Reverse Transcriptase Reaction
Turn heating block to 70-80&deg;C and 42&deg;C. Water baths can also be used in this step. If using heating blocks, put some water so that the heat is uniform.
<ol>
<li>Priming reaction<ol>
<li>Take 3 &mu;g of RNA</li>
<li>Adjust volume to 13 &mu;l with RNase free water</li>
<li>Add 2.5 &mu;l of random hexamer primers (2mg/mL)</li>
</ol></li>
<li>Mix well and incubate at 70-80&deg;C for 8 minutes. Then, place on ice for 5 min.</li>
<li>Prepare RT cocktail (14.5 &mu;l/ rxn): 
<table border="0">
<tbody>
<tr>
<td>Component </td>
<td>Volume</td>
</tr>
<tr>
<td>5X First Strand buffer</td>
<td>6 &micro;l</td>
</tr>
<tr>
<td>0.1 M DTT </td>
<td>3 &micro;l</td>
</tr>
<tr>
<td>25X aminoallyl-dNTP mix</td>
<td>1.2 &micro;l</td>
</tr>
<tr>
<td>SuperScript II RT (200U/&micro;l)</td>
<td>2.3 &micro;l</td>
</tr>
<tr>
<td>Water </td>
<td>2.0 &mu;l</td>
</tr>
</tbody>
</table>
 * For n reactions, prepare Master Mix for n+0.5 aliquots of cocktail to assure that you will not run out. </li>
<li>Add 14.5 &mu;l to cooled reactions.</li>
<li>Mix (do not vortex) and incubate at 42&deg;C for 3-4 hours</li>
<li>Samples can be stored at -20&deg;C if necessary.</li>
</ol>
Hydrolyze RNA
<ol>
<li>To each sample, add 3 &micro;l of 1 N NaOH and 0.6 &micro;l of 0.5 M EDTA. (Final conc.s = 100mM NaOH, 10mM EDTA)</li>
<li>Mix and incubate at 65&deg;C for 10 minutes.</li>
<li>Neutralize by adding 33.6 &mu;l 1M HEPES, pH=7.0 (final conc.= 500mM HEPES)</li>
<li>Samples can be stored at -20&deg;C, if necessary.</li>
</ol>
Reaction Purification: Removal of unincorporated aa-dUTP and free amines
Note: This purification protocol is modified from the Qiagen QIAquick PCRpurification kit protocol. The phosphate wash and elution buffers are substituted for the Qiagen supplied buffers because the Qiagen buffers contain free amines that compete with the Cy dye coupling reaction. The columns from the mini-prep kit can also be used.
<ol>
<li>Mix cDNA reaction with 300 &micro;l (or 5X reaction volume = 336 &mu;l) buffer PB (Qiagen supplied) and transfer to QIAquick column.</li>
<li>Place the column in a 2 ml collection tube (Qiagen supplied) and centrifuge at ~13,000 rpm for 1 minute. Empty collection tube.</li>
<li>To wash, add 750 &micro;l phosphate wash buffer (not Qiagen's) to the column and centrifuge at ~13,000 rpm for 1 minute.</li>
<li>Empty the collection tube and repeat the 750 &micro;l wash and centrifugation step.</li>
<li>Empty the collection tube and centrifuge the column an additional 1 minute at maximum speed.</li>
<li>Transfer column to a new 1.5 mL microfuge tube and carefully add 30 &micro;l phosphate elution buffer. (see solution preparation section)</li>
<li>Incubate for 1 minute at room temperature.</li>
<li>Elute by centrifugation at ~13,000 rpm for 1 minute.</li>
<li>Elute a second time (with another 30 &micro;l of phosphate elution buffer) into the same tube by repeating steps 6-8. The final elution volume should be ~60 &micro;l.</li>
<li>The amount of cDNA can be quantified at this stage: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ng cDNA = (dilution factor)(OD260)(37)(total volume eluted from column)</li>
<li>Dry sample in a speed vac at 45&deg;C.</li>
<li>Samples can be stored at -20&deg;C if necessary.</li>
</ol>
Coupling aa-cDNA to Cy Dye Ester
<ol>
<li>Resuspend aminoallyl-labeled cDNA with 10 &mu;l 50 mM Na-bicarbonate, pH=9.0 if necessary. (This buffer should be made fresh every two weeks)</li>
<li>Working in a dark place, add the probe to a tube of the appropriate Cy dye. Pipet up and down several times to resuspend the dye.</li>
<li>Incubate the reaction for 1 hour in the dark at room temperature. (Incubation can go up to two hours)</li>
</ol>
Reaction Purification II: Removal of uncoupled dye
<ol>
<li>To the reaction add 35 &micro;l 100 mM NaOAc pH 5.2. Mix thoroughly.</li>
<li>Add 250 &micro;l ( or 5X reaction volume = 225 &mu;l) Buffer PB (Qiagen supplied) to each sample and mix by pipetting up and down.</li>
<li>Place a QIAquick spin column in a 2 mL collection tube (Qiagen supplied), apply the sample to the column, and centrifuge at ~13,000 for 1 minute. Empty collection tube.</li>
<li>To wash, add 0.75 mL Buffer PE (Qiagen supplied) to the column and centrifuge at ~13,000 for 1 minute.</li>
<li>Repeat step 4.</li>
<li>Empty collection tube and centrifuge column for an additional 1 minute at maximum speed.</li>
<li>Place column in a clean 1.5 mL microfuge tube and carefully add 30 &micro;l Buffer EB (Qiagen supplied) to the center of the column membrane.</li>
<li>Incubate for 1 minute at room temperature.</li>
<li>Elute by centrifugation at ~13,000 rpm for 1 minute.</li>
<li>Elute a second time into the same tube by repeating steps 7-9. The final eluted volume should be ~60 &micro;l.</li>
</ol>
Analysis of Labeling Reaction
The amount of cDNA and label can be quantified at this stage:
<ul>
<li>Measure OD 260, 550 and 650 (Blank is water.) Use nanodrop microarray program.</li>
<li>Calculate the pmol of dyes incorporation and pmol DNA and nuc/dye ratio with given equations below: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; pmol nucleotides = [OD260 * volume (&mu;L) * 37 ng/&mu;L * 1000 pg/ng] / 324.5 pg/pmol</li>
</ul>
Note: 1 OD260 = 37 ng/&mu;L for cDNA; 324.5 pg/pmol average molecular weight of a dNTP) &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; pmol Cy3 = OD550 * volume (&mu;L)/0.15 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; pmol Cy5 = OD650 * volume (&mu;L)/0.25 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; nucleotides/dye ratio = pmol cDNA/pmol Cy dye
If you are using nanodrop, it already gives pmol/&mu;l for each dye. You only need to multiply this number by volume to get total pmol dye incorporation.
Note: &gt;150-200 pmol of dye incorporation per sample and a ratio of less than 20 nucleotides/dye molecules is optimal for hybridizations.
<ul>
<li>Samples can be stored at -20&deg;C at dark. 
<ul>
<li>Combine probes to be hybridized together and dry down in speedvac at 42&deg;C.</li>
<li>Samples can be stored at -20&deg;C until ready to hyb.</li>
</ul>
</li>
</ul>
Pre-hyb slides
<ol>
<li>Place slide face down over RT 100 mL water in a clean red slide holder for 3-5 minutes</li>
<li>Snap dry the slide by placing face up on a 100&deg;C heating block for a few seconds - watch for the condensation to vanish</li>
<li>UV cross-link the slide at 250 mJoules ( Enter 2500 to the UV crosslinker)</li>
<li>Hydrate slides by dipping in pre-warmed 42&deg;C water. Spin 5 min at 700 RPM at RT.</li>
<li>Incubate slides in 50ml Coplin jar containing pre-warmed pre-hyb (5XSSC, 1% BSA, 0.1% SDS) for at least 45 minutes @ 42&deg;C, and incubation can go longer if necessary. 50 mL pre-hyb buffer: 12.5 mL 20x SSC, 10 ml 5% BSA, 0.5 ml 10 % SDS, 27 mL water </li>
<li>Dip slides in pre-warmed water (42&deg;C) and agitate a couple of minutes to remove pre-hyb buffer.</li>
<li>Rinse in isopropanol and spin 5 min at 700 rpm, room temp, to dry.</li>
</ol>
Preparing samples for hybridization
<ol>
<li>Resuspend probe in water (volumes are given below) 
<table border="0">
<tbody>
<tr>
<td>For total vol. = 30&mu;l </td>
<td>For total vol. = 35&mu;l </td>
<td>For total vol.= 40&mu;l </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>19.8 &mu;l water </td>
<td>23.55 &mu;l water </td>
<td>27.2 &mu;l water </td>
<td> </td>
</tr>
<tr>
<td>1.5 &mu;l</td>
<td>1.5 &mu;l</td>
<td>1.5 &mu;l</td>
<td>10 mg/ml salmon sperm DNA (Invitrogen)</td>
</tr>
<tr>
<td>1.2 &mu;l</td>
<td>1.2 &mu;l</td>
<td>1.2 &mu;l</td>
<td>12.5 mg/ml tRNA</td>
</tr>
<tr>
<td>4.5 &mu;l</td>
<td>5.25 &mu;l</td>
<td>6 &mu;l</td>
<td>20XSSC (3X final)</td>
</tr>
<tr>
<td>3 &mu;l</td>
<td>3.5 &mu;l</td>
<td>4 &mu;l</td>
<td>1% SDS</td>
</tr>
</tbody>
</table>
 Note: The order of addition of the reagents are important. Do not prepare master mix. </li>
<li>Boil for&nbsp;5 minutes, then spin 5 minute at 14,000 rpm. Cool for&nbsp;2 min at room temp.</li>
</ol>
Hybridization
<ol>
<li>Add 10-12 &micro;l of 3X SSC to the wells of the hybridization chamber on each edge and in the middle.</li>
<li>Add a clean lifter slip to the appropriate area of the slide and gently load in hybridization solution (30 -40 &mu;l). There should be extra sample on either edge of the cover slip.</li>
<li>Place the slide in the chamber. Tighten the screws and place in a 65&deg;C water bath overnight. Do not place the chamber directly on the bottom of the water bath. Place a block on top of the uppermost chamber to hold everything in place. Keep the lid on the water bath to protect the light sensitive samples.</li>
</ol>
Hyb Washes
<ol>
<li>Prepare the following buffers: 
<ul>
<li>1X SSC, 0.05 % SDS</li>
<li>0.06 X SSC</li>
</ul>
</li>
<li>After the incubation unseal hybridization chamber. Remove the slide from the chamber, taking care not to disturb the coverslip.</li>
<li>To remove coverslips, submerge slides in a dish containing warm 1X SSC, 0.05 % SDS wash buffer coverslip facing the bottom of the dish. The coverslip will fall off by moving the slide side to side.</li>
<li>After the coverslip is removed, place the slide in a rack a fresh bath with warm 1XSSCm 0.05% SDS. Agitate for a couple of minutes</li>
<li>Transfer slides plus rack to a bath of 0.06 X SSC.</li>
<li>Transfer slides to another bath of 0.06 X SSC containing a fresh rack, blotting the slide on a paper towel to remove as much SDS-containing buffer as possible before placing in the fresh bath.</li>
<li>Agitate slides for a couple of minutes.</li>
<li>Spin immediately at&nbsp;room temperature for 5 min @ 700 rpm, then slides are ready to scan. Store in the dark until scanned.</li>
</ol>

<ol>
	<li>Hasseman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIGR+Aminoallyl+Labeling+of+RNA+for.">TIGR Aminoallyl Labeling of RNA for.</a> Microarrays &amp; TIGR Microarray Labeled Probe Hybridization. , .</li><li>Gilbert, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIGR+Microbial+RNA+Aminoallyl+Labeling+for+Microarrays+&amp;+Hybridization+of+probed+labels.">TIGR Microbial RNA Aminoallyl Labeling for Microarrays &amp; Hybridization of probed labels.</a> , .</li><li>Hedge, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+concise+guide+to+cDNA+Microarray+analysis-II.">A concise guide to cDNA Microarray analysis-II.</a> , .</li></ol>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>AA-dUTP</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0410</td>
<td>5-(3-aminoallyl)-2&#x2019;deoxyuridine-5&#x2019;-triphosphate</td>
</tr>
<tr>
<td>dNTP</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>27-2035-01</td>
<td>100 mM dNTP Set PCR grade</td>
</tr>
<tr>
<td>Random Hexamer primers</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>27-2166-01</td>
<td>3mg/mL</td>
</tr>
<tr>
<td>SuperScript III RT</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>80800444</td>
<td>200U/&#x03BC;L</td>
</tr>
<tr>
<td>CyDye&#x2122;</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN5661</td>
<td>Post-Labelling Reactive Dye Pack</td>
</tr>
<tr>
<td>QIAquick</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>PCR Purification kit</td>
</tr>
<tr>
<td>1M K2HPO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1M KH2PO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1M KPO4</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To make a 1M Phosphate buffer (KPO4, pH 8.5-8.7) combine:
9.5 mL 1M K2HPO4 and 0.5 ml 1M KH2PO4
</td>
</tr>
<tr>
<td>Phosphate wash buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 100 mL Phosphate wash buffer (5 mM KPO4, pH 8.0, 80% EtOH) mix: 0.5 ml 1 M KPO4 pH 8.5 + 15.25 mL MilliQ water + 84.25 mL 95% ethanol. Wash buffer will be slightly cloudy.
** IMPORTANT: Phosphate wash buffer should be prepared daily.</td>
</tr>
<tr>
<td>Phosphate elution buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Diluting 1 M KPO4, pH 8.5 to 4 mM with sterile water</td>
</tr>
<tr>
<td>Sodium Carbonate Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>(Na2CO3): 0.5M, pH 9.0. Dissolve 4.2 g NaHCO3 in 80 mL of sterile water and adjust pH to 9.0 with 10 N NaOH; bring volume up to 100 mL with sterile water. To make a 50 mM solution for the dye coupling reaction dilute 1:10 with water.
Note: Carbonate buffer changes composition over time; make it fresh every couple of weeks to a month.</td>
</tr>		</tbody>
		</table>
		</div>

