这项工作是由本科学生在西南俄克拉荷马州立大学，威德福确定。这项工作是支持由瑞0963258美国国家科学基金会，美国国立卫生研究院确定的INBRE 2P20RR016478，GCAT OCAST HR09 - 137S。 vsm1（ok1468）突变体中分离俄克拉荷马基因敲除联盟。 丹斯特凡诺维奇和唐纳惠勒作者感谢他们在项目执行过程中的宝贵帮助。

1。总RNA的分离<ol><li>健康同步线虫收获室温M9培养基洗的盘子，每盘用2-3毫升。悬浮线虫被转移到15 ml锥形管和沉淀下来，在4 ° C，4分钟离心，3200克。收获线虫清理漂浮的细菌resuspending 7毫升0.1M氯化钠和冰冷的60％W / V蔗糖7毫升沉淀。在冰上孵育15分钟，干净的线虫，游泳通过蔗糖溶液，在4 ° C离心3200克的混合物在4分钟后，收集悬浮混合物。无菌蠕虫被转移到一个新的锥形管，用无菌移液器高达15毫升无RNase的水由3200 g离心4℃2分钟洗两次。松散压实的清洁颗粒被转移到1.5 ml离心管，离心30秒，最高速度（约10000克）。最后，最无RNase水被拆除，10卷RNAlater添加到颗粒，并保存在4 ° C，直到准备进行。 </li><li>为了准备总RNA样品，离心收获线虫4分钟，最大速度（约10000克）RNAlater被丢弃。然后，捏分子研磨树脂（G生物科学）添加到颗粒，用液氮冷冻混合物。需要添加研磨成细粉，用杵和液氮冷冻蠕虫悬挂保持粉冷。研磨后，提取物被保存在冰5分钟，然后用700μL的RLT / BME（比100卷的RLT:1生物医学工程量）混合（QIAGEN）和472μL100％的乙醇。 </li><li>使用的RNeasy试剂​​盒（QIAGEN），并按照制造商建议的协议的总RNA。最后分离出的RNA量是60％生物样品液。 </li></ol> 2。 DNA消化和RNA的清理<ol><li> 50微克可能与基因组DNA污染的总RNA用DNase我（QIAGEN）消化。精华含有0.1 U DNA酶/ 1微克的RNA和1X缓冲液的RDP的反应在室温下孵育10分钟。 </li><li>我清理使用的RNeasy MinElute清理试剂盒（Qiagen），RNA样品用DNase处理。制造商建议的协议之后和60μL最终洗脱体积。 </li></ol> 3。定性和定量分析的RNA <ol><li> RNA浓度测定采用紫外分光光度计Nanodrop（Thermo Scientific的）。 </li><li>使用琼脂糖凝胶电泳RNA样品的完整性进行评估。简言之，无RNase的1％琼脂糖凝胶准备使用1X北方最大甘氨酸凝胶PREP /运行缓冲液（Ambion公司），无RNase的1％琼脂糖LE（Ambion公司）和0.5微克/ ml溴化乙锭。凝胶聚合，RNA样品中加入5μLGlyoxyl样品载入染料/样品和孵化样品在50℃下30分钟glyoxylated。 RNA的梯子也glyoxylated和加载的大小比较。 </li></ol> 4。 cDNA合成<ol><li>对于每个合成反应中，与1μLRT引物（1 pmole /μL）8.4微克的RNA样品混合。一个样本（WT或突变）收到的Cy5的捕获RT引物;其他样品收到Cy3标记捕获RT引物（阵列350标签检测试剂盒，Genisphere提供）。样品混合物加热到75-80 ° C 10分钟，并转移到冰2-3分钟。 </li><li>虽然RNA样品孵育，MasterMix（Genisphere）准备如下:每个RT反应，我们结合4μL的RT 5X反应缓冲液，1μL的dNTP（10毫米每个的dATP，dCTP，dGTP，dTTP的），1μLSuperase在核糖核酸酶抑制剂（阵列350标签检测试剂盒，Genisphere提供），1μL逆转录酶（200 U /μL）。最后，7μLMasterMix被添加到每个RNA样品中，轻轻混合（不使用涡）和孵育2小时42 ° C。 </li></ol> 5。 RNA降解和纯化cDNA样本<ol><li>加入3.5μL0.5M NaOH/50mM EDTA（终浓度）的cDNA合成停止，并在65 ° C下孵育15分钟。然后，反应使用1M的Tris - HCl，pH值8.0至终浓度达到了850毫米的Tris - HCl的瓦解。最后，野生型和突变体的cDNA被合并成一个的离心管。空管73μL1X TE缓冲液冲洗和补充的cDNA含管结合的cDNA管的最终体积为130μL。 </li><li>混合cDNA的纯化以下制造商的协议和QIAquick PCR纯化试剂盒（QIAGEN）。结果纯化的cDNA洗脱体积为60μL。 </li></ol> 6。第一芯片杂交<ol><li> 线虫芯片幻灯片通过GCAT（基因组协会积极开展教学）获得被封锁室温为1小时，使用0.1毫克/毫升超声鲑鱼精子DNA在3X SSC，0.1％SDS。封锁的幻灯片冲洗ddH2O和2000克50毫升锥形管在底部KimWipe的离心1分钟旋转干燥浸渍。 </li><li>然后，基于2X甲酰胺杂交缓冲液（Genisphere）孵育55 ° 10分钟，充分混合溶解结晶，离心1分钟在10000克接下来，25μLcDNA样本轻轻弹25 2X甲酰胺的杂交缓冲液混合。稀释的cDNA样本收集闪光旋转15秒，在80 ° C 10分钟的孵育。 </li><li>最后，基因杂交微阵列小心吹打整个加热cDNA样本而不触及幻灯片的幻灯片。轻轻地降低盖玻片，用注射器针头样品均匀扩散到芯片。之前完全降低，盖玻片盖玻片拉备份，然后用针降低再次，允许轻轻落入到位，最大限度地减少气泡的形成。首先杂交成立了高潮幻灯片水平与DDH 2 O 50μL，在50 ml锥形管下面的幻灯片，并在37℃孵育过夜</li></ol> 7。二杂交<ol><li>杂交芯片在不同温度下与2X SSC洗。首先，芯片幻灯片被转移含2X SSC，0.2％SDS室温锥形管和sloshed轻轻地去除盖玻片。然后，芯片幻灯片被转移到含有55 ° C的2X SSC和0.2％SDS的锥形管和孵育15分钟，在55 ° C。下一步，幻灯片被转移到2X SSC，室温孵育15分钟，轻轻摇动定期。最后，幻灯片被转移到0.2X SSC和培养在室温下15分钟，轻轻摇动定期。水洗幻灯片50 ml锥形管引入幻灯片标签的一面朝下，在底部KimWipe的旋转干燥和2000克离心1分钟</li><li>接下来，第二个杂交混合使用2X甲酰胺的杂交缓冲液（Genisphere）准备。杂交缓冲液在55 ° C孵育10分钟，离心1分钟1万克捕捉荧光试剂（Cy3和Cy5）和防褪色试剂在室温下解冻，用铝箔纸覆盖，以保护从漂白试剂。继杂交步骤，在暗的光线照射的最小化。 150 2X甲酰胺的杂交缓冲液1.5μL抗褪色试剂使防褪色处理的杂交混合结合。 </li><li>捕获试剂Cy3和Cy5振荡3秒和15秒离心10,000 g的第二个杂交液75μL防褪色处理的杂交组合，60μL无核酸酶水，7.5μLCy3标记捕获试剂和7.5μLCy5的捕获试剂相结合准备。第二个杂交组合孵育10分钟，在75 ° C和50μL加热混合物滴加入到了非常仔细的洗/干芯片幻灯片。均匀扩散到芯片的样品，盖玻片，轻轻地降低使用注射器针头。之前完全降低，盖玻片盖玻片拉备份，然后用针降低再次，允许轻轻落入到位，最大限度地减少气泡的形成。杂交芯片幻灯片覆盖铝箔和DDH 2 O 50μL50 ml锥形管被放置在水平增加了下面的幻灯片创建一个湿热试验箱。二杂交是在37 ° C孵育2-5小时。 </li><li>第二个杂交芯片是在黑暗的使用室温2X SSC，0.2％SDS，1毫米数码地面电视和洗涤sloshed轻轻取出盖玻片。然后，芯片被转移到含有55 ° C 2X SSC，0.2％SDS，1毫米的DTT覆盖锥形管，并在55℃孵育15分钟下一步，幻灯片被转移到覆盖的锥形管中含有2X SSC，1毫米DTT和孵育在室温下15分钟，轻轻摇动定期。最后，幻灯片被转移到0.2X SSC，1毫米的DTT，室温孵育15分钟，轻轻摇动定期。微阵列玻片为1分钟，2000克，幻灯片标签的一面覆盖50毫升锥形管底部KimWipe离心干燥。干芯片幻灯片进行扫描，利用戴维森学院GenePix个人的扫描仪型号4100A。 </li></ol> 8。微阵列分析<ol><li>扫描后获得的图像进行分析，使用MAGICTool劳丽海耶和她的本科学生在戴维森学院软件开发。简单地说，"项目"标签下，"新建项目"创建和保存。根据"构建表达个人资料"标签，"载入图像对"选择和红色的图像文件（_635.TIF）上传为"红色"和"绿色"绿色的图像文件（_532.tif）上传。然后，使用"生成基因表达谱"选项卡和"加载基因名单"，A C.线虫基因列表从GCAT网站获得的文件被上传。 </li><li>芯片的图像处理和网格使用"构建表达个人资料"标签和"创建/编辑网格"选项。 "网格设置"对话框中编辑使用格数为"48"，"22行"和"22列"为每个网格点编号的"从左到右"和"自上而下"。 "百分比的对比变化"是对电网增加和放大，直到个别点很容易分辨。电网是首先选择"设置左上点"的左侧面板上创建。然后，左上角点的中心，右上角点和底排点击"网格1"。为每48个网格，这种网格化的过程是反复，左右，从上到下进行。当网格完成，目前电网保存在"文件""另存为"当前电网。当电网已成功保存"完成"选项卡被选中。 </li><li>为了消除任何无关的分析点，"构建表达个人资料"标签被打开了。根据"解决网格"选项"点"举报"被选中​​。斑纹的斑点或背景荧光标记，并选择"另存为当前标志"下的"文件"选项卡"保存。 </li><li>标记的文件被分割使用的"构建表达个人资料"标签，并选择"固定半径"作为首选的分割方法。半径设置为所需的大小，并点击"更新数据"。为了确保圆圈内的网格区域（黄色平方米）保持，我们滚动从上到下，从左到右一次检查"创建表达谱"被选中。 </li><li>在分割过程中，软件计算红:绿色荧光比率为每个斑点，后萎靡不振。使用"表情"选项卡，"操纵数据"，"转换"被选中。 "转换数据"对话框出现，其中"logb（X）"选项，和B = 2输入。探索从"表达"标签的"工作表达文件"。一个实验中，野生型Cy3标记（绿色）捕获序列和突变收到的Cy5的（红色）捕获序列，日志转换比率值分别为阳性基因被诱导和抑制负。 </li><li>最后，由指定的因素诱发或压抑的基因进行分析的"表达"标签下选择"浏览"。在"探索"对话框，"查找匹配准则"设置"&gt; X（正数为诱导基因）"或"闽值&lt;X（压抑的基因负数）"的最大价值的基因的基因＃5中描述的一个实验。折叠式的表达等于2倍，其中"x"的甄选准则中输入的号码的价值。 </li></ol> 9。 cDNA合成和Real - Time PCR <ol><li>如前所述提取总RNA。一旦分离出的RNA的质量和数量进行测定，合成cDNA，使用的iScript cDNA合成试剂盒（BioRad公司），500 ng /μL的从野生型和突变体，制造商推荐的协议分离的总RNA。 </li><li>实时PCR反应使用的智商SYBR GREEN Supermix（BioRad公司），100-500 nmoles基因特异性引物（表1）和上一步产生的4μL的cDNA设置。运行时使用的热循环CFX96实时系统（BioRad公司）10μL反应。 </li><li>最初的热循环加热到95℃，3分钟。其次为5秒一步，在95℃和58℃30秒。这个循环完成共四十次，还插入一个65-95℃，熔解曲线坡度0.5度。 ΔΔCT值计算，使用三个看家基因（ACT - 1，CDC - 42，和PMP - 3）作为对照。 </li></ol><table border="1"><tbody><tr><td> 基因 </td><td> 正向引物 </td><td> 反向引物 </td></tr><tr><td> GST - 5 </td><td> CTGCTCCATTCGGACAACTT </td><td> TCCGTTGAGCTTGAACTCAC </td></tr><tr><td> GST - 9 </td><td> GGAAGACAACTGGCACAATC </td><td> ACCGAGAGCATCAACTTGAG </td></tr><tr><td> kcnl - 1 </td><td> TACGCTCGGCATGTGTTGTATC </td><td> CCAATCATCGGCTCCACTATCT </td></tr><tr><td> KSR - 2 </td><td> CGAACTGCTGCTGGAATGCT </td><td> GTGCTGCGTCTCTTCTGCTT </td></tr><tr><td> LIM - 9 </td><td> CTCATCGAGTGGCTCAATCT </td><td> CACCTTGCTCCATCTTCAAC </td></tr><tr><td> LPR - 6 </td><td> CAGCAGTCACTTCTGTTCAC </td><td> TCTCCAATAGCGGTACTCC </td></tr><tr><td> MES - 6 </td><td> TTGTTCCGTTGGCTCACGTACAG </td><td> CGACAGACGCAGATACACGATCA </td></tr><tr><td> MSP - 32 </td><td> GCCGCACAGGTATGATCCAG </td><td> ATCCAATACGGCGAGCCGAG </td></tr><tr><td> MSP - 142 </td><td> GATCCACCATGTGGAGTTCT </td><td> GATTCTTGCGACGGACCATA </td></tr><tr><td> MSP - 38 </td><td> GCCTTCGGACAAGAGGATACC </td><td> CCATACCGTCTCCTTGGAACC </td></tr><tr><td> MSP - 45 </td><td> TCACCGTTGAGTGGACCAAT </td><td> ACGAACCAACCGTCTCCTT </td></tr><tr><td> MSP - 49 </td><td> CGACGACAAGCACACCTACCACATCAA </td><td> TCGAGGACTCCACATGGTGGATCAACT </td></tr><tr><td> MSP - 56 </td><td> CGAAGATCGTCTTCAATGCGCCATAC </td><td> TCCACATGGTGGATCAACTCCAAGTC </td></tr></tbody></table> 表1。