Ters Transcriptase Reaksiyon 70-80 ° C ve 42 ° C ısıtma bloğu açın Su banyoları da bu adımda kullanılabilir. Isıtma bloklar kullanılıyorsa, ısı tekdüze böylece biraz su koyun. <ol><li> Astar tepki <ol><li> RNA 3 mg alın </li><li> 13 ul RNaz ücretsiz su ile ses seviyesini ayarlayın. </li><li> Rastgele hexamer primerler (2mg/mL) 2.5 ul ekle </li></ol></li><li> İyice karıştırın ve 8 dakika 70-80 ° C&#39;de inkübe edin. Daha sonra, 5 dakika buz koyun. </li><li> : RT kokteyl hazırlayın (14.5 ul / RXN) <table border="0"><tbody><tr><td> Bileşen </td><td> Hacim </td></tr><tr><td> 5X İlk Strand tampon </td><td> 6 ul </td></tr><tr><td> 0.1 M DTT </td><td> 3 ul </td></tr><tr><td> 25X aminoallyl dNTP karışımı </td><td> 1.2 ul </td></tr><tr><td> Üst Simge II RT (200U/μl) </td><td> 2.3 ul </td></tr><tr><td> Su </td><td> 2.0 ul </td></tr></tbody></table> * N reaksiyonlar için tükendi sağlamak için kokteyl n 0,5 alikotları Master Mix hazırlamak. </li><li> Soğutmalı reaksiyonlar 14.5 ul ekleyin. </li><li> Mix (vorteks) ve 42 ° C&#39;de 3-4 saat inkübe </li><li> Gerekirse örnekleri -20 ° C&#39;de saklanabilir. </li></ol> Hidroliz RNA <ol><li> Her bir örnek için, 1 N NaOH 3 ul ve 0,5 M EDTA 0.6 ul ekleyin. (Final conc.s = 100 mM NaOH, 10mM EDTA) </li><li> Mix ve 65 ° C&#39;de 10 dakika için. </li><li> Nötralize 33.6 ul 1M Hepes ekleyerek, pH = 7.0 (son kons .= 500mm Hepes) </li><li> Örnekleri, gerekirse, -20 ° C&#39;de saklanabilir. </li></ol> Reaksiyon Arıtma: adi aa-dUTP kaldırılması ve serbest aminler Not: Bu arıtma protokolü Qiagen QIAquick PCRpurification kiti protokol değiştirilmiş. Qiagen tamponlar Cy boya kaplin reaksiyonu ile rekabet ücretsiz aminler içerdiğinden fosfat yıkama ve yıkama tamponları Qiagen sağlanan tamponlar yerine. Mini hazırlık kiti sütunları da kullanılabilir. <ol><li> 300 ul (veya 5X reaksiyon hacmi = 336 ul) tampon PB (Qiagen temin) ile cDNA reaksiyon karışımı ve QIAquick sütun transfer. </li><li> 2 ml toplama tüpü sütun (Qiagen temin) koyun ve 1 dakika için ~ 13.000 rpm&#39;de santrifüj. Boş toplama tüpü. </li><li> Yıkamak için, sütun ve 1 dakika için ~ 13.000 rpm&#39;de santrifüj 750 ul fosfat yıkama tamponu (değil Qiagen) ekleyebilirsiniz. </li><li> Toplama tüpüne boşaltın ve 750 ul yıkama ve santrifüj basamağı tekrarlayın. </li><li> Toplama tüpüne boşaltın ve sütun maksimum hızda bir ek 1 dakika santrifüj. </li><li> Yeni bir 1.5 mL mikrofuge&#39;de tüp sütun aktarın ve 30 ul fosfat elüsyon tampon dikkatlice ekleyin. (Çözelti hazırlama bölümüne bakınız) </li><li> 1 dakika oda sıcaklığında inkübe edin. </li><li> Için ~ 1 dakika 13.000 rpm&#39;de santrifüj Zehir. </li><li> Zehir adımları yineleyerek 6-8 ikinci kez aynı tüpe (30 ul fosfat elution tampon başka bir). Son elüsyon hacmi ~ 60 ul olmalıdır. </li><li> CDNA miktarı bu aşamada sayısal olabilir: ng cDNA = (seyreltme faktörü) (OD260) (37) (toplam hacmi sütun yıkandı,) </li><li> 45 vac hız Kuru örnek ° C </li><li> Gerekirse örnekleri -20 ° C&#39;de saklanabilir. </li></ol> Boya Ester Cy aa-cDNA Kaplin <ol><li> Yeniden süspanse 10 ul 50 mM Na-bikarbonat, pH = 9.0 gerekirse cDNA aminoallyl etiketli. (Bu tampon her iki hafta taze yapılmalıdır) </li><li> Karanlık bir yerde çalışmak, uygun Cy boya bir tüp prob ekleyin. Pipetleyin yukarı ve aşağı birkaç kez boya tekrar süspansiyon. </li><li> 1 saat reaksiyon oda sıcaklığında karanlıkta inkübe edin. (Kuluçka iki saat kadar gidebilir) </li></ol> Reaksiyon Arıtma II: Kuplajsız boya kaldırılması <ol><li> Reaksiyon için 35 ul 100 mM NaOAc pH 5.2 ekleyin. Iyice karıştırınız. </li><li> 250 ul ekleme (veya 5X