We thank Michelle Goody, PhD, from Edanz Group (https://en-author-services.edanzgroup.com/) for editing a draft of this manuscript.

All animal experimental protocols were approved by the Animal Ethics Committee of Jichi Medical University and were performed in accordance with the Use and Care of Experimental Animals guidelines from the Jichi Medical University Guide for Laboratory Animals.
1. The sham surgery
<ol>
	<li>Prepare the following items: isoflurane, cork sheet, depilatory cream, laboratory wipes, Petri dish with phosphate-buffered saline (PBS), 4-0 nylon, tweezers, surgical scissors, cotton swabs, and 8-week-old C57BL/6 male mice.</li>
	<li>Anesthetize a mouse with 1.5% isoflurane and maintain at 1.5%. Then, apply depilatory cream to the mouse&#39;s abdomen. After a few minutes, wipe the depilatory cream off with a PBS-soaked laboratory wipe.</li>
	<li>Inject 70% ethanol into the mouse&#39;s abdomen and then place the mouse on the cork sheet in the supine position.</li>
	<li>Using surgical scissors and tweezers, make an incision in the skin at the abdomen and cut the muscle and peritoneal membrane from the bladder to the left lower edge of the ribs.</li>
	<li>Moisten two cotton swabs with PBS and then pull the intestines carefully to the side. Place the moistened swabs to identify the left kidney and ureter.</li>
	<li>Close the peritoneal membrane and then close the incision with 4-0 nylon.</li>
</ol>
2. The UUO surgery
<ol>
	<li>Prepare the following items: isoflurane, cork sheet, depilatory cream, laboratory wipes, Petri dish with PBS, 4-0 silk, 4-0 nylon, a 2.5 mL syringe, cotton swabs, tweezers, surgical scissors, and 8-week-old C57BL/6 male mice.</li>
	<li>Anesthetize a mouse with 1.5% isoflurane and maintain at 1.5%. Then apply depilatory cream to the mouse&#39;s abdomen. After a few minutes, wipe the depilatory cream off with a PBS-soaked laboratory wipe.</li>
	<li>Inject 70% ethanol into the mouse&#39;s abdomen mouse and then place the mouse in the supine position on the cork sheet.</li>
	<li>Using surgical scissors and tweezers, make an incision in the skin at the abdomen and cut the muscle and peritoneal membrane from the bladder to the left lower edge of the ribs.</li>
	<li>Place the 2.5 mL syringe underneath the mouse. Take two cotton swabs and moisten them with PBS. Pull the intestines carefully to the side with the tweezers, and place the moistened swabs appropriately to identify the left ureter. Using the tweezers, lift the left kidney.</li>
	<li>Use the 4-0 silk to ligate the left ureter in two places approx. 1 cm apart. Cut the ureter at the center point of the two ligations, and then use 4-0 nylon sutures to close the peritoneal membrane and incision.</li>
</ol>
3. Collection of kidney samples
<ol>
	<li>Prepare the following: 1.5 mL microcentrifuge tubes, isoflurane, cork sheet, 70% ethanol, Petri dish with PBS, tweezers, and surgical scissors.</li>
	<li>Anesthetize a mouse with 1.5% isoflurane and maintain at 1.5%. Inject 70% ethanol into its abdomen and put the mouse on the cork sheet in the supine position.</li>
	<li>Using surgical scissors and tweezers, make an incision in the skin at the abdomen and cut the muscle and peritoneal membrane from the bladder to the left lower edge of the ribs.</li>
	<li>Lift up the peritoneal membrane with the tweezers. With the surgical scissors, make a sideways incision at the upper edge of the peritoneal membrane, and continue the incision along the lowest edge of the ribs.</li>
	<li>Next, identify the left kidney, reflux it with PBS until the kidney turns yellow-white to wash out blood from vessels, and remove the kidney by cutting the left renal artery and vein with the surgical scissors. Place the kidney in the Petri dish and wash it carefully with PBS.</li>
	<li>Cut the kidney into 10 mg samples with the surgical scissors and tweezers (10 mg is an appropriate size for the next step). Put each piece of the kidney in its own 1.5 mL microcentrifuge tube and close the tube&#39;s cap.</li>
	<li>Transfer each microcentrifuge tube into liquid nitrogen, and keep the tubes at &#8722;80 &#176;C for long-term storage before use.</li>
</ol>
4. Extraction of total RNA from the kidney samples
<ol>
