Name: detection of microrna expression in peritoneal membrane of rats using quantitative real-time pcr
Uploaded: 2017-06-27T14:00:00.000+00:00
Duration: 8 min 56 s
Description: Watch this Scientific Journal Video about Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR at JoVE.com

We would like to thank Miyako Shigeta for her excellent technical support. This work was partially supported by JSPS KAKENHI (grant number 25461252).

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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Make a longitudinal incision in the abdominal skin, muscle, and peritoneal membrane using the scissors and forceps.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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).</li>
	<li>Put a 20 mg peritoneal membrane sample into a glass homogenizer and add 700 &#181;L of the phenol/guanidine-based lysis reagent.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>Transfer the homogenized lysate to a new microcentrifuge tube.</li>
	<li>Add 140 &#181;L of chloroform to the homogenized lysate and cap the tube securely. Then, mix the tube by inversion for 15 s.</li>
	<li>Incubate the samples for 2-3 min at room temperature. Then, centrifuge them at 12,000 x g for 15 min at 4 &#176;C.</li>
	<li>Transfer the supernatant (usually 300 &#181;L) to a new microcentrifuge tube without disturbing the precipitate, and add 1.5x its volume (usually 450 &#181;L) of 100% ethanol. Then, mix the sample by vortexing for 5 s.</li>
	<li>Pipette up to 700 &#181;L 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.</li>
	<li>Add 700 &#181;L 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.</li>
	<li>Add 500 &#181;L 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.</li>
	<li>Repeat 2.11.</li>
	<li>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.</li>
	<li>Place the silica-membrane-based spin column in a new 1.5 mL collection tube. Then, transfer 25 &#181;L 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.</li>
	<li>Transfer the 25 &#181;L eluate containing total RNA to a new microcentrifuge tube. Then, store it at &#8722;80 &#176;C before use.</li>
</ol>
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 &#181;g of isolated RNA is reverse-transcribed using the reverse transcriptase, poly(A) polymerase, and oligo-dT primer.
<ol>
	<li>Collect the following items: 1.5 mL microcentrifuge tubes; eight-well strip tubes; RNase-free water; ice; reverse transcriptase kit (see materials table)11.</li>
	<li>Prepare a master mix solution that contains 2.0 &#181;L of 10x nucleic acid mix, 2.0 &#181;L of reverse transcriptase mix (from the kit), and 4.0 &#181;L of 5x hi-spec buffer for a total of 8.0 &#181;L per tube.</li>
	<li>Dispense 8.0 &#181;L of master mix solution (from the kit) into each tube.</li>
	<li>Measure the quantity of total RNAs using a spectrophotometer. Add 1.0 &#181;g 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.</li>
	<li>Add RNase-free water to each tube to a total of 20 &#181;L. Then, mix thoroughly by pipetting and centrifuge it for 15 s.</li>
	<li>Incubate the samples for 60 min at 37 &#176;C. Immediately incubate for 5 min at 95 &#176;C. 
	NOTE: This step can be performed in a thermal cycler.</li>
	<li>Transfer cDNA to a new microcentrifuge tube and dilute ten times (1:10) with RNase-free water.</li>
	<li>Store the diluted cDNA on ice temporarily, and at &#8722;80 &#176;C for long term before use.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Prepare a master mix containing 12.5 &#181;L of 2x PCR master mix, 2.5 &#181;L of 5 &#181;M each miRNA primer dissolved in nuclease-free water, and 1.25 &#181;L of 10x universal primer for each well.</li>
	<li>Dispense 22.5 &#181;L of master mix into each well of the 96-well plate.</li>
	<li>Add 2.5 &#181;L of template cDNA to each well.</li>
	<li>Seal the plate with adhesive film for the 96-well reaction plate. Then, centrifuge the plate at 1,000 x g for 30 s.</li>
	<li>Run the PCR cycling program with the real-time PCR Instrument and software as follows.
	<ol>
		<li>Set the plate in the real-time PCR instrument. In the real-time PCR software, define experimental properties (input experimental name, choose &#34;96-wells&#34; for the experimental type of instrument, choose &#34;2&#8722;&#916;&#916;CT method&#34; for the quantitation method, choose &#34;SYBR green reagents&#34; for the reagent to detect the target sequence, choose &#34;standard ramp speed&#34; for the instrument run).</li>
		<li>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 &#34;none&#34; for the dye to use as the passive reference.</li>
		<li>Input a reaction volume of &#34;20 &#181;L&#34; and the following PCR cycling conditions in the real-time PCR software in accordance with the instructions: preincubation at 95 &#176;C for 15 min, and then 40 cycles of denaturation at 94 &#176;C for 15 s, annealing at 55 &#176;C for 30 s, and extension at 70 &#176;C for 30 s.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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&#8722;&#916;&#916;CT 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.</li>
</ol>

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The authors declare that they have no conflicts of interest.

