We acknowledge the help provided by Lourdes Delta Arrueta Antequera (Department of Entomology, Ohio State University/OARDC, Wooster, OH) in the setup of the experiments. We thank Dr. Therese Poland (USDA, Forest Services, NRS) for send EAB larval samples. Help provided by Dr. Luis A Canas and Nuris M Acosta with the microscope setup is appreciated. Funding for this project was provided by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.

The complete protocol with different steps is depicted in the flow chart in Figure 1. Individual steps constituting dissection, RNA extraction, First Strand cDNA synthesis and qRT-PCR are detailed below.
I. Larval Dissection
<ol>
 <li>Prior to the start of this procedure, place Emerald ash borer, or EAB, larvae on moist tissue paper until dissections are performed. Keep freshly prepared 1X phosphate buffered saline, or PBS, on ice to keep the buffer cold throughout the dissection.</li>
 <li>To begin, fix the EAB larvae on the dissection plate with fine dissection pins. Then, add cold 1X PBS to the dissection plate.</li>
 <li>First, decapitate larvae with a fine pair of dissection scissors. Subsequently remove the last abdominal segment of the larvae. </li>
 <li>Using a watchmaker pair of forceps, lift the cuticle from the larval carcass. Then, make an incision along the length of the carcass using a pair of scissors. Take care not to rupture the gut as the incision is made.</li>
 <li>Carefully isolate the midgut tissue from other tissues such as fat bodies and connective tissue. Then, rinse the tissue in fresh 1X PBS buffer to ensure that there is no contamination of fatbody. Transfer the isolated midgut to 500 &mu;L of pre-chilled Trizol reagent in a 1.5 mL eppendorf tube.</li>
 <li>Next, isolate the fat body tissue using forceps and transfer it into a 1.5 mL eppendorf tube containing 500 &mu;L of chilled Trizol reagent. </li>
 <li>Finally, isolate the cuticle tissue from the larval carcass by scrapping off any adhering tissue, taking care not to damage tissue integrity. Rinse the isolated cuticle tissue in fresh PBS buffer and transfer it to a 1.5 mL eppendorf tube with 500 &mu;L of chilled Trizol reagent. </li>
 <li>The isolated fat body, cuticle and midgut tissues can be stored at -80 &deg;C until RNA extraction. </li>
 <li>In preparation for RNA extraction, sort the various EAB samples according to the developmental stages. These stages include 1st-, 2nd-, 3rd-, and 4th-instars, prepupae and adults .</li>
</ol>
II. RNA Extraction (As per manufacturer s protocol for using Trizol Reagent)
Homogenization
<ol>
	<li>To begin RNA Extraction, first use plastic pestles to homogenize the tissues in the Trizol containing 1.5 mL eppendorf tubes. After homogenization, bring the total volume of each sample to1 mL with the Trizol reagent.</li>
 <li>For the developmental stages, homogenize whole animal in liquid nitrogen with a mortar and pestle. Then add an aliquot of the homogenate (50 &mu;g) to 1 mL of Trizol.</li>
</ol>
Phase separation:
<ol start="3">
 <li>Incubate the sample tubes with 1 mL Trizol for 15 minutes at room temperature. </li>
 <li>Following incubation, add 200&mu;L of chloroform to each tube. Immediately after adding chloroform, shake the tubes vigorously for about 15 seconds. Then, incubate the tubes at room temperature for an additional 2-3 minutes.</li>
 <li>Next, centrifuge the samples at 12,000Xg for 15 minutes at 2 &deg;C. After centrifugation, the RNA will be in the aqueous phase. The volume of the aqueous phase should be 600 &mu;l, or 60% of the total Trizol volume.</li>
</ol>
RNA precipitation
<ol start="6">
 <li>Carefully remove only the aqueous phase. Presence of substances below the aqueous phase will cause contamination of the extracted RNA. Then, transfer the aqueous phase into an appropriately labeled 1.5 mL eppendorf tube. </li>
 <li>To precipitate the RNA from the aqueous phase, mix 0.5 mL of isopropyl alcohol to each tube. Incubate the samples at room temperature for 10 minutes. After the incubation, centrifuge the tubes at 12,000Xg for 10 minutes at 2&deg;C.</li>
</ol>
RNA Wash
<ol start="8">
 <li>Following centrifugation, discard the supernatant from the tubes. RNA is present in the gel-like pellet formed at the bottom of the tube. Wash the pellet with 75% ethanol, adding at least 1 mL of 75% ethanol per 1 mL of Trizol. Mix by vortexing. Then, centrifuge the tubes at 7500Xg for 5 minutes at 2 &deg;C. </li>