reaksiyon hacmi = 225 ul) Buffer her bir örnek ve yukarı ve aşağı pipetleme karışım PB (Qiagen temin) </li><li> 2 ml toplama tüpüne bir QIAquick spin kolon (Qiagen temin) yerleştirin, sütun örnek uygulamak ve 1 dakika için ~ 13.000 santrifüj. Boş toplama tüpü. </li><li> Yıkamak için, 1 dakika için ~ 13.000 kolon ve santrifüj 0.75 ml Tampon PE (Qiagen temin) ekleyin. </li><li> 4. adımı yineleyin. </li><li> Boş toplama tüpü ve ek olarak 1 dakika boyunca maksimum hızda santrifüj sütun. </li><li> Dikkatlice yerleştirin temiz bir 1.5 mL mikrofuge&#39;de tüp, sütun ve sütun membran merkezine 30 ul Tampon EB (Qiagen temin) ekleyin. </li><li> 1 dakika oda sıcaklığında inkübe edin. </li><li> Için ~ 1 dakika 13.000 rpm&#39;de santrifüj Zehir. </li><li> Ikinci kez tekrar aynı tüpe Zehiradımları 7-9. Son yıkandı, hacmi ~ 60 ul olmalıdır. </li></ol> Etiketleme Reaksiyon Analizi CDNA miktarı ve etiket, bu aşamada sayısal olabilir: <ul><li> OD 260, 550 ve 650 (Boş sudur.) Nanodrop mikroarray programı kullanın ölçün. </li><li> Boyalar dahil edilmesi ve aşağıda verilen denklemler ile pmol DNA ve boya / nük oranı pmol hesaplayın: pmol nükleotidler = [OD260 * hacmi (mcL) * 37 ng / mcL * 1000 pg / ng / 324,5 pg / pmol </li></ul> Not: 1 OD260 = cDNA için 37 ng / ml; bir dNTP 324,5 pg / pmol ortalama molekül ağırlığı) pmol Cy3 = OD550 * hacmi (mcL) / 0.15 pmol Cy5 = OD650 * hacmi (mcL) / 0.25 nükleotidler / boya oranı = pmol pmol / cDNA Cy boya Nanodrop kullanıyorsanız, zaten her boya için ul / pmol verir. Sadece toplam pmol boya birleştirmek için hacim itibariyle bu sayıyı çarpmak gerekir. Not: boya dahil edilmesi ve örnek başına, 20&#39;den az nükleotidler / boya molekülleri bir oran&gt; 150-200 pmol döllemeler için en uygunudur . <ul><li> Örnekleri koyu -20 ° C&#39;de saklanabilir. <ul><li> Birlikte melezleşmiştir probları birleştirin ve 42 speedvac kuru ° C </li><li> Örnekler HYB için hazır olana kadar -20 ° C&#39;de saklanabilir. </li></ul></li></ul> Pre-HYB slaytlar <ol><li> 3-5 dakika temiz bir kırmızı slayt tutucuya yerleştirin slayt yüzü aşağı RT üzerine 100 ml su </li><li> Birkaç saniye için 100 ° C ısıtma bloğu yüzü yukarı yerleştirerek slayt kuru Snap - yoğunlaşma kaybolmaya izlemek </li><li> 250 mJoules UV çapraz bağ slayt (UV çapraz bağlayıcı 2500 girin) </li><li> Önceden ısıtılmış 42 ° C su daldırma Hidrat slaytlar. RT 700 RPM 5 dk Spin. </li><li> Gerekirse en az 45 dakika önceden ısıtılmış ön-HYB (5XSSC,% 1 BSA,% 0.1 SDS) içeren 50ml Coplin kavanoza inkübe slaytlar @ 42 ° C ve inkübasyon uzun gidebilirsiniz. 50 ml-HYB ön tamponu: 12.5 ml 20x SSC, 10 ml% 5 BSA, 0.5 ml% 10 SDS, 27 ml su </li><li> Dip önceden ısıtılmış su (42 ° C) slaytlar ve HYB ön tampon kaldırmak için bir kaç dakika ajitasyon. </li><li> Isopropanol durulayın ve 700 rpm, oda sıcaklığında, kuru 5 dakika döndürün. </li></ol> Hibridizasyon için numunelerin hazırlanması <ol><li> Su içinde süspanse edin prob (birimler aşağıda verilmiştir) <table border="0"><tbody><tr><td> Toplam vol. = 30μl </td><td> Toplam vol. = 35μl </td><td> Toplam vol .= 40μl </td><td></td></tr><tr><td> 19.8 ul su </td><td> 23.55 ul su </td><td> 27.2 ul su </td><td> </td></tr><tr><td> 1.5 ul </td><td> 1.5 ul </td><td> 1.5 ul </td><td> 10 mg / ml somon sperm DNA (Invitrogen) </td></tr><tr><td> 1.2 ul </td><td> 1.2 ul </td><td> 1.2 ul </td><td> 12.5 mg / ml tRNA </td></tr><tr><td> 4,5 ul </td><td> 5.25 ul </td><td> 6 ul </td><td> 20XSSC (3X son) </td></tr><tr><td> 3 ul </td><td> Ul 3.5 </td><td> 4 ul </td><td> 1% SDS </td></tr></tbody></table> Not: reaktiflerin ek sırası önemlidir. Ana karışımı hazırlamak etmeyin. </li><li> 5 dakika kaynatılır, sonra 14.000 rpm&#39;de 5 dakika dönerler. 