Using the protocol presented in this manuscript, miRNAs in rat peritoneal membrane were successfully purified and detected using qRT-PCR. The reliability of qRT-PCR data analysis depends on the quality of purified miRNAs. Therefore, the purity of miRNAs may be checked before qRT-PCR by the ratio of absorbance at 260 nm to that at 280 nm, which can be measured using a spectrophotometer. When significant amplification of miRNA cannot be obtained using qRT-PCR, the concentration of template cDNA may be increased. The concentration of each miRNA-specific primer may also be increased.
There are several alternative methods to determine the level of miRNA expression, including microarray, Northern blotting, and ribonuclease protection assay. qRT-PCR is a sensitive, high-throughput procedure that requires a minimal amount of sample as compared with Northern blotting or ribonuclease protection assay. Microarrays enable investigation of the expression of numerous miRNAs simultaneously, and the data generally show a strong correlation with the data obtained by qRT-PCR17; however, no consensus has been reached regarding a methodology that can confirm the validity of comparisons of microarray data between research groups18.
This protocol has the following limitations. First, the validation of miRNA detection by this protocol has not been verified in other organs such as liver, lung, and kidney. Second, it also has not been verified in other experimental animals, including mouse, hamster, dog, and pig. The platform of this protocol for the purification of miRNAs using a biopolymer-shredding spin column and a silica-membrane-based spin column, and detection of miRNAs using qRT-PCR has been reported to be able to purify high-quality RNA from tissues and shows high accuracy and sensitivity for the detection of miRNA expression10,11. We also showed that the detection of miRNA expression in rat peritoneal membrane can be successfully conducted using this protocol. This protocol can be used for studies investigating the profile of miRNA expression in the peritoneal membrane of rats in a wide range of pathological conditions. In addition, many samples can be treated together using this protocol because it involves a simple process. Therefore, this protocol is useful for studies that require analysis of the expression profile of many miRNAs in various pathological conditions of the peritoneal membrane.
Several critical points should be kept in mind when following this protocol. First, samples containing purified RNAs should be placed on ice to avoid degradation. In addition, the step of homogenization should be performed on ice to protect RNAs from heat degradation. Furthermore, peritoneal membrane samples should be adequately homogenized until they have completely dissolved in lysis reagent, and the use of a column shredder for further homogenization is recommended because peritoneal membrane samples contain substantial connective tissue that is resistant to dissolution by homogenization. Second, the stability of the expression level of endogenous control miRNA should be verified across the experimental set-up in qRT-PCR. This is necessary because various pathological conditions in which this procedure can be used may affect the expression levels of endogenous control miRNAs, which can compromise the results.
In conclusion, we have described a protocol for the purification and detection of microRNA expression in rat peritoneal membrane using qRT-PCR.

MicroRNAs (miRNAs) are short, noncoding RNAs that post-transcriptionally regulate messenger RNA (mRNA) expression1. Changes in the expression of miRNAs regulate the expression of many mRNAs that play pivotal roles in various pathological conditions, including cancer, inflammation, metabolic disorders, and fibrosis2,3,4,5,6,7,8. Therefore, miRNAs have potential as novel biomarkers and therapeutic targets2,3,4,5,6,7,8. The miRNA expression profile has been determined in various rat organs and tissues, including liver, heart, lung, and kidney9. However, standard methods for the purification and detection of miRNAs in rat peritoneal membrane have not been well established.
The overall goal of this protocol is to successfully purify and detect miRNAs in the rat peritoneal membrane. First, the peritoneal membrane sample was homogenized using a glass homogenizer, followed by exposure to a biopolymer-shredding system in a microcentrifuge spin column10. Next, total RNA including miRNA was purified from the peritoneal membrane sample using a silica-membrane-based spin column10. Then, cDNA was synthesized from the purified total RNA using reverse transcriptase, poly(A) polymerase, and oligo-dT primer11. Finally, the expression of miRNA was determined by qRT-PCR using an intercalating dye11. The rationale of this protocol is based on previous studies that showed significant purification and detection of miRNA in tissues by a simple process8,10,11. It has been reported that the use of a biopolymer-shredding system in a microcentrifuge spin column and silica-membrane-based spin column can purify high-quality total RNA from tissues10. The method of synthesizing cDNA from purified total RNA using reverse transcriptase, poly(A) polymerase, and oligo-dT primer, and the method of detecting miRNA expression by qRT-PCR using intercalating dye in this protocol have been reported to show high accuracy and sensitivity11. In addition, this is a simple process, which saves time and prevents technical error. Therefore, this protocol is useful in studies that require highly accurate and sensitive detection of miRNA in rat peritoneal membrane in a wide range of pathological conditions.