</ol>
RNA elution
<ol start="9">
 <li>Discard the supernatant from the tubes. Centrifuge the tubes again in the small table centrifuge for 1 minute. Remove any excess supernatant from the tube by careful pipetting. Leave the tube open and let the pellet air dry for 5-10 minutes. Be careful not to over dry the pellet as this will significantly decrease its solubility.</li>
 <li>After air-drying, resuspend the pellet in <a href="http://en.wikipedia.org/wiki/Diethylpyrocarbonate">diethylpyrocarbonate</a>, or DEPC, treated water. The amount of water used to resuspend the pellet will depend on the size of the pellet. Use 50 &mu;L of DEPC treated water for a small pellet or 100 &mu;L for a big pellet. Let the pellet completely dissolve in DEPC treated water by moving the pipette tip up and down several times. </li>
 <li>Once the pellet has dissolved, place the sample at 55 &deg;C for 10 minutes. </li>
 <li>Then, measure the concentration of each RNA sample using a Nanodrop spectrophotometer (2 &mu;L of the RNA sample). </li>
 <li>Store the RNA samples at -80&deg;C until further use. </li>
</ol>
III. First-Strand cDNA Synthesis (As per manufacturer s protocol using SuperScript first strand Synthesis kit by Invitrogen.)
<ol>
	<li>For each RNA sample add the following in a PCR tube:
	<table border="0">
 <tbody>
 <tr><td>RNA</td><td>x &mu;l</td></tr>
 <tr><td>10mM dNTP mix M</td><td>1&mu;l</td></tr>
 <tr><td>Oligo(dt) (0.5 &mu;g/ &mu;l)</td><td>1 &mu;l</td></tr>
 <tr><td>DEPC treated water</td><td>(8-x) &mu;l</td></tr>
 	<tr><td>Total Volume:</td><td>10 &mu;l</td></tr>
 <tr><td colspan="2">Note: x is the volume of RNA used for cDNA synthesis. Depending on the concentration of the RNA, 2-3 &mu;g of RNA will be used for each reaction and the total volume of each reaction should be 10 &mu;l.</td></tr>
	</tbody>
 </table>
 </li>
 <li>Place the above reaction mix in a thermocycler at 65&deg;C for 4 minutes.</li>
	<li>Remove the tubes from the theromocyler and place them on ice immediately for at least 2 minutes.</li>
 <li>Prepare the first-strand master mix by adding the following:
	<table border="0">
 <tbody>
 <tr><td>10X RT Buffer</td><td>2&mu;l</td></tr>
 <tr><td>25mM MgCl2</td><td>4 &mu;l</td></tr>
 <tr><td>0.1M DTT</td><td>2 &mu;l</td></tr>
 <tr><td>RNase out</td><td>1 &mu;l</td></tr>
 </tbody>
 </table>
	</li>
 <li>Add 9 &mu;l of master mix to each tube containing the RNA sample, mix by pipeting and centrifuge the mixture briefly.</li>
 <li>Place the tubes back in the thermocycler and incubate for 2 minutes at 42&deg;C.</li>
 <li>Pause the thermocycler and add 1 &mu;l of Superscript II reverse transcriptase enzyme (Invitrogen) to each tube. This step should be done quickly.</li>
 <li>Incubate the samples at 42&deg;C for 1.5 hours.</li>
 <li>Terminate the reaction at 70&deg;C for 15 minutes.</li>
 <li>Remove the tubes from the thermocycler and place them on ice.</li>
 <li>Add 1 &mu;l of RNase H to each tube and place them back in the thermocycler at 37&deg;C for 20 minutes. </li>
 <li>After taking the samples out of the thermocycler, add 20 &mu;l of nuclease-free water in each tube. The volume of each sample is doubled at this stage.</li>
 <li>Check the concentration of each sample by using 2 &mu;l of the sample. Take aliquot of each sample, to make the concentration of 20ng/&mu;l for use in qRT-PCR. The amount of water to make up the samples to a concentration of 20ng/&mu;l will depend on the initial concentration of the cDNA synthesized.</li>
</ol>
IV. qRT-PCR:
Primer Design and Determination of reference gene
<ol>
 <li>Design primers with a melting temperature (Tm) of 60&deg;C and a product size of ~100bp.</li>
 <li>AP-PERI1 is used as the gene of interest for this experiment. A reference gene or internal control is needed for later analysis and normalization of the data. Ribosomal protein (AP-RP1) is used as the reference gene for this experiment.</li>
 <li>Prepare 5uM working stocks of the primers that will be used, including your reference gene.</li>
</ol>
Preparation of standards
<ol start="4">
 <li>In order to calculate the efficiency of the primers being used standards should be made. </li>
 <li>Make a mix of the cDNA from the different samples that will be tested.</li>