	<li>Prepare the following items: 1.5 mL microcentrifuge tubes, 2.0 mL microcentrifuge tubes, 100% ethanol, chloroform, silicon homogenizer, ice, a vortex mixer, biopolymer spin columns in 2.0 mL collection tubes11,12, membrane-anchored spin columns in 2.0 mL collection tubes12,13, phenol/guanidine-based lysis reagent, wash buffer containing guanidine and ethanol (wash buffer 1), wash buffer containing ethanol (wash buffer 2), and RNase-free water.</li>
	<li>Put a 10 mg kidney sample in the silicon homogenizer. Add 700 &#181;L of the phenol/guanidine-based lysis reagent in the homogenizer.</li>
	<li>Prepare the homogenizer and then slowly press/twist the homogenizer&#39;s pestle against the kidney sample to homogenize the sample. Repeat the pressing/twisting until the kidney sample is completely dissolved in the phenol/guanidine-based lysis reagent.</li>
	<li>To further homogenize the sample, transfer the homogenized lysate (in a 2.0 mL collection tube) to the biopolymer spin column.</li>
	<li>Spin the homogenized lysate at 14,000 x g for 3 min at room temperature (RT), and then transfer the precipitated lysate to an unused 1.5 mL microcentrifuge tube.</li>
	<li>Combine the lysate in the tube with 140 &#181;L of chloroform and then close the tube cap tightly. To mix the lysate and chloroform, invert the tube 15 times. 
	NOTE: The chloroform can be used without a hood.</li>
	<li>Incubate each sample for 2&#8211;3 min at RT and then spin each sample at 12,000 x g for 15 min at 4&#176;C.</li>
	<li>Without disturbing the precipitate, transfer the supernatant (which is typically ~300 &#181;L) to a new 1.5 mL microcentrifuge tube, and then add 1.5x its volume (typically ~450 &#181;L) of 100% ethanol. Vortex the mixture for 5 s.</li>
	<li>Load 700 &#181;L of the sample onto a membrane-anchored spin column in a 2.0 mL collection tube. Close the column cap and spin the column at 15,000 x g for 15 s. Throw away the precipitated lysate in the collection tube.</li>
	<li>Wash the sample thoroughly by adding 700 &#181;L of wash buffer 1 to the membrane-anchored spin column in a 2.0 mL collection tube. Close the column cap, and spin the column at 15,000 x g for 15 s. Throw away the precipitated lysate in the collection tube.</li>
	<li>Load 500 &#181;L of wash buffer 2 onto the membrane-anchored spin column in a 2.0 mL collection tube to remove trace salts. Close the column cap, and spin the column at 15,000 x g for 15 s. Throw away the precipitated lysate in the collection tube. 
	NOTE: A membrane-anchored spin column can separate RNA and DNA.</li>
	<li>Perform step 4.11 again.</li>
	<li>Spin the membrane-anchored spin column in a 2.0 mL collection tube again at 15,000 x g for 1 min. Throw away the precipitated lysate in the collection tube.</li>
	<li>Transfer the membrane-anchored spin column to a new 1.5 mL collection tube. Dissolve total RNA by adding 30 &#181;L of RNase-free water to the column. Close the column cap, and wait 5 min at room temperature. Then, spin the column at 15,000 x g for 1 min.</li>
	<li>Transfer the total amount of sample containing total RNA to a new microcentrifuge tube. Put each of the tubes on ice, and measure the concentration of total RNA by spectrophotometry.</li>
	<li>Keep the tubes with samples at &#8722;80 &#176;C for long-term storage before use.</li>
</ol>
5. Synthesis of cDNA with the reverse transcription of total RNA
NOTE: The MIQE (Minimum Information for the Publication of Quantitative Real-Time PCR Experiments) guidelines were issued to encourage better experimental practices and help obtain reliable and unequivocal results19. In this protocol, cDNA is synthesized from 1.0 &#181;g of purified total RNA in a two-step procedure using reverse transcriptase, poly(A) polymerase, and oligo-dT primer.
<ol>
	<li>Prepare the following: 1.5 mL microcentrifuge tubes, eight-well strip tubes with caps, the cap of each eight-strip tube, distilled water, ice, a reverse transcriptase kit (see the Table of Materials)14,15 in the melted state, a thermal cycler, and a vortex mixer.</li>
	<li>Start the thermal cycler.</li>
	<li>Prepare a master mix solution: Add 2.0 &#181;L of reverse transcriptase mix (included in the kit) and 2.0 &#181;L of 10x nucleic acid mix into 4.0 &#181;L of 5x hi-spec buffer (to obtain a total of 8.0 &#181;L master mix per tube).</li>
	<li>Put 8.0 &#181;L of the master mix solution into each tube of an eight-well strip tube.</li>
