Transcriptase 반응을 역방향 70-80로 가열 블록 돌려 ° C 42 ° C. 워터 온천도이 단계에서 사용할 수 있습니다. 가열 블록을 사용하는 경우, 열이 균일되도록 물을 넣어. <ol><li> 마중물 반응 <ol><li> RNA 3 μg을 가지고 </li><li> RNase 무료 물을 13 μl로 볼륨을 조정 </li><li> 랜덤 hexamer primers (2mg/mL) 2.5 μl 추가 </li></ol></li><li> 잘 믹스 8 분 동안 70-80 ° C에서 알을 품다. 5 분 얼음 그런 다음, 장소. </li><li> : RT 칵테일 (14.5 μl / rxn) 준비 <table border="0"><tbody><tr><td> 구성 요소 </td><td> 음량 </td></tr><tr><td> 배 첫 스트랜드 버퍼 </td><td> 6 μl </td></tr><tr><td> 0.1 M DTT </td><td> 3 μl </td></tr><tr><td> 25X aminoallyl - dNTP 믹스 </td><td> 1.2 μl </td></tr><tr><td> 윗첨자 II RT (200U/μl) </td><td> 2.3 μl </td></tr><tr><td> 물 </td><td> 2.0 μl </td></tr></tbody></table> * N 반응의 경우, 밖으로 실행되지 않습니다 보증하기위한 칵테일 N 0.5 aliquots에 대한 마스터 믹스를 준비합니다. </li><li> 냉각 반응 14.5 μl를 추가합니다. </li><li> 혼합 (와동하지 않음)과 ° C 3-4시간에 대한 42 알을 품다 </li><li> 필요한 경우 샘플은 -20 ° C에 저장할 수 있습니다. </li></ol> Hydrolyze RNA <ol><li> 각 샘플하려면, 1 N NaOH 3 μl와 0.5 M EDTA (에틸렌 다이아 민 테트라 초산) 0.6 μl를 추가합니다. (최종 conc.s = 100mM NaOH, 10mM EDTA (에틸렌 다이아 민 테트라 초산)) </li><li> 믹스 65에서 품어 ° C를 10 분. </li><li> 33.6 μl 1M HEPES를 추가하여 중화, 산도 = 7.0 (최종 CONC .= 500mM의 HEPES) </li><li> 샘플이 필요한 경우, -20 ° C에 저장할 수 있습니다. </li></ol> 반응 정제 : 비법인 AA - dUTP의 제거와 무료 아민 참고 :이 정화 프로토콜은 Qiagen QIAquick의 PCRpurification 키트 프로토콜에서 수정됩니다. Qiagen 버퍼가 싸이 염료 커플링 반응과 경쟁 무료 아민을 포함하기 때문에 인산염 세척 및 용출 버퍼는 Qiagen 제공된 버퍼에 대한 대체됩니다. 미니 준비 키트에서 열이도 사용할 수 있습니다. <ol><li> 300 μl (또는 배 반응 볼륨 = 336 μl) 버퍼 PB (Qiagen 공급)와 cDNA 반응을 믹스하고 QIAquick 열로 전송할 수 있습니다. </li><li> 2 ML 수집 관에서 열 (Qiagen 공급)을 놓고 1 분 ~ 13,000 rpm으로 원심 분리기. 빈 컬렉션 튜브. </li><li> 세척하려면 1 분 ~ 13,000 rpm으로 열 및 원심을 750 μl 인산염 세척 버퍼 (안 Qiagen의)을 추가합니다. </li><li> 수집 튜브를 비우기와 750 μl 세척과 원심 분리의 단계를 반복합니다. </li><li> 수집 튜브를 비우기 및 열에게 최대 속도에서 추가로 1 분 원심 분리기. </li><li> 새로운 1.5 ML의 microfuge 튜브에 열을 전송 조심스럽게 30 μl 인산 용출 버퍼를 추가합니다. (솔루션 준비 부분을 참조) </li><li> 실온에서 1 분 알을 품다. </li><li> 1 분 ~ 13,000 rpm으로 원심 분리에 의해 Elute. </li><li> 반복 단계 6-8하여 동일한 튜브에 두 번 (인산 용출 버퍼의 다른 30 ​​μl)를 Elute. 최종 용출 량 ~ 60 μl해야합니다. </li><li> cDNA의 양은이 단계에서 계량 수 있습니다 : NG cDNA = (희석 계수) (OD260) (37) (총 볼륨이 칼럼에서 eluted) </li><li> 45 VAC 속도 드라이 샘플 ° C. </li><li> 필요한 경우 샘플은 -20 ° C에 저장할 수 있습니다. </li></ol> 다이 에스터을 싸이에 AA - cDNA 커플링 <ol><li> Resuspend 10 μl 50 MM 나 - 중탄산염, 산도 = 9.0 필요한 경우와 cDNA aminoallyl - 분류. (이 버퍼가 격주 신선한하여야한다) </li><li> 어두운 장소에서 작업, 해당 싸이의 염료의 튜브에 프로브를 추가합니다. Pipet 위아래로 여러 번은 염료를 resuspend 수 있습니다. </li><li> 상온에서 어둠 속에서 1 시간 동안 반응을 품어. (부화 최대 2 시간이 갈 수 있습니다) </li></ol> 반응 정제 II : uncoupled 염료의 제거 <ol><li> 반응 35 μl 100 MM NaOAc의 산도 5.2을 추가합니다. 완전히 섞는다. </li><li> 250 μl 추가 (또는 배 반응 볼륨을 = 225 μl) 버퍼 각각의 샘플 및 최대 pipetting 아래로 섞어 PB (Qiagen 공급). </li><li> 2 ML 수집 관에 QIAquick의 스핀 컬럼 (Qiagen 공급)을 장소, 그 결과 해당 컬럼에 대한 샘플을 신청하고, 1 분 ~ 13,000에서 원심 분리기. 빈 컬렉션 튜브. </li><li> 세척하려면 1 분 ~ 13,000에서 열 및 원심 분리기에 0.75 ML 버퍼 PE를 (Qiagen 제공)을 추가합니다. </li><li> 4 단계를 반복합니다. </li><li> 빈 수집 튜브와 최대 속도로 추가 1 분 원심 분리기 칼럼. </li><li> 신중하게 플레이스 깨끗한 1.5 ML의 microfuge 튜브의 열 및 열 막의 중심 30 μl 버퍼 EB을 (Qiagen 제공)을 추가합니다. </li><li> 실온에서 1 분 알을 품다. </li><li> 1 분 ~ 13,000 rpm으로 원심 분리에 의해 Elute. </li><li> 반복하여 동일한 튜브에 두 번 Elute단계 7-9. 최종 eluted 볼륨 ~ 60 μl해야합니다. </li></ol> 라벨링 반응 분석 cDNA의 금액과 레이블은이 단계에서 계량 수 있습니다 : <ul><li> OD 260, 550 및 650 (빈 물을 수 있습니다.) nanodrop microarray 프로그램을 사용을 측정합니다. </li><li> 염료의 설립 및 아래 방정식과 pmol DNA와 염료 / nuc 비율 pmol을 계산할 수 : pmol의 세포핵 = [OD260 * 볼륨 (μL) * 37 μL / NG * 1000 PG / NG] / 324.5 PG / pmol </li></ul> 주 : 1 OD260 cDNA를 위해 = 37 NG / μL, dNTP의 324.5 PG / pmol 평균 분자량) pmol Cy3 = OD550 * 볼륨 (μL) / 0.15 pmol Cy5 = OD650 * 볼륨 (μL) / 0.25 세포핵 / 염색 비율 = pmol pmol / cDNA 싸이 염료 당신이 nanodrop를 사용하는 경우, 이미 μl / pmol의 각 색소 있습니다. 당신은 전체 pmol 염​​료의 설립을 위해 볼륨을하여이 수를 모두 곱하면해야합니다. 참고 :&gt; 염료 샘플 당 정관 이하 20 세포핵 / 염료 분자의 비율은 150-200 pmol가 hybridizations 최적입니다. <ul><li> 샘플은 암흑에서 -20 ° C에 저장할 수 있습니다. <ul><li> 함께 hybridized하는 프로브를 결합하고 42 speedvac 아래로 건조 ° C.를 </li><li> 샘플은 hyb 준비 때까지 -20 ° C에 저장할 수 있습니다. </li></ul></li></ul> 사전 hyb 슬라이드 <ol><li> 3~5분을위한 깨끗한 빨간색 슬라이드 홀더에 넣어 슬라이드 땅에 얼굴 RT 100 ML 물 </li><li> 몇 초 동안 100 ° C 가열 블록에 얼굴을 배치하여 슬라이드를 건조 스냅 - 결로가 사라져 위해 감시 </li><li> 250 mJoules에서 UV는 상호 링크 슬라이드 (UV crosslinker에 2500 입력) </li><li> 사전 예열 42 ° C의 물 속에서 수영의 수화물 슬라이드. RT에서 700 rpm으로 5 분 스핀. </li><li> 필요한 경우 적어도 45 분 동안 미리 예열 사전 hyb (5XSSC, 1 % BSA, 0.1 % SDS)를 포함 50ml 코플린있는 항아리에 품어 슬라이드는 @ 42 ° C, 그리고 부화 더 오래 갈 수 있습니다. 50 ML 사전 hyb 버퍼 : 12.5 ML 20x SSC, 10 ML 5% BSA, 0.5 ML 10% SDS, 27 ML 물 </li><li> 딥 사전 예열 물 (42 ° C)에서 슬라이드와 사전 hyb 버퍼를 제거하는 몇 분을 선동하다. </li><li> 이소프로판올에 씻어 700 RPM, 온도는 실온, 건조시 5 분 스핀. </li></ol> 하이브 리다이 제이션 준비 샘플 <ol><li> 물에 Resuspend의 프로브 (볼륨이 아래에 있습니다) <table border="0"><tbody><tr><td> 총 권하십시오. = 30μl </td><td> 총 권하십시오. = 35μl </td><td> 총 권 .= 40μl에 대한 </td><td></td></tr><tr><td> 19.8 μl 물 </td><td> 23.55 μl 물 </td><td> 27.2 μl 물 </td><td> </td></tr><tr><td> 1.5 μl </td><td> 1.5 μl </td><td> 1.5 μl </td><td> 10 MG / ML 연어 정자 DNA (Invitrogen) </td></tr><tr><td> 1.2 μl </td><td> 1.2 μl </td><td> 1.2 μl </td><td> 12.5 MG / ML tRNA </td></tr><tr><td> 4.5 μl </td><td> 5.25 μl </td><td> 6 μl </td><td> 20XSSC (배 최종) </td></tr><tr><td> 3 μl </td><td> μl 3.5 </td><td> 4 μl </td><td> 1퍼센트 SDS </td></tr></tbody></table> 참고 : 시약의 이외의 순서가 중요합니다. 마스터 믹스를 준비하지 마십시오. </li><li> 5 분 삶아 후 14,000 rpm으로 5 분만 스핀. 온도는 실온에서 2 분 쿨. </li></ol> 이종 교잡 <ol><li> 각 모서리와 중앙에 하이브리드화 챔버의 우물에 배 SSC의 10-12 μl를 추가합니다. </li><li> 슬라이드의 해당 영역에 깨끗한 리프터 전표를 추가하고 부드럽게 (30 -40 μl) 하이브 리다이 제이션 솔루션에로드합니다. 커버 슬립 중 하나를 가장자리에 여분의 샘플이 있어야합니다. </li><li> 챔버에있는 슬라이드를 놓습니다. 하룻밤 65 ° C 물 목욕에있는 나사와 장소를 조이십시오. 물 목욕의 바닥에 직접 챔버을 올려 놓지 마십시오. 장소에 모든 잡아 맨 위에 챔버 위에 블록을 배치합니다. 빛을 민감한 샘플을 보호하기 위해 물을 욕조에 뚜껑을 유지. </li></ol> Hyb은 세척 <ol><li> 다음 버퍼를 준비 : <ul><li> 1X SSC, 0.05 % SDS </li><li> 0.06 X SSC </li></ul></li><li> 부화 풀어봐 하이브리드화 챔버 후. coverslip을 방해하지 않도록 돌봐, 챔버에서 슬라이드를 제거합니다. </li><li> coverslips을 제거하려면, 따뜻한 1X SSC, 접시의 바닥을 향하고 0.05 % SDS 세척 버퍼 coverslip을 포함 접시에 잠수함 슬라이드. coverslip는쪽으로 슬라이드 사이드를 이동하여 떨어질 것입니다. </li><li> coverslip가 제거되면 따뜻한 1XSSCm 0.05 % SDS와 랙 신선한 목욕탕에서 슬라이드를 놓으십시오. 몇 분 동안 선동 </li><li> 0.06 X SSC의 목욕에 슬라이드 플러스 랙을 전송합니다. </li><li> 다른 바로 슬라이드를 전송0.06 X SSC는 신선한 욕조에 넣어 전에 가능한 한 많은 SDS - 포함된 버퍼를 제거하는 종이 타월에있는 슬라이드를 모래 바닥, 신선한 랙을 포함의 일. </li><li> 몇 분 동안 슬라이드를 선동. </li><li> 5 분 @ 700 RPM에 상온에서 바로 스핀, 그리고 슬라이드는 스캔 준비가되었습니다. 스캔까지 어둠 속에 보관하십시오. </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

Microarrays을 사용하여 세균의 유전자 발현 분석

bacterial-gene-expression-analysis-using-microarrays

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

비디오: 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.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

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

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생물학

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.

우두 바이러스 감염 및 바이러스 유전자 발현의 시간적 분석 : 1 부

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.

우두 바이러스 감염 및 바이러스 유전자 발현의 시간적 분석 : 제 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.

Zebrafish Oligonucleotide Microarray의 플랫폼을 사용하여 글로벌 유전자 발현 분석

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.

척수 전기 생리학

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.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

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.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

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.

에메랄드 애쉬 보러에서 유전자 발현 분석 ( Agrilus planipennis)를 사용하여 정량 리얼 타임 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.

Microarrays을 사용하여 tRNA의 Aminoacylation (충전) 레벨의 게놈 - 와이드 분석

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.

의 파라핀 - 임베디드 및 냉동 섹션 Drosophila 성인 근육

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