum van muizen in aandoeningen zoals leeftijdsafhankelijke nierinsufficiëntie. De techniek maakt de detectie van microRNA-expressie met hoge nauwkeurigheid en gevoeligheid mogelijk door een eenvoudig proces dat tijd bespaart en technische fouten voorkomt. Om te beginnen, snijd de huid van de buik met behulp van een pincet en een chirurgische schaar.Snijd de spieren in het peritoneale mem...

Video: Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Bioengineering

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

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

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

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&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;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.

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.

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Medicine

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

Immunology and Infection

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Biology

A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, standard protocols for the purification of miRNAs and detection of their expression in the kidneys of acute kidney injury (AKI) mice have not been well established. This study developed an effective and simple protocol to purify and quantify miRNAs in the kidneys of an AKI mouse model induced by renal ischemia-reperfusion using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). This protocol comprises five steps: 1) induction of AKI by renal ischemia-reperfusion, 2) harvesting of kidneys, 3) purification of total RNA, including miRNAs, from kidneys, 4) cDNA synthesis by reverse transcription of miRNA, and 5) qRT-PCR to detect miRNA expression. Using this protocol, the renal ischemia-reperfusion injury model can be generated with mild to severe forms of AKI. Additionally, if the procedure is followed properly, a consistent AKI model with minimal individual differences can be obtained. This qRT-PCR assay shows a very wide dynamic range and enables the discrimination of mature miRNAs, which can be accurately quantified with high specificity. This protocol can be used to study the miRNA expression profile in AKI kidneys.

This article presents a protocol for detecting microRNA expression in the kidneys of an acute kidney injury mouse model using quantitative real-time reverse-transcription polymerase chain reaction. This protocol emphasizes an ischemic kidney injury mouse model and the careful extraction of microRNA samples.

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...

Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment

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