Name: detection of microrna expression in peritoneal membrane of rats using quantitative real-time pcr
Uploaded: 2017-06-27T14:00:00
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>Eut...

<ol>
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M., Shaw, G., Larner, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+mRNA+and+protein+extraction+from+the+same+experimental+sample+using+a+commercially+available+column-based+RNA+preparation+kit.">Concurrent mRNA and protein extraction from the same experimental sample using a commercially available column-based RNA preparation kit.</a> Biotechniques. 40 (1), 54-58 (2006).</li><li>Mestdagh, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+quantitative+miRNA+expression+platforms+in+the+microRNA+quality+control+(miRQC)+study.">Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.</a> Nat Methods. 11 (8), 809-815 (2014).</li><li>Bustin, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MIQE+guidelines:+minimum+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.</a> Clin Chem. 55 (4), 611-622 (2009).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nat Protoc. 3 (6), 1101-1108 (2008).</li><li>Krediet, R. T., Lindholm, B., Rippe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+peritoneal+membrane+failure.">Pathophysiology of peritoneal membrane failure.</a> Perit Dial Int. 20, S22-S42 (2000).</li><li>Devuyst, O., Margetts, P. J., Topley, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+the+peritoneal+membrane.">The pathophysiology of the peritoneal membrane.</a> J Am Soc Nephrol. 21 (7), 1077-1085 (2010).</li><li>Hirahara, I., Ishibashi, Y., Kaname, S., Kusano, E., Fujita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylglyoxal+induces+peritoneal+thickening+by+mesenchymal-like+mesothelial+cells+in+rats.">Methylglyoxal induces peritoneal thickening by mesenchymal-like mesothelial cells in rats.</a> Nephrol Dial Transplant. 24 (2), 437-447 (2009).</li><li>Dallas, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+levels+assessed+by+oligonucleotide+microarray+analysis+and+quantitative+real-time+RT-PCR+--+how+well+do+they+correlate?.">Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR -- how well do they correlate?.</a> BMC Genomics. 6, 59 (2005).</li><li>Rockett, J. C., Hellmann, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirming+microarray+data--is+it+really+necessary?.">Confirming microarray data--is it really necessary?.</a> Genomics. 83 (4), 541-549 (2004).</li></ol>

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 concen...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>includes 10&#215; 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&#160;&#160;</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&#39;-UGUAGUGUUUCC 
			UACUUUAUGGA-3&#39;&#160;&#160;</td>
		</tr>
		<tr>
			<td>miRNA-21-5p primer</td>
			<td>Qiagen</td>
			<td>MS00009079</td>
			<td>5&#39;-UAGCUUAUCAG 
			ACUGAUGUUGA-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-221-3p primer</td>
			<td>Qiagen</td>
			<td>MS00003857</td>
			<td>5&#39;-AGCUACAUUGU 
			CUGCUGGGUUUC-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-223-3p primer</td>
			<td>Qiagen</td>
			<td>MS00033320</td>
			<td>5&#39;-UGUCAGUUUG 
			UCAAAUACCCC-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-34a-5p primer</td>
			<td>Qiagen</td>
			<td>MS00003318</td>
			<td>5&#39;-UGGCAGUGUCU 
			UAGCUGGUUGU-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-327 primer</td>
			<td>Qiagen</td>
			<td>MS00000805</td>
			<td>5&#39;-CCUUGAGGGG 
			CAUGAGGGU-3&#39;</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&#160;</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&#160;</td>
			<td>peritoneal dialysis fluid</td>
		</tr>
		<tr>
			<td>Sprague&#8211;Dawley rats</td>
			<td>SLC</td>
			<td>not assign&#160;</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 fibro...

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 ...