<table><tbody><tr><td>QIA shredder</td><td>Qiagen</td><td>79654</td><td>biopolymer-shredding system in a micro centrifuge spin-column</td></tr><tr><td>miRNeasy Mini kit</td><td>Qiagen</td><td>217004</td><td>silica-membrane based spin column</td></tr><tr><td>QIAzol Lysis Reagent</td><td>Qiagen</td><td>79306</td><td>phenol/guanidine-based lysis reagent</td></tr><tr><td>Buffer RLT</td><td>Qiagen</td><td>79216</td><td>wash buffer 1</td></tr><tr><td>Buffer RWT</td><td>Qiagen</td><td>1067933</td><td>wash buffer 2</td></tr><tr><td>miScript II RT kit&amp;nbsp;</td><td>Qiagen</td><td>218161</td><td>includes 10&amp;times; Nucleics Mix containing deoxynucleotides, ribonucleotide triphosphates, and oligo-dT primers; miScript Reverse Transcriptase Mix containing poly(A) polymerase and reverse transcriptase and; miScript HiSpec buffer</td></tr><tr><td>miScript SYBR Green PCR kit</td><td>Qiagen</td><td>218073</td><td>includes QuantiTect SYBR Green PCR Master Mix and miScript Universal Primer</td></tr><tr><td>RNU6-2 primer&amp;nbsp;&amp;nbsp;</td><td>Qiagen</td><td>MS00033740</td><td>not disclosed</td></tr><tr><td>miRNA-142-3p primer</td><td>Qiagen</td><td>MS00031451</td><td>5&#039;-UGUAGUGUUUCC&lt;br/&gt; UACUUUAUGGA-3&#039;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>miRNA-21-5p primer</td><td>Qiagen</td><td>MS00009079</td><td>5&#039;-UAGCUUAUCAG&lt;br/&gt; ACUGAUGUUGA-3&#039;</td></tr><tr><td>miRNA-221-3p primer</td><td>Qiagen</td><td>MS00003857</td><td>5&#039;-AGCUACAUUGU&lt;br/&gt; CUGCUGGGUUUC-3&#039;</td></tr><tr><td>miRNA-223-3p primer</td><td>Qiagen</td><td>MS00033320</td><td>5&#039;-UGUCAGUUUG&lt;br/&gt; UCAAAUACCCC-3&#039;</td></tr><tr><td>miRNA-34a-5p primer</td><td>Qiagen</td><td>MS00003318</td><td>5&#039;-UGGCAGUGUCU&lt;br/&gt; UAGCUGGUUGU-3&#039;</td></tr><tr><td>miRNA-327 primer</td><td>Qiagen</td><td>MS00000805</td><td>5&#039;-CCUUGAGGGG&lt;br/&gt; CAUGAGGGU-3&#039;</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&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>4311971</td><td>adhesive film for 96-well reaction plate</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>methylglyoxal</td><td>Sigma-Aldrich</td><td>M0252</td><td></td></tr><tr><td>Midperic</td><td>Terumo</td><td>not assign&amp;nbsp;</td><td>peritoneal dialysis fluid</td></tr><tr><td>Sprague&amp;ndash;Dawley rats</td><td>SLC</td><td>not assign&amp;nbsp;</td><td></td></tr></tbody></table>

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.

The results presented here are based on our previously reported study8. We investigated the miRNA expression profile in peritoneal fibrosis. Peritoneal fibrosis is a major complication in peritoneal dialysis. It is characterized by loss of the mesothelial cell monolayer and the excess accumulation of extracellular matrix components, and is associated with peritoneal membrane failure14,15. A peritoneal fibrosis rat model was produced by the intraperitoneal injection of 100 mL/kg peritoneal dialysis fluid (2.5% glucose, 100 mM NaCl, 35 mM sodium lactate, 2 mM CaCl2, and 0.7 mM MgCl2 containing 20 mM methylglyoxal) for 5 days a week for 3 weeks to male rats, aged 12 weeks and with body weights of 230-250 g16. The following groups served as controls: rats without any treatment (mock rats) and rats injected with peritoneal dialysis fluid without methylglyoxal (control rats). Based on miRNA microarray screening, we found that the levels of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) significantly increased in the peritoneal membrane of peritoneal fibrosis rats compared with those in mock rats and control rats, using qRT-PCR following the protocol presented in this manuscript (Figure 1)8.
<img alt="Figure 1" src="/files/ftp_upload/55505/55505fig1.jpg" /> 
Figure 1: Upregulated miRNAs in the Peritoneal Membrane of Peritoneal Fibrosis Rats. qRT-PCR determination of the expression of miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p in mock rats (n = 6), control rats (n = 6), and peritoneal fibrosis rats (n = 6). Values are the mean &#177; standard error (error bars). Analysis of variance (ANOVA) was employed to investigate differences among groups. If statistical significance was detected by ANOVA, Tukey&#39;s test was performed as a post hoc analysis to compare the means of two different groups. Differences with a p-value &#60;0.05 were considered significant. Abbreviations: qRT-PCR, quantitative real-time reverse-transcription polymerase chain reaction; miRNAs, microRNAs; NS, not significant; *, p &#60;0.05. This figure has been modified from Morishita et al.8 (with permission). <a href="http://cloudflare.jove.com/files/ftp_upload/55505/55505fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR at JoVE.com

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

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 ...

detection-microrna-expression-peritoneal-membrane-rats-using

Nanyang Technological University

Research

JoVE Journal

Genetics

7.8K Views.  Saitama Medical Center, Jichi Medical University. 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. 

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

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

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Medicine

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Immunology and Infection

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 ...

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.

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 ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

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 ...

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 ...