Part 1: Fluorescent Labeling of cDNA
The Cy3 dye used in the microarray experiment is sensitive to photo-degradation. The procedure should take place in a minimal amount of light. The labeling protocol was adapted from the BioPrime Array CGH Genomic Labeling System Manual1.
<ol><li>The reagents for the labeling reaction are stored at -20&deg;C. From the BioPrime Array CGH Genomic Labeling System thaw 2.5X Random Primer Buffer, 10X dCTP mix, and nuclease-free water. Also thaw the Cy3-dCTP dye. After thawing, briefly centrifuge all reagents before using.</li>
<li>A separate labeling reaction will need to be completed for each cDNA sample to be analyzed. Into a 0.6 ml thick-walled tube add 500 ng cDNA, 40 &mu;l 2.5X Random Primer Buffer and nuclease-free water to achieve a total volume of 86 &mu;l.</li>
<li>Mix well and briefly centrifuge. Incubate reaction for 5 minutes at 100&deg;C on a thermocycler.</li>
<li>Immediately transfer reaction into an ice bath slurry for 10 minutes.</li>
<li>To each tube then add 10 &mu;l 10X dCTP mix, 2 &mu;l 1 mM Cy3-dCTP and 2 &mu;l Exo Klenow DNA polymerase. The Exo Klenow DNA polymerase should be left in the freezer until needed and should always be kept on ice.</li>
<li>Mix reaction well and briefly centrifuge.</li>
<li>Incubate reaction at 37&deg;C for 16 hours in a thermocycler.</li>
</ol>
Part 2: Precipitation of Labeled cDNA
<ol>
<li>Before continuing in the protocol turn on the NimbleGen Hybridization System and allow the system to equilibrate to 42&deg;C.</li>
<li>For each reaction, place a Microcon column into a 1.5 ml tube. Add 300 &mu;l 0.1X SSC to each column followed by the contents of the labeling reaction (100 &mu;l).</li>
<li>Centrifuge the tube containing the column at 16,100 x g for 10 minutes at 4&deg;C.</li>
<li>Discard the flow through, which may be slightly pink in color, and add another 300 &mu;l 0.1X SSC to column.</li>
<li>Repeat centrifugation at 16,100 x g for 10 minutes at 4&deg;C.</li>
<li>Discard the flow through and add an additional 300 &mu;l 0.1X SSC to column.</li>
<li>Centrifuge at 16,100 x g for 12 minutes at 4&deg;C.</li>
<li>Discard flow through. The flow through should be clear after this last wash and you should be able to see the pink labeled cDNA on the column.</li>
<li>Apply 100 &mu;l nuclease-free water to column. Remove column from the 1.5 ml tube and invert column into a new 1.5 ml tube.</li>
<li>Centrifuge at 2,300 x g for 2 minutes at 4&deg;C. The labeled cDNA should be rehydrated in the water that collects at the bottom of the tube.</li>
<li>Use a NanoDrop ND-1000 spectrophotometer according to the manufacturer's instructions to calculate DNA concentration and evaluate dye incorporation by calculating the base to dye ratio where the nucleotide to Cy3 ratio equals the A260/A550 ratio multiplied by 23.15. The base to dye ratio should be between 50 and 80.</li>
<li>If cDNA if labeled sufficiently, aliquot 6 &mu;g of labeled cDNA into a new 1.5 ml tube.</li>
<li>Mix in 0.1X volume of 3 M NaOAc, pH 5.2 and 1X volume isopropanol. Allow mixture to incubate in the dark for 30 minutes at room temperature.</li>
<li>Next centrifuge the sample at 16,100 x g for 20 minutes at room temperature.</li>
<li>Carefully discard the supernatant without disturbing the DNA pellet, which should be pink in color.</li>
<li>Perform an ethanol wash by adding 500 &mu;l 70% EtOH and mix by gentle inversion.</li>
<li>Centrifuge sample at 16,100 x g for 5 minutes at room temperature.</li>
<li>Remove the supernatant, invert the tube, and allow the pellet to dry for 5 minutes. At this step the pellet may be stored at -20&deg;C if desired or the protocol can be continued to the hybridization step.</li>
</ol>
Part 3: Hybridization
The hybridization procedure is adapted from the Roche NimbleGen Arrays User's Guide for Gene Expression Analysis2.
<ol>
<li>Dissolve the pellet in 5 &mu;l of nuclease-free water.</li>
<li>Once the pellet is dissolved add hybridization components listed in Table 1. Hybridization components are from the Roche NimbleGen Hybridization Kit. After all components are added the total volume should now total 18 &mu;l. Mix well and briefly spin the tube to collect the contents at the bottom.</li>
<li>The next step is to denature the labeled cDNA by placing it on a heat block at 95&deg;C for 5 minutes.</li>
<li>Immediately transfer the tube to the NimbleGen Hybridization System while the arrays are prepared in the following steps.</li>
<li>Microarrays should always be handled carefully and stored according to manufacturer's instructions. Roche NimbleGen recommends storing their expression arrays in the dark with desiccant at room temperature.</li>
<li>Load the array into the Precision Mixer Alignment Tool (PMAT) provided in the NimbleGen array processing accessories.</li>
<li>Load an X1 mixer onto the PMAT and remove the adhesive strip with forceps. Close the PMAT securely. Apply pressure to the mixer through the hole in the PMAT and slowly open so that the array and mixer disengage from the PMAT.</li>
<li>Firmly apply pressure with the brayer around the outside of the mixer to ensure the seal to the array is complete and to remove any bubbles. Place the array-mixer complex onto the hybridization system to warm.</li>
<li>Using a displacement pipette, slowly add 15 &mu;l of the labeled sample into the fill port. This is the hole located in the center of the mixer. Apply slow and steady pressure to the pipette so that the sample spreads evenly through the mixer without any air bubbles. Wipe off any excess sample from the port holes and seal port holes with adhesive strips.</li>
<li>Close the latch and start mixing of the hybridization solution using program B on the NimbleGen Hybridization System. Allow microarrays to hybridize for 16-20 hours.</li>
</ol>
Part 4: Post-hybridization Washes and Scanning of Microarray
The washing and scanning procedure is adapted from the Roche NimbleGen Arrays User's Guide for Gene Expression Analysis2.
<ol>
<li>Once hybridization is near completion prepare wash buffers as outlined in Table 2. Pre-warm Wash Buffer IA to 42&deg;C in a water bath.</li>
<li>Turn on GenePix 4000B array scanner and allow the scanner to warm for about 15 minutes before use.</li>
<li>Pour the pre-warmed Wash Buffer IA into a dish.</li>
<li>Remove the array-mixer complex from the NimbleGen Hybridization system and slide into the jig (supplied in the NimbleGen array processing accessories). Submerge assembly into pre-warmed Wash Buffer IA and carefully remove mixer by peeling away from slide.</li>
<li>Remove array from jig and agitate for 15 seconds in Wash Buffer IA.</li>
<li>Quickly transfer array into a slide rack in the room temperature Wash Buffer IB. Agitate the slide rack vigorously for 2 minutes.</li>
<li>Transfer the slide rack into Wash Buffer II and agitate for 1 minute.</li>
<li>Transfer the slide rack into the Wash Buffer III and agitate for 15 seconds.</li>
<li>Transfer the array into the slide drying centrifuge and spin for 2 minutes to remove all moisture. Proceed to scanning.</li>
<li>It is recommended to scan the array immediately since the dye is sensitive to both light and ozone.</li>
<li>Load the array, barcode down, into the GenePix 4000B array scanner.</li>
<li>Open the GenePix Pro software and proceed to scanning of array. The Roche NimbleGen gene expression arrays utilize a one color hybridization strategy. As a result only one wave length is required for scanning. Scan Cy3 fluorescence signal using the 532 nm laser. Open the hardware setting dialog box and set PMT value of the 532 nm laser to a value of 500.</li>
<li>In the hardware setting dialog box, set power to 100%, pixel size to 5 microns, lines to average to 1, and focus position to 0 microns.</li>
<li>Perform a preview scan and set scan area.</li>
<li>Perform scan.</li>
<li>Once scanning is complete check the image histogram. Ideally 1e-5 normalized counts should be at 65,000 saturation limit meaning that ~1-2% of the features are saturated. If the normalized counts are less than 1e-5, increase the PMT gain and rescan the slide. If the normalized counts are greater than 1e-5, decrease the PMT gain and rescan the slide.</li>
<li>Once scanning is complete save the image. It is recommended to choose a standard naming convention for images for ease in reference. The image can now be loaded into the data analysis program of preference for data normalization and calculation of intensity values.</li>
</ol>
Representative Results
You can first begin to assess the efficiency of the Cy3 fluorescence labeling reaction of the cDNA when beginning the washes with 0.1X SSC. During each subsequent wash the flow through should become less pink in color with the last flow through clear. In addition, the Cy3-labeled cDNA product should be visible on the column during these wash steps. If the flow through is bright pink in color or there is no pink product on the column, this is a good indication that the labeling reaction did not proceed correctly. The labeling reaction can be assessed quantitatively using the NanoDrop ND-1000 spectrophotometer to determine concentration of the labeled DNA and base to dye ratio (Figure 1). The labeling reaction routinely yields at least 10 &mu;g of labeled DNA in our laboratory. Dye incorporation can be calculated by using a simple base/dye ratio where the nucleotide to Cy3 ratio equals the A260/A550 ratio multiplied by 23.15. Values ranging from 50-80 indicate sufficient dye incorporation in the labeling reaction. If the labeling reaction yields a low concentration of DNA or poor dye incorporation, hybridization of the sample onto the microarray is not recommended and labeling reaction should be repeated.
Assessment of the quality of the hybridization can be conducted by evaluating the image histogram and the scanned image of the microarray (Figure 2). Acceptable hybridizations will have 1-2% of the features saturated (i.e., yielding 1e-5 normalized counts at the 65,000 saturation limit) with the PMT value set near 500. If the PMT value has to be adjusted drastically from 500 to attain 1e-5 normalized counts at the 65,000 saturation limit, this generally indicates poor hybridization of the labeled cDNA to the microarray. Data quality will be comprised if this data is continued through subsequent analysis. Hybridization quality can also be qualitatively assessed by viewing images of the scanned array (Figure 3). The Roche NimbleGen Hybridization Kit contains an alignment oligo mixture of Cy3- and Cy5-labeled 48mer oligonucleotides that hybridize to alignment features on the arrays. Qualitative assessment of the intensity of the alignment oligos to other features on the array permits a quality check on the hybridization efficiency of the fluorescently labeled cDNA onto the array. Following further analysis, a scatter plot of your analyzed data can be used to determine the relative levels of gene expression and to identify genes that are differentially expressed between your samples (Figure 4).
<img src="/files/ftp_upload/1471/1471figure1thumb.jpg" alt="Thumbnail of Figure 1" /> Figure 1. A NanoDrop spectrum from cDNA labeled with Cy3. Labeled cDNA yield and dye incorporation can be assessed using a NanoDrop ND-1000 spectrophotometer. Please <a href="https://www.jove.com/files/ftp_upload/1471/1471figure1.jpg">click here</a> to see a larger version of figure 1.
<img src="/files/ftp_upload/1471/1471figure2thumb.jpg" alt="Thumbnail of Figure 1" /> Figure 2. A GenePix histogram for microarray scan. Quality of the hybridization can be assessed by evaluating the image histogram. Acceptable hybridizations will have 1-2% of the features saturated with 1e-5 normalized counts at the 65,000 saturation limit with the PMT value set near 500. Please <a href="https://www.jove.com/files/ftp_upload/1471/1471figure2.jpg">click here</a> to see a larger version of figure 2.
<img src="/files/ftp_upload/1471/1471figure3.jpg" border="0" alt="Figure 3" /> Figure 3. A scanned microarray image. Hybridization quality can be assessed by viewing the scanned image of the microarray. The image should be examined for the presence of dust or other interfering factors prohibiting visualization of each feature and thus, preventing accurate calculation of the fluorescence intensity of the feature. The intensity of the alignment oligos can be compared to the other features on the array to assess hybridization efficiency. 
<img src="/files/ftp_upload/1471/1471figure4.jpg" border="0" alt="Figure 4" /> Figure 4. A scatter plot of an analyzed microarray experiment. The goal of this procedure is to identify genes that are differentially expressed in your samples. Microarray data can be further analyzed and viewed in a scatter plot to visualize genes that are differentially expressed.
Table 1. Components from Roche NimbleGen Hybridization Kit to add for hybridization step 2.
<table border="1">
<tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>Sample (dissolved in water)</td>
 <td>5 &mu;l</td>
 </tr>
 <tr>
 <td>2X Hybridization Buffer </td>
 <td>9 &mu;l</td>
 </tr>
 <tr>
 <td>Hyb A</td>
 <td>3.6 &mu;l</td>
 </tr>
 <tr>
 <td>Alignment Oligo</td>
 <td>0.4 &mu;l</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>18 &mu;l</td>
 </tr>
</tbody> 
</table>
Table 2. Wash buffers to be prepared for washing of arrays following hybridization. 
<table border="1">
<tbody>
 <tr>
 <td>Component</td>
 <td>Wash Buffer IA*</td>
 <td>Wash Buffer IB</td>
 <td>Wash Buffer II</td>
 <td>Wash Buffer III</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>225 ml</td>
 <td>225 ml</td>
 <td>225 ml</td>
 <td>225 ml</td>
 </tr>
 <tr>
 <td>10X Wash Buffer I, II, or III</td>
 <td>25 ml</td>
 <td>25 ml</td>
 <td>25 ml</td>
 <td>25 ml</td>
 </tr>
 <tr>
 <td>1 M DTT</td>
 <td>25 &mu;l</td>
 <td>25 &mu;l</td>
 <td>25 &mu;l</td>
 <td>25 &mu;l</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>250 ml</td>
 <td>250 ml</td>
 <td>250 ml</td>
 <td>250 ml</td>
 </tr>
 </tbody>
</table>
*Pre-warm to 42&deg;C

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> BioPrime Array CGH Genomic Labeling System Manual. , (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Roche NimbleGen Arrays User's Guide for Gene Expression Analysis. , (2008).</li></ol>

Protocolos microarray muitos, incluindo o detalhado aqui, use corantes fluorescentes como meio de medir e quantificar a hibridização. O corante fluorescente Cy3 é sensível a foto e degradação de ozônio. Recomenda-se que o procedimento seja realizado em uma iluminação mínima. Além disso, pequenas partículas de poeira pode interferir com a hibridação e digitalização. Especial atenção deve ser dada para garantir que a área de trabalho está limpo e livre de poeira. Há muitas...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Array Microcentrifuge Dryer</td>
<td>&nbsp;</td>
<td>ISC Bioexpress</td>
<td>C-1303-T</td>
<td>&nbsp;</td>
<tr>
<td>BioPrime Array CGH Genomic Labeling System</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18095-011</td>
<td>&nbsp;</td>
<tr>
<td>Cyanine 3-dCTP</td>
<td>&nbsp;</td>
<td>PerkinElmer</td>
<td>NEL576</td>
<td>&nbsp;</td>
<tr>
<td>GenePix 4000B</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>GENEPIX 4000B</td>
<td>&nbsp;</td>
<tr>
<td>Microcon YM-30</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>42411</td>
<td>&nbsp;</td>
<tr>
<td>NimbleGen Array Processing Accessories</td>
<td>&nbsp;</td>
<td>Roche NimbleGen</td>
<td>5223539001</td>
<td>&nbsp;</td>
<tr>
<td>NimbleGen Hybridization Kit</td>
<td>&nbsp;</td>
<td>Roche NimbleGen</td>
<td>5223474001</td>
<td>&nbsp;</td>
<tr>
<td>NimbleGen Hybridization System 4</td>
<td>&nbsp;</td>
<td>Roche NimbleGen</td>
<td>5223652001</td>
<td>&nbsp;</td>
<tr>
<td>NimbleGen Wash Buffer Kit</td>
<td>&nbsp;</td>
<td>Roche NimbleGen</td>
<td>5223504001</td>
<td>&nbsp;</td>
<tr>
<td>X1 Mixers</td>
<td>&nbsp;</td>
<td>Roche NimbleGen</td>
<td>5223725001</td>
<td>&nbsp;</td>		</tbody>
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global gene expression analysis using a zebrafish oligonucleotide microarray platform

Gene microarray tecnologia permite a medição quantitativa e perfil de expressão gênica dos níveis de transcrição em uma base de todo o genoma. Gene microarray tecnologia é utilizada em numerosas disciplinas biológicas em uma variedade de aplicações, incluindo análise global de expressão gênica em relação ao estágio de desenvolvimento, para um estado de doença, e em respostas tóxicas. Aqui, nós incluímos uma demonstração de análise global de expressão gênica utilizando um abrangente zebrafish específicos plataforma de microarranjos de oligonucleotídeos. O peixe-zebra plataforma de microarray expressão contém 385.000 sondas, 60 pares de bases de comprimento, interrogando 37.157 alvos com até 12 sondas por alvo. Para esta plataforma, todas as informações de cDNA e genômica disponível para o peixe-zebra foi coletado a partir de bases de dados genômicos diversos, incluindo Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC ( http://genome.ucsc.edu), e ZFIN (http://www.zfin.org). Como resultado dessa matriz de expressão fornece cobertura completa do transcriptoma zebrafish atual. O microarray expressão zebrafish foi impresso pela Roche NimbleGen (Madison, WI). Esta demonstração técnica inclui a marcação fluorescente de um produto cDNA, hibridação do produto marcado para a plataforma de cDNA microarray, e digitalização de matriz para a aquisição de sinal usando a estratégia de uma análise de cor.