The overall goal of this procedure is to successfully purify and detect miRNAs in the rat peritoneal membrane. To begin this procedure, after inhalational anesthesia with 4%isoflurane. Spray the abdominal skin of the rat with 70%ethanol and mount it on the cork sheet, on its'back.Next make a longitudinal incision in the animal's abdominal skin, muscle, and peritoneal membrane using scissors and forceps. 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 PPS in a petri dish. Cut out and trim 20 milligrams of peritoneal membrane sample.In this procedure, place a 20 milligram peritoneal membrane sample into a glass homogenizer. And add 700 microliters phenol guanidine based lysis reagent. After that, homogenize the peritoneal membrane sample on ice, by slowing pressing a pestle onto the sample with twisting.Repeat this process several dozen times, until the peritoneal membrane sample has completely dissolved into the phenol guanidine based lysis reagent. for further homogenization, transfer the homogenized lysate to a bio-polymer shredding system in the micro centrifuge spin column in a two milliliter collection tube. Then centrifuge it at 14, 000 times G for three minutes at four degrees Celsius.Next, transfer the homogenized lysate to a new micro centrifuge tube. Add 140 microliters of chloroform to the homogenized lysate and cap the tube securely. Subsequently, mix the tube by inversion for 15 seconds.Afterward, incubate the sample for two to three minutes at room temperature. Following that, centrifuge it at 12, 000 times G for 15 minutes at four degrees Celsius. Transfer 300 microliters of the supernatant to a new micro centrifuge tube, without disturbing the precipitant.And add 450 micro liters of 100%ethanol. Then mix the sample by vortexing for five seconds. Next pipette up to 700 micro liters of the sample into a silicone membrane based spin column, placed in a two milliliter collection tube, and close the cap.Then centrifuge it at 15, 000 times G for 15 seconds. After centrifugation, discard the flow through in the collection tube. Then add 700 microliters of wash buffer 1, to the silicon membrane based spin column to stringently wash the sample and close the cap.Centrifuge it at 15, 000 times G for 15 seconds. After centrifugation, discard the flow through in the collection tube. Add 500 microliters of wash buffer 2, to the silica membrane based spin column, to remove any trace of salt, and close the cap.Then centrifuge it at 15, 000 times G for 15 seconds. After centrifugation, discard the flow through and the collection tube and repeat this procedure again. Next, centrifuge the silica membrane based spin column again at 15, 000 times G for one minute.After centrifugation, discard the collection tube and any flow through. Place the silica membrane based spin column in a new, one point five milliliter collection tube. Then transfer 25 microliters of RNase free water onto the column and close the cap.Leave the sample at room temperature for five minutes. Centrifuge it at 15, 000 times G for one minute. Following that, transfer the 25 microliter alouette containing the total RNA to a new micro centrifuge tube and store it at minus 80 degrees Celsius before use.In this procedure, prepare a master mix solution that contains two microliters of 10x nucleic acid mix, two microliters of reverse transcriptase mix, and four microliters of 5x Hispec buffer, for a total of 8 microliters per tube. Then dispense 8 microliters of the master mix solution into each tube. Measure the quantity of the total RNAs using a Spectrophotometer.Afterward, add one microgram of the isolated total RNAs, purified from the peritoneal membrane sample, to each tube. Following that, add RNase free water to each tube to bring it to a total of 20 microliters. Then mix it thoroughly by pipetting and centrifuge it for 15 seconds.Following that, incubate the sample for 60 minutes at 37 degrees Celsius. Then incubate it for five minutes at 95 degrees Celsius. Transfer the newly generated cDNA to a new micro centrifuge tube and dilute it 10 times with RNase free water.In this procedure, prepare a master mix containing 12 point five microliters of 2x PCR master mix, two point five microliters of five micromolar of each miRNA primer dissolved in nuclease free water, and one point two five microliters of 10x universal primer, for each well. Dispense 22 point five microliters of this master mix into each well of a 96 well plate. Then add two point five microliters of template cDNA to each well.Seal the 96 well reaction plate with a piece of film. Then centrifuge it at 1, 000 times G for 30 seconds. To run the PCR cycling program, set the plate in the real time PCR instrument.In the real time PCR software, define the experimental properties. Next, assign names to the samples and target miRNAs in each well. Then in the real time PCR software input a reaction volume of 20 micro liters and the following PCR cycling conditions in accordance with the instructions.Pre incubation at 95 degrees Celsius for 15 minutes. And then 40 cycles of denaturation at 94 degrees Celsius for 15 seconds, and kneeling at 55 degrees Celsius for 30 seconds, And extension at 70 degrees Celsius for 30 seconds. Based on miRNA, micro array screening, the levels of six miRNAs have been found to be significantly increased in the peritoneal membrane of peritoneal fibrosis rats, compared with those in mock rats and control rats, using QRTCPR, following the protocol presented in this manuscript.

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Genetics

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

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Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Detection of Copy Number Alterations Using Single Cell Sequencing

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Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

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This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

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High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

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 ...

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.

Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that ...

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Accurate Telomere Length Assessment in Human Population Studies

Monochrome Multiplex Quantitative PCR Telomere Length Measurement 

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. Telomere length (TL) has been associated with various exposures, biological processes, and health outcomes. This article describes the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay protocol routinely conducted in our laboratory for measuring relative mean TL from human DNA. There are several different PCR-based TL measurement methods, but the specific protocol for the MMqPCR method presented in this publication is repeatable, efficient, cost-effective, and suitable&#160;for population-based studies. This detailed protocol outlines all information necessary for investigators to establish this assay in their laboratory. In addition, this protocol provides specific steps to increase the reproducibility of TL measurement by this assay, defined by the intraclass correlation coefficient (ICC) across repeated measurements of the same sample. The ICC is a critical factor in evaluating expected power for a specific study population; as such, reporting cohort-specific ICCs for any TL assay is a necessary step to enhance the overall rigor of population-based studies of TL. Example results utilizing DNA samples extracted from peripheral blood mononuclear cells demonstrate the feasibility of generating highly repeatable TL data using this MMqPCR protocol.

Author Spotlight: Exploring the Impact of Trauma on Cellular Aging

Here we present a protocol for the measurement of relative telomere length (TL) using the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay. The MMqPCR assay is a repeatable, efficient, and cost-effective method for measuring TL from human DNA in population-based studies.

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. ...

Optimizing Low-Frequency Mutation Detection with Wild-Type Blocking PCR

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.

Author Spotlight: Advancing the Detection of Low-Frequency Mutations in Cancer Tissues

This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other diseases.

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting ...