Name: methods to investigate the regulatory role of small rnas and ribosomal occupancy of plasmodium falciparum
Uploaded: 2015-12-04T14:00:00.000+00:00
Duration: 10 min 22 s
Description: Watch this Scientific Journal Video about Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum at JoVE.com

This research was funded by Doris Duke Charitable Foundation, Burroughs Wellcome Fund, NIH R21DK080994, Duke Chancellor's pilot project fund, the Roche Foundation for Anemia Research and The Bill and Melinda Gates Foundation. G.L. is supported by Duke's UPGG and the NIH (Grant # 5R01AI090141-03) and K.A.W. by the NSF Graduate Research Fellowship Program.

1:隔离从P.小型的RNA 疟原虫在IDC <ol><li>获得疟原虫无性文化29。 注:所需要的培养规模将有所不同根据所需的应用;然而，10毫升培养在3-5％寄生虫血症和5％的血细胞比容提供了充足的RNA进行实时PCR（RT-PCR）。该技术最初是为异步培养物进行了优化，但是当感染周期中的特定时间点需要的话，环阶段寄生虫可以通过山梨醇同步不迟超过10-12小时入侵后同步。同步应该一个周期（约48小时）29后重复。 </li><li>池感染细胞一起（〜5×10 9的RBCs在3-5％的寄生虫血症）和填充冷PBS到管和离心机的顶部，在800×g离心5分钟，无制动以沉淀红细胞。 </li><li>离心后，小心地用吸吸管除去上清液，附到真空中，由于红细胞​​粒料很容易去除。用冷的1×PBS洗涤沉淀一次。 </li><li>在寒冷的0.15％皂苷悬浮细胞沉淀（以1×PBS中），并置于冰上30分钟。 </li><li>离心机的细胞在1500星光12分钟在4°C，除去冷PBS上清（这将是暗红色），并重新悬浮颗粒。再一次重复此步骤。 注意:为了确保存在的微RNA是反射寄生虫内微RNA，而不是红细胞污染，一些寄生虫分离条件进行了测试:1）皂苷裂解，2）皂苷裂解结合RNA酶A处理的宿主细胞的miRNA和3 ）甲基-β-环糊精处理，除去从主机红细胞的寄生虫。所有的处理，得到类似的结果。 </li><li>裂解使用推荐600微升裂解缓冲液分离的寄生虫沉淀。的裂解缓冲液体积这是足够的培养物高达约30 ml的在2％的血细胞比容和5％寄生虫血症。 注:对于较大的寄生虫培养物，该体积能够增加。以确保寄生虫粒料的完全裂解通过彻底涡旋至少1分钟是重要的。此外，为便于提取，裂解的样品可以被转移到一个1.7 ml离心管。 </li><li>添加匀浆添加剂（醋酸酯2钠，pH 4）的60微升，或1/10体积在步骤6中使用的裂解缓冲液，。 </li><li>离开样品在冰上10分钟。 </li><li>添加600微升，或一个体积等于裂解缓冲液，在步骤6中加入酸苯酚choloroform，向每个样品并涡旋1分钟的量。 </li><li>通过离心样品以10,000×g离心5分钟分离水相和有机相。此自旋长于由该试剂盒建议，以确保所有的血红蛋白/疟原虫色素是在有机相中。 </li><li>收集上层（水溶液层），并将其传输至新管。重复步骤9和10，如果水相为白色或（更可能）褐色，这表明亲蛋白质污染。 </li><li>通过移液管确定得到的上清液的体积，然后添加1.25体积的100％乙醇（〜800微升），并通过反相1.7 ml离心管5次混合。 </li><li>穿过一个设置RNA结合滤筒各样品（若干不同，其中是市售的）通过离心在14,000rpm xg离心1分钟或通过使用真空歧管中。 </li><li>洗滤筒700微升所述miRNA的洗涤缓冲液1使用微量，以14000×g离心纺丝1分钟。 </li><li>通过在14000 XG离心每1分钟两次清洗过滤器滤芯500微升洗涤液的2/3。 </li><li>洗脱的RNA用100μlDEPC水，这已被预热到95°C下，在50μl的洗涤两次，以14000×g离心离心每1分钟。测量的RNA通过UV吸光度在260nm处的浓度。 注:一种RNA凝胶（或TBE - 尿素丙烯酰胺或变性甲醛琼脂糖凝胶）可以bë用于评估所述分离的RNA的尺寸分布。 注:这种RNA是适合于通过微阵列，RNA测序，Northern印迹和核糖核酸酶保护测定分析。 </li></ol> 2:定量从P.小型的RNA 疟原虫在IDC <ol><li>确定分离在步骤1使用紫外分光光度法在260nm处个别样本的浓度。 </li><li>生成的实时PCR引物为所需的RNA种类。对于单独的miRNA，我们使用了预制miRNA的qPCR实验，而mRNA的或融合的miRNA表达我们设计引物进行SYBR绿色。