	<li>Adjust the total RNA density. To isolate 1.0 &#181;g of total RNA from the kidney samples in 12 &#181;L of RNase-free water, transfer the appropriate amount of total RNA into distilled water, using the density data measured as described in step 4.15. 
	NOTE: If any DNA contamination is present, the contaminated DNA will be co-amplified in the qRT-PCR.</li>
	<li>Place a 12 &#181;L aliquot of total RNA into each tube and close the tube&#39;s cap. Centrifuge the tube for 15 x.</li>
	<li>Put the tube in the thermal cycler and incubate the sample for 60 min at 37 &#176;C. Next, immediately incubate the sample for 5 min at 95 &#176;C for the synthesis of cDNA.</li>
	<li>When the incubation is complete, transfer the cDNA into a new 1.5 mL microcentrifuge tube and dilute the cDNA ten times (1:10) with distilled water. Vortex and centrifuge the tube for 5 s.</li>
	<li>Temporarily store the diluted cDNA on ice and move the samples to &#8722;80 &#176;C for long-term storage before use.</li>
</ol>
6. qRT-PCR of miRNA
NOTE: We used the intercalator method to perform the qRT-PCR of miRNA. Primers for RNA are U6 small nuclear 2 (RNU6-2), miRNA-3070-3p, miRNA-6401, miRNA-7218-5p, and miRNA-7219-5p were used.
<ol>
	<li>Prepare the following: 1.5 mL microcentrifuge tubes, a vortex mixer, a 96-well reaction plate for the qRT-PCR, adhesive film for the 96-well reaction plate, an adhesive film applicator, a 96-well centrifuge rotor, miRNA-specific primers, a real-time PCR instrument, and a green dye-based PCR kit (see the Table of Materials)14,15 containing 2x PCR master mix and 10x universal primer.</li>
	<li>After mixing them in 1.5 mL microcentrifuge tubes, vortex the following items: 6.25 &#181;L of distilled water, 1.25 &#181;L of each 5 &#181;M miRNA primer dissolved in nuclease-free water, 12.5 &#181;L of 2&#215; PCR master mix, and 2.5 &#181;L of 10x universal primer.</li>
	<li>Prepare the cDNA synthesized as described in step 5, and melt it. Vortex and centrifuge the cDNA for 5 s.</li>
	<li>Put a 22.5 &#181;L aliquot of the reagent (created as described in step 6.2) in each well of the 96-well plate.</li>
	<li>Put a 2.5 &#181;L aliquot of cDNA in each well of the plate.</li>
	<li>Using the adhesive film applicator, secure the adhesive film tightly on the 96-well plate. Centrifuge the plate with the 96-well centrifuge rotor at 1,000 x g for 30 s to settle the reactions at the bottom of each well.</li>
</ol>
7. PCR cycling
<ol>
	<li>Start the real-time PCR system, and place the plate created as described in step 6.6. in the real-time PCR system. Set the experiment properties next; identify the experiment name. Select the following: &#34;96-well (0.2 mL)&#34; as the system&#39;s experiment type, &#34;Comparative CT (&#916;&#916;CT)&#34; as the quantitation method, &#34;SYBR Green Reagents&#34; as the reagents to detect the target sequence, and &#34;standard&#34; as the system&#39;s run.</li>
	<li>Assign a name to the sample and the target miRNA, and then assign a name to the sample and target miRNA in each well. Samples should be assigned in duplicate in order to obtain appropriate data for confirmation of the results. Select a reference sample and an endogenous control, and select &#34;none&#34; for the dye to be used as the passive reference. Make sure to set the negative reverse transcriptase and non-template control for the miRNA expression in order to eliminate the cross-contamination of reagents.</li>
	<li>Make sure the reaction volume setting is &#34;20 &#181;L&#34; and the PCR cycling conditions are set at 95 &#176;C for 15 min, followed by 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>
	<li>After the qRT-PCR process is complete, use the system&#39;s software program to analyze the qRT-PCR data. Ensure that the threshold line that is automatically selected by the software program is appropriate for each well.</li>
	<li>Check the threshold cycle (CT) value of the endogenous control and target miRNA analyzed in each sample. The CT value is determined by the intersection of the amplification curve and the threshold line. In the present study, we used RNU6-2 as an endogenous control for the target miRNA expression level, and we used the &#916;&#916;CT method to determine the relative expression level of each target miRNA20.</li>
</ol>