 <li>Make 5X serial dilutions for each desired point in the graph. Four dilutions should be enough but five are highly recommended.</li>
 <li>The volume will vary depending on how many points and how many technical replicates are being used. There should be enough to make a standard for each gene being tested including the reference gene.</li>
</ol>
Plate Template
<ol start="8">
 <li>Design a template indicating the sample name for each well in the qRT-PCR plate. Remember to include standards for each gene and at least two technical replicates per sample.</li>
</ol>
Set up the cycling parameters
<ol start="9">
 <li>Turn the qRT-PCR machine on and enter the cycling parameters. The cycling parameters for this experiment is as follows:
 Step 1:
 <ul>
 	<li>1 cycle: 95&deg;C 3 min</li>
 </ul>
 Step 2:
 <ul>
 <li>40 cycles: 95&deg;C 15 sec followed by 60&deg;C 30 sec (Optional: for determining the melting curve include the following steps)</li>
 </ul>
	Step 3:
	<ul>
 <li>1 cycle: 95&deg;C 1 min</li>
 </ul>
	Step 4:
	<ul>
	<li>1cycle: 55&deg;C 1 min</li>
 </ul>
	Step 5:
	<ul>
 <li>81 cycles: 55&deg;C 30 sec</li>
 </ul>
 </li>
</ol>
	Plate set up for CFX96 (BioRad) machine
<ol start="11">
<li>Prepare the master mix for each gene (including the reference gene) in separate tube. For each well, the master mix consists of
	<table border="0">
 <tbody>
 <tr><td>Nuclease free water</td><td>2&mu;l</td></tr>
 <tr><td>2.5 X SYBR green</td><td>4 &mu;l</td></tr>
 <tr><td>Primer forward</td><td>1 &mu;l</td></tr>
 <tr><td>Primer reverse</td><td>1 &mu;l</td></tr>
 <tr><td>Total volume</td><td>8 &mu;l</td></tr>
	</tbody>
 </table>
 </li>
 <li>While preparing the master mix include 1-2 extra reactions to account for pipetting errors.</li>
 <li>According to the previously designed template, add 2 &mu;L (20 ng/ &mu;L) of cDNA to each well. Then, add 8 &mu;L of the master mix to each well, resulting in a total volume of 10 &mu;L per reaction.</li>
 <li>Pipette up and down (2-3X) to make sure the sample is well mixed. Check that no liquid stays on the tip. To avoid cross contamination, use a new tip for each sample.</li>
 <li>Once the entire plate is setup, cover the plate with optical tape. Avoid touching the tape as the presence of grease can affect the reading. Then, centrifuge the plate at 500 rpm for 1 minute to ensure that all the products in the wells are at the bottom of the plate.</li>
 <li>Following centrifugation, check that there is no ice or water on the bottom of the plate. Finally, place the plate in the qRT-PCR machine and run the PCR program.</li>
</ol>
V. Diagrammatic Representation of the Experiment
<img src="/files/ftp_upload/1974/1974fig1.jpg" alt="Figure 1" /> Figure 1: Flow chart depicting the sequential order of steps for the gene expression study.
<img src="/files/ftp_upload/1974/1974fig2.jpg" alt="Figure 2" /> Figure 2: A) larval EAB dissection showing midgut in the middle. B) isolated midgut of larval EAB
<img src="/files/ftp_upload/1974/1974fig3.jpg" alt="Figure 3" /> Figure 3: A) Mean relative expression values (REVs) for a peritrophin gene (AP-PERI1) in different larval tissues including cuticle (Cu), midgut (MG) and fat bodies (FB). An EAB specific ribosomal protein (herein named, AP-RP1) was used as the internal control for normalizing the data obtained for the gene of interest, AP-PERI1. B) Relative fold change of AP-PERI1 in larval tissues. The cuticle tissue showed the least expression and therefore was taken as the calibrator (1X) sample (Pfaffl, 2001).
<img src="/files/ftp_upload/1974/1974fig4.jpg" alt="Figure 4" /> Figure 4: A) Mean relative expression values (REVs) for a peritrophin gene (AP-PERI1) in different developmental stages of EAB including larval instars (1st, 2nd, 3rd and 4th), prepupae (PP) and adults (A). An EAB specific ribosomal protein (AP-RP1) was used as the internal control for normalizing the data obtained for the gene of interest, AP-PERI1. B) Relative fold change of AP-PERI1 in various developmental stages. The PP sample showed the least level of mRNA levels and therefore was taken as the calibrator (1X sample). 
VI. Conclusion
Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. Further, qRT-PCR has mostly been the key tool to validate data generated from high throughput gene expression analyses such as microarrays and the new-generation RNA-Seq.

<ol>
	<li>Lehane, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritrophic+matrix+structure+and+function.">Peritrophic matrix structure and function.</a> Annu Rev Entomol. 42, 525-550 (1997).</li><li>Mittapalli, O., Sardesai, N., Shukle, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cDNA+cloning+and+transcriptional+expression+of+a+peritrophin-like+gene+in+the+Hessian+fly,+Mayetiola+destructor+[Say].">cDNA cloning and transcriptional expression of a peritrophin-like gene in the Hessian fly, Mayetiola destructor [Say].</a> Arc of Insect Bio. 64, 19-29 (2007).</li><li>Pauchet, Y., Wilkinson, P., van Munster, M., Augustin, S., Pauron, D., ffrench-Constant, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing+of+the+midgut+transcriptome+of+the+poplar+leaf+beetle+Chrysomela+tremulae+reveals+new+gene+families+in+Coleoptera.+Ins+Bio+and+Mol.">Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera. Ins Bio and Mol.</a> 39, 403-413 (2009).</li><li>Pfaffl, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mathematical+model+for+relative+quantification+in+real-time+RT-PCR.">A new mathematical model for relative quantification in real-time RT-PCR.</a> Nucleic Acids Res. 29, 2002-2007 (2001).</li><li>Tellam, R. L., Lehane, M. J., Billingsley, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of the Insect Midgut. , 86-114 (1996).</li></ol>

No conflicts of interest declared.

The threshold cycles (Ct) value is obtained for each sample in the qRT-PCR plate. For making the standard curve, the Ct value obtained for each dilution was plotted against the log of its concentration. The Ct values for the experimental samples were then plotted onto this dilution series standard curve. Target quantities were calculated from separate standard curves generated for each experiment i.e. tissue and developmental expression. The relative expression values (REVs) were then determined by dividing the quantitie...

analysis of gene expression in emerald ash borer (agrilus planipennis) using quantitative real time-pcr

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Watch this Scientific Journal Video about Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR at JoVE.com

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

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10.6K Views.  The Ohio State University. Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Video: Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Bacterial Gene Expression Analysis Using Microarrays

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...