Watch this Scientific Journal Video about Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform at JoVE.com

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarrays são ferramentas poderosas no perfil de expressão gênica ao nível do genoma. Esta tecnologia tem aplicação em uma variedade de disciplinas biológicas, incluindo biologia do desenvolvimento e toxicologia. Neste vídeo, detalhamos um protocolo para análise global de expressão gênica utilizando uma plataforma de microarray abrangente de oligonucleotídeos para o peixe-zebra.

Análise da expressão global de Gene usando uma plataforma Microarray Zebrafish Oligonucleotide

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

global-gene-expression-analysis-using-zebrafish-oligonucleotide

Research

JoVE Journal

Biology

11.6K visualizações.  Purdue University. Gene microarrays são ferramentas poderosas no perfil de expressão gênica ao nível do genoma. Esta tecnologia tem aplicação em uma variedade de disciplinas biológicas, incluindo biologia do desenvolvimento e toxicologia. Neste vídeo, detalhamos um protocolo para análise global de expressão gênica utilizando uma plataforma de microarray abrangente de oligonucleotídeos para o peixe-zebra.

Vídeo: Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Bacterial Gene Expression Analysis Using Microarrays

Análise de Expressão Gênica Utilizando bacteriana Microarrays

Biologia

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.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Isolamento de RNA Síntese Zebrafish e cDNA embrionárias para Análise de Expressão Gênica

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

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.

Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células NAMALWA

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 ...

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.

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.

Análise de Expressão Gênica em Emerald Borer Ash ( Agrilus planipennis) Usando Quantitativa em Tempo Real-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 ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Seções incluído em parafina e congelados de Drosophila Músculos Adulto

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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.

Determinar perfis de expressão genética em C. elegans Usando Microarray e PCR em tempo real

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.

Na reconstituição in vitro da atividade T. Telomerase castaneum

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

Gene Trapping Using Gal4 in Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional mutagenesis using fluorescent reporters to tag expression of mutated genes. Several laboratories have constructed and tested enhancer- and gene-trap vectors in zebrafish, using fluorescent proteins, Gal4- and lexA- based transcriptional activators as reporters 1-7. These vectors had two potential drawbacks: suboptimal stringency (e.g. lack of ability to differentiate between enhancer- and gene-trap events) and low mutagenicity (e.g. integrations into genes rarely produced null alleles). Gene Breaking Transposon (GBTs) were developed to address these drawbacks 8-10. We have modified one of the first GBT vectors, GBT-R15, for use with Gal4-VP16 as the primary gene trap reporter and added UAS:eGFP as the secondary reporter for direct detection of gene trap events. Application of Gal4-VP16 as the primary gene trap reporter provides two main advantages. First, it increases sensitivity for genes expressed at low expression levels. Second, it enables researchers to use gene trap lines as Gal4 drivers to direct expression of other transgenes in very specific tissues. This is especially pertinent for genes with non-essential or redundant functions, where gene trap integration may not result in overt phenotypes. The disadvantage of using Gal4-VP16 as the primary gene trap reporter is that genes coding for proteins with N-terminal signal sequences are not amenable to trapping, as the resulting Gal4-VP16 fusion proteins are unlikely to be able to enter the nucleus and activate transcription. Importantly, the use of Gal4-VP16 does not pre-select for nuclear proteins: we recovered gene trap mutations in genes encoding proteins which function in the nucleus, the cytoplasm and the plasma membrane.

This protocol describes the method of gene trap insertional mutagenesis using Gal4-VP16 as the primary reporter and GFP/RFP as secondary reporters in zebrafish. Approximately one in ten high-expressing F0 fish yield gene trap progeny co-expressing GFP and RFP. The screening procedure can be readily scaled to adapt to the size of the laboratory performing the insertional mutagenesis screen.

Gene Trapping Usando Gal4 em Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional ...