Name: Bacterial Gene Expression Analysis Using Microarrays (Video) | JoVE Journal
Uploaded: 2007-05-28T00:00:00.000+00:00
Duration: 29 min 41 s
Description: Learn bacterial gene expression profiling using indirect labeling and microarray hybridization with Cy3 and Cy5 fluorescent dyes. This protocol compares wild type and mutant samples to detect differential gene expression accurately.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

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/pubmed?term=((TIGR+Aminoallyl+Labeling+of+RNA+for%5BTitle%5D)+AND+%22Microarrays+%26+TIGR+Microarray+Labeled+Probe+Hybridization%22%5BJournal%5D)">TIGR Aminoallyl Labeling of RNA for.</a> Microarrays & TIGR Microarray Labeled Probe Hybridization. , Forthcoming.</li>
<li>Gilbert,, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TIGR+Microbial+RNA+Aminoallyl+Labeling+for+Microarrays+%26+Hybridization+of+probed+labels%5BTitle%5D)+AND+%22%22%5BJournal%5D)">TIGR Microbial RNA Aminoallyl Labeling for Microarrays & Hybridization of probed labels.</a> , Forthcoming.</li>
<li>Hedge,, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+concise+guide+to+cDNA+Microarray+analysis-II%5BTitle%5D)+AND+%22%22%5BJournal%5D)">A concise guide to cDNA Microarray analysis-II.</a> , Forthcoming.</li>
</ol>

<table><tbody><tr><td>AA-dUTP</td><td></td><td>Sigma-Aldrich</td><td>A0410</td><td>5-(3-aminoallyl)-2&amp;rsquo;deoxyuridine-5&amp;rsquo;-triphosphate</td></tr><tr><td>dNTP</td><td></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></td><td>Amersham</td><td>27-2166-01</td><td>3mg/mL</td></tr><tr><td>SuperScript III RT</td><td></td><td>Invitrogen</td><td>80800444</td><td>200U/&amp;#x03BC;L</td></tr><tr><td>CyDye&amp;#8482;</td><td></td><td>Amersham</td><td>RPN5661</td><td>Post-Labelling Reactive Dye Pack</td></tr><tr><td>QIAquick</td><td></td><td>Qiagen</td><td>28104</td><td>PCR Purification kit</td></tr><tr><td>1 M K2HPO4</td><td></td><td></td><td></td><td></td></tr><tr><td>1 M KH2PO4</td><td></td><td></td><td></td><td></td></tr><tr><td>1 M KPO4</td><td>Buffer</td><td></td><td></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></td><td></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></td><td></td><td>Diluting 1 M KPO4, pH 8.5 to 4 mM with sterile water</td></tr><tr><td>Sodium Carbonate Buffer</td><td></td><td></td><td></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>

bacterial gene expression analysis using microarrays

Bacterial Gene Expression Analysis Using Microarrays

Hi, my name is Ena and We routinely do gene expression profiling in the lab. And we use this technique to monitor differential regulated genes between meth and wild type. And today I have a wild type and meth RNA samples and we all do C first.We use a indirect labeling methods and the advantage of this technique is to prevent eye bias that can occur in direct labeling. And to do that we have RNA samples and we opt obtain three microgram of RNA in 13 microliter of to total volume. And we add 2.5 micro of random he in our parks.And we'll incubate sample at seven five degree to have to the nature of the RNA for eight minutes. And now we'll add the, we'll add reverse transcription cocktail that contains AM L-D-L-D-U-T-P continu mix, reverse transcriptase buffer and also ET TT Big it down And incubate the sample at 42 degree for four hours. So after four hours we just take the samples out.And now in the tube we have CDNA and RNA mix. And what we want to do is do hydrox RNA by adding sodium hydroxide and EDTA and final concentration for sodium hydroxide should be a hundred molar. And final concentration for e BT should be five molar.And now we will incubate samples at 65 degree water bat

bacterial-gene-expression-analysis-using-microarrays

Research

JoVE Journal

Biology

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.

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

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.

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Microarray Analysis for Saccharomyces cerevisiae

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

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

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

This manuscript describes the use of state-of-the-art technology provided by DNA-microarrays. Microarrays provide an overview of the transcriptomic changes in bacteria incurred under a specific condition. Moreover, we highlight the ease by which large amounts of data can be analyzed by using convenient in-house developed software packages.

A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

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.

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

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

Learn bacterial gene expression profiling using indirect labeling and microarray hybridization with Cy3 and Cy5 fluorescent dyes. This protocol compares wild type and mutant samples to detect differential gene expression accurately.

Bacterial Gene Expression Analysis Using Microarrays

Explore More Videos