正向和反向的实时PCR引物序列 10。代表性的成果: RNA分离和分析 活动区前体囊泡在突触前网站的融合是一个强制性的步骤（3）在突触。 VSM - 1，最近发现的V - SNARE掌握蛋白质，提出了一个未定的机制（4，我们的未发表的作品），以抑制酵母和线虫中的突触囊泡融合（见图1）。为了更好地了解底层的分子通路VSM - 1调控线虫和进入突触领域带来一些光，我们开始了全基因组的屏幕和确定的主要精子蛋白基因（MSP）是功能齐全的VSM - 1的情况下诱导。 为了调查在VSM - 1（ok1468）C.突变株诱导基因线虫 ，我们进行了微阵列基因分析。这是通过测试从野生型中分离的成绩单和VSM - 1突变株，并确定其浓缩。具体来说，我们第一次分离同步L4和L3野生型和VSM - 1突变体幼虫线虫总RNA。然后，我们对待用DNase我得到的总RNA，并考察了提取的RNA用琼脂糖凝胶电泳质量。在图2所示的实验中，一梯是用在第一线代表的碱基对数量的标准。野生型样品前处理和后处理用DNase我被装在2和4车道，VSM - 1突变体样品前处理和后用DNase处理我被装在泳道3和5，分别。完整，良好的质量和RNA凝胶电泳分析表明，野生型和VSM - 1突变体RNA样品。这是通过观察两个频段，这两个亚单位核糖体RNA的决定。 芯片杂交 一旦确定RNA的质量，我们进行cDNA合成。要做到这一点，我们反转录mRNA和捕获序列的尾部。生产含有Cy3和Cy5捕捉序列的cDNA杂交芯片的幻灯片和扫描发送。芯片的图像，然后进入到一个开放源码的计算机软件程序称为MAGICTool劳丽海耶和她的本科学生在戴维森学院开发。使用这个软件，我们覆盖Cy3和Cy5的图像，网格化的芯片，并分析荧光剖面（见图3）。一旦网格完成，我们每一个人的正方形，然后分段确保整个印刷寡核苷酸现正研究。然后，我们分析的诱导比率和创建的基因谱表达，诱导增强突触的线虫基因编码的MSP系列。 （见表3）。 验证和使用实时PCR基因表达的定量分析。 为了进一步了解在VSM - 1突变体的遗传档案，我们分析了基因的MSP家族成员的代码使用实时PCR。首先，提取总RNA从野生型和突变株VSM - 1（ok1468）。 RNA样品反转录的cDNA用于实时PCR分析。实时PCR表达谱的评价表明，似乎是在VSM - 1（ok1468）突变体诱导的MSP系列的一个成员。此外，数据实时PCR验证结果从芯片和收窄搜索一个MSP基因的候选（图4）。 <img src="/files/ftp_upload/2777/2777fig1.jpg" alt="图1"/> 图1。A。 UNC - 17免疫染色显示，VSM - 1突变体表现出沿背神经索突触密度较高。 63X放大倍率，后（右），外阴图像进行分析。 B.分析的WT和VSM - 1（ok1468）突变体的突触表示在VSM - 1突变体（** P &lt;0.01）有统计学显着增强的突触密度。二十线虫各基因型的图像。 <img src="/files/ftp_upload/2777/2777fig2.jpg" alt="图2"/> 图2提取RNA的质量是用琼脂糖凝胶电泳确定。野生型和VSM - 1（ok1468）样本中提取DNA酶I治疗前，后两个完整的核糖体亚基的RNA明显。 <img src="/files/ftp_upload/2777/2777fig3.jpg" alt="图3"/> 图3。A。芯片控制标记为红色（Cy5的）的实验和VSM - 1突变体的形象标记为绿色（ Cy3标记）。 B.代表图像显示48％微阵列分析的电网1。小广场上的每一个网格是一个单一的印刷寡核苷酸的代表，每个格子是22行和22列组成。 <img src="/files/ftp_upload/2777/2777table2.jpg" alt="表2"/> 表2微阵列分析，从幼虫4（A）和（二）C.幼虫3孤立的成绩单 。 线虫线虫表明，突触增强的突变体诱导的主要精子蛋白编码基因。突出显示的基因表明，在幼虫3和幼虫4诱导的基因。 <img src="/files/ftp_upload/2777/2777fig4.jpg" alt="图4"/> 图4。实时PCR分析表明，一个MSP基因家族成员，MSP -32 VSM - 1（ok1468）突变体诱导。代表正常化倍表达图显示VMS - 1（ok1468）MSP - 32ΔΔCT值相比有明显不同的野生型（WT）和3个看家基因用于正常化（ACT - 1，CDC - 42，和PMP -3）。平均3副本绘制+ / - 标准差。

<ol>
	<li>Chai, A., Withers, J., Koh, Y. H., Parry, K., Bao, H., Zhang, B., Budnik, V., Pennetta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hVAPB,+the+causative+gene+of+a+heterogeneous+group+of+motor+neuron+diseases+in+humans,+is+functionally+interchangeable+with+its+Drosophila+homologue+DVAP-33A+at+the+Neuromuscular+Junction.">hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the Neuromuscular Junction.</a> Hum Mol Genet. 17, 266-280 (2008).</li><li>Tsuda, H., Han, S. M., Yang, Y., Tong, C., Lin, Y. Q., Mohan, K., Haueter, C., Zoghbi, A., Harati, Y., Kwan, J., Miller, M., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Amyotrophic+Lateral+Sclerosis+8+protein+VAPB+is+cleaved,+secreted,+and+acts+as+a+ligand+for+Eph+receptors.">The Amyotrophic Lateral Sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors.</a> Cell. 133, 963-977 (2008).</li><li>Dresbach, T., Torres, V., Wittenmayer, N., Altrock, W. D., Zamorano, P., Zuschratter, W., Nawrotzki, R., Ziv, N. E., Garner, C. C., Gundelfinger, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+Active+Zone+Precursor+Vesicles.">Assembly of Active Zone Precursor Vesicles.</a> Journal of Biological Chemistry. 281, 6038-6047 (2005).</li><li>Lustgarten, V., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+VSM1+Encodes+a+v-SNARE+Binding+Protein+That+May+Act+as+a+Negative+Regulator+of+Constitutive+Exocytosis.">Yeast VSM1 Encodes a v-SNARE Binding Protein That May Act as a Negative Regulator of Constitutive Exocytosis.</a> Molecular and Cellular Biology. 19, 4480-4494 (1999).</li><li>Vi&#241;uela, A., Snoek, L., Riksen, J., Kammenga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Gene+Expression+Analysis+in+Response+to+Organophosphorous+Pesticide+Chlorpyrifos+and+Diazinon+in+C.+elegans.">Genome-Wide Gene Expression Analysis in Response to Organophosphorous Pesticide Chlorpyrifos and Diazinon in C. elegans.</a> PloS one. 5, 1-8 (2010).</li><li>Niwa, R., Hada, K., Moliyama, K., Ohniwa, R., Tan, Y., Olsson-Carter, K., Chi, W., Reinke, V., Slack, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=elegans+sym-1+is+downstream+target+of+the+hunchback-like-1+developmental+timing+transcription+factor.">elegans sym-1 is downstream target of the hunchback-like-1 developmental timing transcription factor.</a> Cell Cycle. 8, 4147-4154 (2009).</li><li>Kershner, A., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+mRNA+targets+for+Caenorhabditis+elegans+FBF,+a+conserved+stem+cell+regulator.">Genome-wide analysis of mRNA targets for Caenorhabditis elegans FBF, a conserved stem cell regulator.</a> PNAS. 107, 3963-3941 (2010).</li><li>Hammock, E., Eagleson, K., Barlow, S., Earls, L., Miller, D., Levitt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologs+of+genes+expressed+in+Caenorhabditis+elegans+GABAergic+neurons+are+also+found+in+the+developing+mouse+forebrain.">Homologs of genes expressed in Caenorhabditis elegans GABAergic neurons are also found in the developing mouse forebrain.</a> Neural Development. 5, 1-14 (2010).</li></ol>

没有利益冲突的声明。

在这项研究中，我们开发了一个简单的，用户友好的协议，它可以被用来确定基因的表达谱，底层的各种生物现象。在这个协议中，首次分离的总RNA和对待我们的RNA分离方法已大大简化省略酚的提取和使用干净了5,6亲和树脂使用DNA酶一。简单地说， 角由用液态氮和高分子树脂研磨冷冻线虫线虫 cuticule和细胞膜破裂开。接下来，提取的RNA用DNase我治疗前cDNA合成。后面的步骤，以尽量减少非特异性杂交​​，基因组DNA污染的RNA样品中可能引入的干扰，并产生许多误报。然后，野生型和VSM - 1（ok1468）标记Cy3和Cy5捕获序列和突变体的cDNA杂交到C。线虫芯片获得通过的GCAT方案。这个系统是比同类产品更敏感，和两色的设计，减少所需的阵列数量，造成更大的时间和成本效率7,8。此外，该系统并不需要其他类似的系统的专用设备，因此，此过程可在任何标准的实验室环境中完成7。第三，荧光杂交阵列图像进行分析，使用开放源码软件MAGICTool，基因表达谱创建。 MAGICTool是一个用户友好的的程序，很容易利用个人所有经验水平的，从本科到博士后。然而，最佳的性能，需要大量的内存可用性。最后，候选基因的获得，使用基因芯片分析与实时PCR验证。 虽然基因组的方法是一种有效的方法，为研究生物现象，存在一定的局限性，应该考虑。首先，尽管这种芯片协议的特殊性，在一个家庭内的成绩单是从彼此难以区分，如果打印的寡核苷酸是采取保守序列。实时PCR在基因家族的相似性进行补偿，甚至可能会区分相同基因的具体亚型。然而，与实时PCR找到真正的控制，同样是一个有机体的整个寿命表示，这是一个挑战。正因为如此，结果将是相对的。一个蛋白质组学的方法是一种替代方法，以我们的技术。可能是孤立的个别蛋白质，利用二维凝胶电泳和质谱鉴定。 我们的研究明确显示，主要精子蛋白（MSP）家族的许多成员都在VSM - 1具有增强的突触密度的突变线虫引起的。中型项目是独特的为 C。 线虫和卵母细胞成熟和排卵负责。湾仔1表明，在果蝇的神经肌肉接头处突触前布顿数量和大小的调节可能是由包含主要精子蛋白域的分子控制。津和他的同事们研究2表明主要精子蛋白域可作为Ephrine受体的配体，受体酪氨酸激酶信号级联触发。此外，实时PCR分析验证，并收窄芯片结果MSP系列，MSP - 32的成员之一。两者合计，这里介绍的工作的中央神经两难全基因组的分析，以及本科生科研在科学事业的力量。

<table border="1"><tbody><tr><td> 组件 </td><td> 目录＃ </td><td> 公司 </td></tr><tr><td> RNA的后来</td><td> AM7020 </td><td> Ambion公司</td></tr><tr><td>分子研磨树脂</td><td> 786-138 </td><td> Gbiosciences </td></tr><tr><td>的RNeasy迷你包</td><td> 74104 </td><td> Qiagen公司</td></tr><tr><td>无RNase琼脂糖乐</td><td> AM9040 </td><td> Ambion公司</td></tr><tr><td> NorthernMax甘氨酸10X凝胶PREP /缓冲</td><td> 8678 </td><td> Ambion公司</td></tr><tr><td> RNA千年标记</td><td> AM7150 </td><td> Ambion公司</td></tr><tr><td>乙二醛样品上样染料</td><td> 8551 </td><td> Ambion公司</td></tr><tr><td> DNA酶设置</td><td> 79254 </td><td> Qiagen公司</td></tr><tr><td>的RNeasy MinElute套件</td><td> 74204 </td><td> Qiagen公司</td></tr><tr><td>逆转录酶和5X反应缓冲液</td><td> RT300320 </td><td> Genisphere </td></tr><tr><td>阵列350 </td><td> W300180 </td><td> Genisphere </td></tr><tr><td> QIAquick PCR纯化试剂盒</td><td> 28104 </td><td> Qiagen公司</td></tr><tr><td> 20X SSC </td><td> 82021-4846 </td><td> VWR </td></tr><tr><td>剪切鲑鱼精子DNA </td><td> AM9680 </td><td> Ambion公司</td></tr><tr><td> SDS溶液</td><td> V6551 </td><td> Promega公司</td></tr><tr><td>数码地面电视的EM </td><td> 3860 </td><td> VWR </td></tr><tr><td> iScript的cDNA合成试剂盒</td><td> 170-8890 </td><td> BioRad公司</td></tr><tr><td> IQ的SYBR绿色Supermix </td><td> 170-8880 </td><td> BioRad公司</td></tr></tbody></table>

determining genetic expression profiles in c. elegans using microarray and real-time pcr

突触是由突触前的信号细胞和靶细胞中的一个突触后终端的活跃区。在化学突触的情况下，消息释放终端突触前和突触后细胞上的受体的神经递质。我们线虫以往的研究表明，VSM - 1负调控胞外分泌。此外，分析VSM - 1突变体的突触表明，动物缺乏一个VSM - 1增加了全功能的突触连接。根据这些初步调查结果，我们推测，C 。 线虫 VSM - 1可能起到至关重要的作用在突触。为了检验这一假设，进行双标记基因芯片分析，测定和基因表达谱。首先，提取总RNA，反转录为cDNA，DNA微阵列杂交。然后，在硅片的荧光探针杂交分析显示显着的诱导许多突触增强的突变体基因编码的主要精子蛋白家族（MSP）的成员。中型精子在 C中的重要组成部分线虫和线虫的卵母细胞成熟和排卵的信号。在果蝇中，湾仔和他的同事1表明，MSP类似的分子调节神经肌肉接头处突触前布顿的数量和规模。此外，津和他的同事进行分析2，中型可作为Eph受体的配体和触发受体酪氨酸激酶信号级联。最后，实时PCR分析证实，MSP - 32的基因编码是在VSM - 1（ok1468）突变引起的。两者合计，我们的实验室进行的研究表明，VSM - 1突变体在突触密度的显着增加，这可能是由MSP - 32信号介导的。

Watch this Scientific Journal Video about Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR at JoVE.com

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

进行微阵列分析，以确定基因的表达谱<em&gt; C。线虫</em&gt;，并实时PCR是用来验证和量化的微阵列数据。

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

determining-genetic-expression-profiles-c-elegans-using-microarray

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

23.1K 次观看.  Southwestern Oklahoma State University. 进行微阵列分析，以确定基因的表达谱 C。线虫，并实时PCR是用来验证和量化的微阵列数据。

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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

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.

全球基因表达分析，使用斑马鱼的寡核苷酸芯片平台

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

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

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.

白蜡窄吉丁的基因表达分析（ Agrilus planipennis）使用实时定量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 ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用"冻结开裂"

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.

线性扩增介导的PCR  - 未知侧翼DNA的遗传物质和表征本地化

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

确定基因表达概况 C。线虫使用微阵列和实时PCR

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