作者感谢维持拟南芥女士珍妮弗M.洛梅尔和塔拉北路Rognan。这项工作是从美国圣路易斯大学和美国国立卫生赠款1R15GM086846 - 01和3R15GM086846 - 01S1 W.小的启动资金支持。

一，遗传隧道 1。 Emasculating的母本为了区分使用的DNA序列多态性，两个不同生态型，如哥伦比亚0（COL - 0）和LER，将作为女性和男性的父母选择的母亲和父亲的等位基因。植物应年轻和健康。一个可以阉割母本，用解剖显微镜，放大镜遮阳帽，或肉眼。找到阶段- 12花（史密斯等人，1990年）和删除任何鲜花或角果上面和下面他们的基地花梗用剪刀裁剪。消毒镊子轻轻浸泡成95％乙醇的烧杯中，这将删除任何钳花粉和杀死花粉以及尖端的基础。轻轻撬除了使用产钳的花蕾，轻轻取出4个萼片，4个花瓣，6个雄蕊，使雌蕊裸露和完整。尽量避免在此过程中损坏的心皮。 2。采摘的花粉供体和开展授粉在一个90度角的雌蕊（史密斯等人，1990年），在其中大量的花粉脱落的延长花瓣最好的花粉捐助者anthesed阶段14开的花。抓住花基地，只是上面的花梗，这将导致流传开的花。灰尘的准备雌蕊柱头与花药。柱头授粉后，将覆盖着黄色的花粉，可以很容易地在显微镜下观察。 3。标签的十字架上所有的植物阉割雌蕊授粉后，我们的标签与珠宝的女性和男性的父母和日期的标签和信息交叉。接近的植物在土壤中的股份，使用一个字符串，以配合花序干的股权，并用塑料袋罩在授粉的雌蕊。 二。在拟南芥中分离胚乳 1。准备材料我们通常会得到必要的材料，准备收获前胚乳和胚胎组织：液氮罐，液氮，在解剖显微镜，细尖镊子（5杜蒙生物学INOX福斯特，瑞士），显微镜载玻片的两个新的双（ 3“X 1”X 1.0毫米），pH值5.7的解决方案0.3米的山梨醇和5毫米的MES。 2。收集角果在7 - 8天后授粉（DAP），种子准备在中旬后期胚胎发育的鱼雷阶段，会有收获的，胚乳和胚胎解剖。有时，它可能需要9-10磷酸二铵，如果被阉割的花是太年轻了。 3。隔离的胚乳和胚由于拟南芥种子是微小的，我们用解剖显微镜隔离胚乳和胚。 8磷酸二铵丝丽放在显微镜下，使用一对镊子举行丝丽花梗，并用另一双钳尖端利润率两个心皮保险丝滑动打开丝丽。使用一对镊子拿起pH值5.7到0.3米山梨醇和5毫米的MES解决方案的一个种子，在珠孔端的一个小切口滑出胚胎，挤推胚乳胚乳和种皮中分离出完整无缺的结束（木下等人 ，2004年）。放入液氮中的单独的微管胚和胚乳。继续此，直到我们积累了从10-15角果胚或胚乳，然后储存在​​-80 ° C冰箱管。在某些情况下，胚乳没有从种皮，即分离，可以分离出基因印迹分析胚乳和种皮的混合物从总RNA。 三。重亚硫酸盐测序 1。所需的试剂十六烷基三甲基溴化铵（CTAB）为准备基因组DNA，限制性内切酶，3 M氢氧化钠（新鲜的），6.42 M尿素/ 4米的亚硫酸氢钠（Sigma - Aldrich公司，2米的焦亚硫酸钠，S9000，Na2S2O5，分子量：190） 10毫米的氢醌，DNA纯化试剂盒（Promega公司向导DNA清理系统，猫＃A7280），TE缓冲液，6.3米的NaOH（新鲜的），10米NH 4醋酸，20微克/μL的tRNA，和100％的乙醇。 2。亚硫酸氢钠治疗协议详情<ol><li>隔离胚乳基因或胚胎的DNA“（Rogers和Bendich，1988）使用CTAB方法。 </li><li>精华100纳克 - 2微克的基因组DNA在20μL到100μL，限制性内切酶，要分析区域外的总量。多边环境协定的发起人，我们使用，NdeI，XhoI位和PstI或HindIII位。 </li><li>煮沸五分钟的DNA限制性内切酶变性，然后冰解渴。 </li><li> 15分钟，加入1 / 9 3 M NaOH和孵化的体积（2.2μL，20μL酶切的DNA）在37 ° C。 </li><li>转移250μLPCR管的解决方案。 </li><li> 7.5克的尿素溶解在10毫升无菌蒸馏水慢慢加入7.6克焦亚硫酸钠，过1-2小时加热通常有助于溶解;新鲜的10米氢氧化钠调整pH值至5;最后添加无菌蒸馏水容至20毫升。这是6.24 M尿素/ 4米的亚硫酸氢钠溶液。 </li><li>新增6.24 / 4米的亚硫酸氢钠溶液的终浓度为5.36中号M尿素和3.44中号分别为（波林等 ，1998）。例如，添加208μL6.42 M尿素/ 4米以上的22.2μL变性基因组DNA（肖等人，2003年）亚硫酸氢钠溶液。 </li><li>加入10毫米的氢醌在终浓度为0.5毫米（12μL，20μL消化）的DNA。 </li><li>进行亚硫酸氢钠治疗，在PCR仪：55 ° C时15分钟，95℃30秒30次。 </li><li>脱盐使用向导DNA清理系统自Promega的亚硫酸氢钠处理DNA，并跟进协议（ 雅各布森等人，2000年） 。 </li><li>测量的TE的确切数量列脱盐后收回，并添加氢氧化钠6.3米的0.3米孵育终浓度在37 ° 15分钟。 </li><li> 10米NH 4醋酸（pH 7.0）中，终浓度为3米，2μL20μg/μL的tRNA的，3卷100％的乙醇，然后混合。在14000转离心15分钟。 </li><li>用70％乙醇洗涤沉淀一次，做一个简短的离心机，并删除多余的乙醇。 </li><li>干颗粒speedvac在5-10分钟和悬浮在25 - 100μLTE缓冲液取决于DNA的量开始。现在的亚硫酸氢钠处理DNA PCR分析。 </li></ol> 3。 PCR扩增<ol><li>由于甲基化的胞嘧啶转化为尿嘧啶，这是很难用亚硫酸氢钠处理的DNA为模板扩增大片段。因此，我们通常设计引物扩增的产品不再大于500 bp。 4 - KB的多边环境协定的启动子序列，我们设计了多套引物扩增14个重叠的碎片，以覆盖整个地区（肖等人，2003年） 。 </li><li>序列顶部链，在设计一个正向引物，我）选择一个G（鸟嘌呤）丰富的地区，以便有没有超长核苷酸引物退火温度较高的;二）改变C（胞嘧啶）为Y（嘧啶在CG和CNG上下文）和改变其余的C T（胸腺嘧啶）。在设计反向引物，我）选择C丰富的地区;二）改变G到R在CG和CNG上下文（嘌呤）和改变剩余的G到A（腺嘌呤）。 </li><li>序列底部股，在设计一个正向引物，我）选择一个C丰富的地区;二）改变G到R在CG和CNG上下文和更改设计反向引物，我）选择其余G到A。一个G -丰富的地区;二）改变C到Y（嘧啶）在CG和CNG上下文和改变其余的C到T </li><li>我们通常使用1-2μL的亚硫酸氢钠处理的DNA为模板，每个PCR扩增（肖等人，2003年） 。 PCR产物凝胶电泳分析，以确认正确的片段大小，然后将凝胶纯化，插入克隆到TOPO TA克隆载体pCR2.1（Invitrogen公司）。单菌落拿起和培养;质粒DNA的提取和测序发送。 </li></ol> 4。序列分析亚硫酸氢钠测序的原理是，将被转换成高浓度亚硫酸氢钠在PH5.0扩增PCR产物将作为胸腺嘧啶尿嘧啶由于水解脱氨甲基化的胞嘧啶5 - 甲基胞嘧啶，而不会由亚硫酸氢钠修改并保持后PCR扩增（Clark 等，1994; Frommer 等，1992）。胞嘧啶。后获得的测序结果，我们比较它与特定链的模板进行PCR扩增。如果模板中的胞嘧啶残基在测序结果胸腺嘧啶读取，它表明，没有甲基化的胞嘧啶。如果模板中的胞嘧啶残基仍然是一个胞嘧啶测序，这意味着是甲基化的胞嘧啶。 四。代表性的成果<img src="/files/ftp_upload/2327/2327fig1.jpg" alt="图1 /" /> 图1。

<ol>
	<li>Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic Acids Res. 22, 2990-2997 (1994).</li><li>Cokus, S. J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C. D., Sriharsa Pradhan, S., Nelson, S. F., Pellegrini, M., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+bisulphite+sequencing+of+the+Arabidopsis+genome+reveals+DNA+methylation+patterning.">Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.</a> Nature. 452, 215-219 (2008).</li><li>Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc Natl Acad Sci USA. 89, 1827-1831 (1992).</li><li>Gehring, M., Huh, J. H., Hsieh, T. F., Penterman, J., Choi, Y., Harada, J. J., Goldberg, R. B., Fischer, R. L. D. E. M. E. T. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+glycosylase+establishes+MEDEA+polycomb+gene+self-imprinting+by+allele-specific+demethylation.">DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation.</a> Cell. 124, 495-506 (2006).</li><li>Henderson, I. R., Chan, S. R., Cao, X., Johnson, L., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+sodium+bisulfite+sequencing+in+plants.">Accurate sodium bisulfite sequencing in plants.</a> Epigenetics. 5, 47-49 (2010).</li><li>Hsieh, T. F., Ibarra, C. A., Silva, P., Zemach, A., Eshed-Williams, L., Fischer, R. L., Zilberman, D. <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+demethylation+of+Arabidopsis+endosperm.">Genome-wide demethylation of Arabidopsis endosperm.</a> Science. 324, 1451-1454 (2009).</li><li>Jacobsen, S. E., Sakai, H., Finnegan, E. J., Cao, X., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+hypermethylation+of+flower-specific+genes+in+Arabidopsis.">Ectopic hypermethylation of flower-specific genes in Arabidopsis.</a> Curr. Biol. 10, 179-186 (2000).</li><li>Kinoshita, T., Miura, A., Choi, Y., Kinoshita, Y., Cao, X., Jacobsen, S. E., Fischer, R. L., Kakutani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-way+control+of+FWA+imprinting+in+Arabidopsis+endosperm+by+DNA+methylation.">One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation.</a> Science. 303, 521-523 (2004).</li><li>Lister, R., O'Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H., Ecker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+integrated+single-base+resolution+maps+of+the+epigenome+in+Arabidopsis.">Highly integrated single-base resolution maps of the epigenome in Arabidopsis.</a> Cell. 133, 523-536 (2008).</li><li>Lister, R., Pelizzola, M., Dowen, R. H., Hawkins, R. D., Hon, G., Tonti-Filippini, J., Nery, J. R., Lee, L., Ye, Z., Ngo, Q. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+DNA+methylomes+at+base+resolution+show+widespread+epigenomic+differences.">Human DNA methylomes at base resolution show widespread epigenomic differences.</a> Nature. 462, 315-322 (2009).</li><li>Paulin, R., Grigg, G. W., Davey, M. W., Piper, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urea+improves+efficiency+of+bisulfite-mediated+sequencing+of+5'-+methylcytosine+in+genomic+DNA.">Urea improves efficiency of bisulfite-mediated sequencing of 5'- methylcytosine in genomic DNA.</a> Nucl. Acids Res. 26, 5009-5010 (1998).</li><li>Rogers, S. O., &amp;amp, B. e. n. d. i. c. h., J, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+plant+tissues.">Extraction of DNA from plant tissues.</a> Plant Molecular Biology Manual. A6, 1-10 (1988).</li><li>Smyth, D. R., Bowman, J. L., Elliot, M., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Flower+Development+in+Arab&#237;dopsis.">Early Flower Development in Arab&#237;dopsis.</a> Plant Cell. 2, 755-767 (1990).</li><li>Xiao, W., Gehring, M., Choi, Y., Margossian, L., Pu, H., Harada, J. J., Goldberg, R. B., Pennell, R. I., Fischer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinting+of+the+MEA+Polycomb+gene+is+controlled+by+antagonism+between+MET1+methyltransferase+and+DME+glycosylase.">Imprinting of the MEA Polycomb gene is controlled by antagonism between MET1 methyltransferase and DME glycosylase.</a> Dev. Cell. 5, 891-901 (2003).</li></ol>

这是比较容易分离胚胎胚乳和种皮，从种皮中分离出来的胚乳，在胚胎发育的早期或中期鱼雷阶段，特别是对种子，但它是单调乏味的。由于种皮不仅有助于某些基因，极少量的组织，例如，MEA和荧光增白剂 ，我们不从种皮中分离出来的胚乳。这意味着，我们可以从胚乳和种皮组织混合物中分离的RNA，检查一个基因的母亲和父亲的等位基因表达，比较，并确定该基因的印迹状态的表...

用品<ul><li>解剖显微镜</li><li>剪刀</li><li>精尖钳</li><li>珠宝标签</li><li>植物锦标</li><li> String或扭曲的关系</li><li> 4“× 2”× 8“聚乙烯袋</li><li> 3“X 1”X 1.0毫米显微镜载玻片</li><li>液氮</li><li>液氮容器</li><li>加热块</li><li> PCR管</li><li>热循环</li><li>离心管</li><li>离心</li><li>凝胶电泳设施</li><li>哥伦比亚0 拟南芥植物</li><li> 拟南芥兰茨贝格万寿菊植物</li></ul>试剂<ul><li> 70％的乙醇</li><li> 95％的乙醇</li><li> 100％的乙醇</li><li>山梨醇0.3米和5毫米的MES，pH值5.7 </li><li>十六烷基三甲基铵（CTAB） </li><li> 100％的乙醇</li><li> Cholorform </li><li>限制性内切酶</li><li> 3 M氢氧化钠</li><li> 6.3米氢氧化钠</li><li> 6.24 M尿素/ 4米的亚硫酸氢钠</li><li>无菌蒸馏水H 2 O </li><li> 10 mM的对苯二酚</li><li>向导DNA清理系统（Promega公司） </li><li> 10米NH 4醋酸</li><li> 20微克/微升的tRNA </li><li> TE缓冲液</li><li> TOPO TA克隆试剂盒（Invitrogen公司） </li></ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

拟南芥是一种优秀的模式生物研究表观遗传机制。其中一个原因是DNA甲基丧失功能的空突变是可行的，从而提供了一个系统来研究如何在基因组DNA甲基化的损失，影响经济增长和发展。印指母系和父系等位基因表达差，在哺乳类动物和植物再生产的发展中起着重要作用。 DNA甲基化是决定是否印迹基因的母亲或父亲的等位基因表达或沉默的关键。在开花植物中，有一个再生产的双受精事件：一个精子细胞受精的卵细胞，形成与中央细胞上升到胚乳的胚胎和精子第二保险丝。胚乳是印迹发生在植物的组织。美狄亚，一个SET域polycomb的组基因和荧光增白剂，一种转录因子调节开花，胚乳和其表达的印迹显示前两个基因是由DNA甲基化和去甲基化控制植物。为了确定印记的基因和胚乳中的甲基化模式的状态，我们需要能够先隔离胚乳。由于种子是在拟南芥中的微小的，但它仍然具有挑战性，隔离拟南芥胚乳，并检查其甲基化。在此视频协议中，我们报告如何进行遗传交叉，隔离胚乳组织，并确定由亚硫酸氢钠测序的甲基化状态。

Watch this Scientific Journal Video about Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm at JoVE.com

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

印记是在植物和哺乳动物繁殖的现象。 DNA甲基化起着重要作用的印迹机制。隔离胚乳和确定的印记基因的甲基化状态<em&gt;拟南芥</em&gt;可以是困难的。在这个协议中，我们介绍了如何隔离胚乳，并确定由亚硫酸氢钠测序的甲基化。

印记基因的DNA甲基化的测定拟南芥胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

determination-dna-methylation-imprinted-genes-arabidopsis

Research

JoVE Journal

Biology

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

17.3K 次观看.  Saint Louis University. 印记是在植物和哺乳动物繁殖的现象。 DNA甲基化起着重要作用的印迹机制。隔离胚乳和确定的印记基因的甲基化状态拟南芥可以是困难的。在这个协议中，我们介绍了如何隔离胚乳，并确定由亚硫酸氢钠测序的甲基化。

视频: Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNA甲基化：亚硫酸氢修改和分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

科研

生物学

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

在所有人类染色体24 chromosomics：检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

种皮床品检测到基因探索<em&gt;体外</em&gt;如何控制胚乳种子萌发的<em&gt;拟南芥</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

增强的减少表示亚硫酸氢盐测序的DNA甲基化的评估，在碱基对决议

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性<em&gt; KCNJ10</em&gt;（Kir4.1）

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...