2 dakika süreyle oda sıcaklığında soğutun. </li></ol> Melezleme <ol><li> Her kenar ve orta hibridizasyon odasının kuyulardan 3X DGM&#39;nin 10-12 ul ekleyin. </li><li> Slayt uygun bir alana, temiz bir kaldırıcı kayma ekleyin ve hafifçe hibridizasyon çözüm yük (30 -40 ul). Kapak kayma iki kenarında ekstra örnek olmalıdır. </li><li> Odasında slayt yerleştirin. Bir gecede 65 ° C su banyosunda vida ve yer sıkın. Su banyosu alt doğrudan odasına koymayın. Her şeyi yerinde tutmak için en üst odasının üstünde bir blok yerleştirin. Işığa duyarlı örnekleri korumak için su banyosu kapağı tutun. </li></ol> HYB yıkar <ol><li> Aşağıdaki tamponlar hazırlayın: <ul><li> 1X SSC,% 0.05 SDS </li><li> 0.06 X SSC </li></ul></li><li> Inkübasyon mührünü kırmak hibridizasyon odasına sonra. Lamel rahatsız etmemek için özen slayt odasından çıkarın. </li><li> Lamelleri kaldırmak için, sıcak 1X SSC, çanak alt bakan% 0.05 SDS yıkama tamponu lamel içeren bir tabak içinde batığın slaytlar. Lamel slayt yan tarafına hareket ettirerek kapalı düşecek. </li><li> Lamel kaldırıldıktan sonra slayt, taze bir banyo, sıcak 1XSSCm% 0.05 SDS bir rafa koyun. Birkaç dakika için kışkırtıcılık </li><li> 0.06 X SSC banyo slaytlar artı rafa aktarın. </li><li> Slaytları başka bir ba aktarın0.06 X SSC, taze hamamı yerleştirmeden önce mümkün olduğunca çok SDS içeren tampon kaldırmak için bir kağıt havlu üzerine slayt blot, taze raf içeren th. </li><li> Birkaç dakika için slaytlar çalkalayın. </li><li> 5 dak 700 rpm için oda sıcaklığında hemen Spin, daha sonra slayt tarama için hazır. Taranan kadar karanlıkta saklayın. </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>

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<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

Bakteriyel Gen İfadesi Analizi Mikroarray&#39;ler Kullanımı

bacterial-gene-expression-analysis-using-microarrays

Research

JoVE Journal

Biology

8.7K Görüntüleme.  University of California Santa Cruz - UCSC. 

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.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

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

Biyoloji

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

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 1 of 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Bölüm 1

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

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Bölüm 3

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.

Zebra balığı Oligonükleotid Mikroarray Platformu kullanarak Global Gen İfadesi Analizi

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

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Omurilik Elektrofizyoloji

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

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.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

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.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

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 Kül Freze Gen İfade Analizi ( Agrilus planipennis) Kullanarak Kantitatif 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 ...

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.

Mikroarray&#39;ler kullanarak tRNA Aminoacylation (Şarj) Seviyeler genom Analizi

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.

Parafin ve Dondurulmuş Bölüm Drosophila Yetişkin Kaslar

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