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The authors declare that they have no conflicts of interest.

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

MicroRNAs (miRNAs)&#8201;&#8212;&#8201;the short, noncoding RNAs that cause the degradation and transcriptional inhibition of messenger RNA (mRNA)1&#8201;&#8212;&#8201;have been shown to regulate the expression of various mRNAs that have crucial roles in both physiology and disease (e.g., inflammation, fibrosis, metabolic disorders, and cancer). Some of the miRNAs may therefore be candidate novel biomarkers and therapeutic targets for a variety of diseases2,3,4,5. Although miRNA expression profiles in mouse organs and tissues including brain, heart, lung, liver, and kidney have been described6,7,8,9,10, there have been no standard methods for the extraction and evaluation of miRNAs in mouse kidney with renal interstitial fibrosis.
We have designed a protocol to reliably purify and detect the expressions of miRNAs in the kidneys of mice with renal interstitial fibrosis. The protocol involves five main steps, as follows. (1) 8-week-old C57BL/6 male mice are divided into groups of mice that undergo a sham-operation (controls) and mice that are subjected to a surgery providing unilateral ureteral obstruction (UUO), which is linked to renal interstitial fibrosis. (2) Kidney samples are extracted from the sham and UUO mice, homogenized separately in a silicon homogenizer, and then transferred to a biopolymer-shredding system on a microcentrifuge spin column11,12. (3) The total RNA containing miRNA is extracted from the kidney samples by a silica membrane-based spin column12,13. (4) Using this extracted total RNA, complementary DNA (cDNA) is synthesized from the total RNA with the use of reverse transcriptase, poly(A) polymerase, and oligo-dT primer14,15. (5) The expressions of miRNAs are evaluated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) using an intercalating dye14,15.
This protocol is based on investigations that obtained meaningful extractions and evaluations of miRNAs in a variety of tissues11,12,13,14,15, and the biopolymer-shredding system used in the protocol was shown to purify high-quality, total RNA from tissues in 200612. In addition, prior studies have confirmed the accuracy and sensitivity of aspects of the protocol (i.e., the cDNA synthesis with reverse transcriptase, poly(A) polymerase, and oligo-dT primers from extracted total RNA) for the determination of miRNA expression by qRT-PCR with an intercalating dye14,15. Since the new protocol has the advantages of simplicity, time-saving, and the reduction of technical errors, the protocol can be used in research that requires the accurate and sensitive identification of the miRNA profile in mouse kidney. Moreover, the protocol could be applied to investigations of many pathological conditions.
We next describe the determination of the miRNA expression profiles in mice with UUO, which is linked to renal interstitial fibrosis. In humans, renal interstitial fibrosis is a common and important feature of both chronic kidney disease and end-stage renal disease, regardless of their etiology16,17. This renal interstitial fibrosis is associated with the progression of renal failure, and it is characterized by increased expressions of extracellular matrix components in the interstitial spaces (e.g., collagen, fibronectin, and &#945;-smooth muscle actin)17,18.

<table>
	<tbody>
		<tr>
			<td>Qiagen</td>
			<td>79216</td>
			<td>Wash buffer 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>1067933</td>
			<td>Wash buffer 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tokyo Laboratory Animals Science</td>
			<td>Not assigned</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific</td>
			<td>4316813</td>
			<td>96-well reaction plate</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>Adhesive film for 96-well reaction plate</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>MS00001701</td>
			<td>5&#39;-UUAAUGCUAAUUGUGAUAGGGGU-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>MS00065141</td>
			<td>5&#39;-UUACACUCCAGUGGUGUCGGGU-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>MS00068067</td>
			<td>5&#39;-UGCAGGGUUUAGUGUAGAGGG-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>MS00068081</td>
			<td>5&#39;-UGUGUUAGAGCUCAGGGUUGAGA-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>217004</td>
			<td>Membrane anchored spin column in a 2.0 mL collection tube</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Reverse transcriptase kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Green dye-based PCR kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>79654</td>
			<td>Biopolymer spin columns in a 2.0 mL collection tube</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>79306</td>
			<td>Phenol/guanidine-based lysis reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>Real-time PCR instrument</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>Real-time PCR instrument software</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>129112</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>MS00033740</td>
			<td>Not disclosed</td>
			<td></td>
		</tr>
		<tr>
			<td>Takara Bio</td>
			<td>9790B</td>
			<td>Silicon homogenizer</td>
			<td></td>
		</tr>
		<tr>
			<td>ASKUL</td>
			<td>GA04SW</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AS ONE</td>
			<td>ER1004NA45-KF2,62 -9968-32</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A UUO mouse model was created by left ureteral ligation as described21 in 8-week-old male mice weighing 20&#8211;25 g. Ureters were completely obstructed by double ligation with 4-0 silk sutures. An analgesic (meloxicam 5 mg/kg, subcutaneous injection) was administered before surgery and also daily on the 2 days post-surgery. At 8 days post-surgery, kidneys were collected, rinsed with PBS, dissected, and stored in liquid nitrogen for further analysis. Sham-operated mice served as controls. The dou...

Watch this Scientific Journal Video about Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction at JoVE.com

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

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 ...

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JoVE Journal

Genetics

6.1K Views.  Jichi Medical University. 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.

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

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

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

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

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

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

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

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

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 ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the ...

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 ...

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 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. ...