bacterial gene expression analysis using microarrays

Watch this Scientific Journal Video about Bacterial Gene Expression Analysis Using Microarrays at JoVE.com

Bacterial Gene Expression Analysis Using Microarrays

bacterial-gene-expression-analysis-using-microarrays

Research

JoVE Journal

Biology

8.7K Views.  University of California Santa Cruz - UCSC. Bacterial Gene Expression Analysis Using Microarrays

Video: 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 ...

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage without prior knowledge of what genes the community might be expressing. Thus it provides the most unbiased perspective on community gene expression in situ. Environmental transcriptomics protocols are technically difficult since prokaryotic mRNAs generally lack the poly(A) tails that make isolation of eukaryotic messages relatively straightforward 1 and because of the relatively short half lives of mRNAs 2. In addition, mRNAs are much less abundant than rRNAs in total RNA extracts, thus an rRNA background often overwhelms mRNA signals. However, techniques for overcoming some of these difficulties have recently been developed. A procedure for analyzing environmental transcriptomes by creating clone libraries using random primers to reverse-transcribe and amplify environmental mRNAs was recently described was successful in two different natural environments, but results were biased by selection of the random primers used to initiate cDNA synthesis 3. Advances in linear amplification of mRNA obviate the need for random primers in the amplification step and make it possible to use less starting material decreasing the collection and processing time of samples and thereby minimizing RNA degradation 4. In vitro transcription methods for amplifying mRNA involve polyadenylating the mRNA and incorporating a T7 promoter onto the 3 end of the transcript. Amplified RNA (aRNA) can then be converted to double stranded cDNA using random hexamers and directly sequenced by pyrosequencing 5. A first use of this method at Station ALOHA demonstrated its utility for characterizing microbial community gene expression 6.

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

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.

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

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.

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 ...

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.

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

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

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.

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