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

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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 ...

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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 ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3&#39; end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.

Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR

RNA hairpins and loops can function as primers for reverse transcription (RT) in absence of sequence-specific primers, interfering with the study of overlapping antisense transcripts. We have developed a technique able to identify strand-specific RNA, and we have used it to study HIV-1 antisense protein ASP.

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). ...

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

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

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 ...