聚变的RNA，正向引物是miRNA序列本身（的miR-451:AAACCGTTACCATTACTGAGTT），而反向是基因特异性引物〜100bp的所述miRNA的下游中的mRNA序列（PKA-R:ATCGAACATGCTGTGAACAT）。 </li><li>使cDNA的使用RNA样品或者发夹结构引物的miRNA（使用20-40纳克的RNA）单独或随机六聚体为mRNA和/或miRNA-mRNA融合物种（使用1微克的RNA）。混合RNA和引物5分钟，在65℃，然后冷却至室温。之后，添加逆转录酶混合物（0.5微升反转录酶，0.5μl的RNase抑制剂，1微升的dNTPs（各2.5mM），4μl的5×缓冲液，2微升0.1M DTT，1微升H 2 O）。 <ol><li>运行样品上能够检测或者SYBR绿色或TaqMan探针热循环。 SYBR绿定量PCR 30，用于特定的基因被运行作为3步PCR，95℃15秒，最低引物退火温度零下5℃，30秒，和68℃，30秒，40个循环，使用商业SYBR Green母液。 miRNA的实时PCR被运行作为二步PCR，95℃15秒和60℃1分钟，进行40个循环。 </li><li>量化使用ΔΔCT方法分析和规范他们对任何饶GTP酶（PF08_0110）或18S rRNA的31。 </li></ol></li></ol> 3:前言小RNA进入疟原虫<ol><li>沉淀300微升每转红血细胞（250微升红细胞，约1.5×10 9细胞，最终将被使用）在完整的疟疾介质离心，在800×g离心5分钟，制动器1。 </li><li>用RPMI重悬在完全cytomix（120毫米氯化钾洗两次红细胞; 0.15毫米氯化钙2; 10mM的K 2 HPO 4 / KH 2 PO 4，pH为7.6; 25毫米的HEPES，pH值7.6; 2mM的EGTA，pH值7.6; 5mM的氯化镁 ; pH值用KOH在800 xg离心在制动器1离心5分钟后调整），在50％的血细胞比容。 </li><li>转移细胞到0.2厘米电穿孔杯中。 </li><li>添加的定制合成的寡核苷酸到反应杯10微克，或适量，并悬浮培养物。 </li><li>调整电穿孔至310 V / 950μF，并提供一个脉冲，每个试管中。 </li><li>重悬在300微升红细胞的比色皿内预热完成MAL咏叹调 ​​媒体和板块它们在24孔板和在密闭容器与疟疾血液混合气体在37℃（3％O 2，5％的CO 2）。 </li><li> 4小时后，感染转染的红细胞与同步晚期滋养体至1％的近似最终的寄生虫血症。 </li><li>加入新鲜红细胞转每4-6天，感染的培养，并确定使用的YoYo-1染色％原虫通过流式细胞仪，如第4所示。 </li></ol> 4:测定红细胞的microRNA效应在寄生虫感染率<ol><li>取100微升重悬的文化通过微量移液器和地点在1.7 ml离心管。用1ml 1×PBS中并离心在台式微量离心机离心800×g离心5分钟洗涤两次。 </li><li>重新悬浮颗粒在1毫升0.025％戊二醛。孵育样品20分钟，在室温。 注:样品在这里可以贮存数周，在4℃。此后，沉淀感染ř离心在800×g离心5分钟的BC。用1ml的1×PBS洗两次。 </li><li>重新悬浮颗粒在1毫升0.015％，皂甙。孵育在4℃下15分钟。粒料感染RBC离心800×g离心5分钟。用1ml的1×PBS洗两次。 Repeat.4.3。重悬细胞1ml含1微升（1,000X）的DNA染色的1×PBS。 </li><li>确定寄生虫血症的程度上的流式细胞仪，在488nm激发和〜530纳米（FL-1）发射。 </li><li>第一，栅极血细胞计数基于FSC和SSC流集中在红细胞。然后，通过荧光在FL-1通道测量门控RBC度原虫的（感染RBC）。 注:后期寄生虫是10倍比早期阶段寄生虫多种荧光，和〜100倍以上的未感染RBC。另外，收集在低速趋于降低噪音。最后，该方法相比已经3 H-次黄嘌呤掺入和Giemsa染色，既如先前报道13，和所有的技术GAVË类似的结果。 </li></ol> 5:的miRNA基因融合产品的生物素标记和洗脱<ol><li>直接订购定制合成的RNA寡核苷酸与脱硫生物素（DB-）共价连接于5&#39;端。 </li><li>染未感染RBC具有脱硫生物素 - 缀合（DB-）miRNA与未修饰的miRNA（阴性对照），如在步骤3中指示。 </li><li>感染红细胞转用P.疟原虫寄生虫至0.5％（步骤1.1）的起始寄生虫血症。 </li><li>提取寄生虫核糖核酸4天（96小时）后，如步骤1所述。 </li><li>孵育10微克的寄生虫的RNA用50μl链霉填充珠，通过微量添加，并在4℃下旋转，在微量离心管中旋转1小时。 </li><li>沉淀小珠，在800×g离心30秒，并用20mM的KCl和1 / 1,000 RNA酶抑制剂。 </li><li>洗脱的RNA从珠通过竞争了2mM生物素和用200μl的RNA捕获洗脱缓冲液（20mM氯化钾，1/1，000酶抑制剂和2mM生物素），在4 ℃过夜轮换。 </li><li>决定指出P的富集程度使用SYBR绿定量RT-PCR和微RNA富集（阳性对照）通过定量PCR荧光定量如步骤2所示疟原虫成绩单。 </li></ol> 6:核糖体分离，以确定P的核糖体占用疟原虫 <ol><li>放置5毫升15％的蔗糖溶液（400毫乙酸钾，25mM的钾的HEPES，pH值7.2，15mM的醋酸镁，200μM的放线菌酮，1mM的DTT，1mM的PMSF，40 U / ml的RNA酶抑制剂，和15％的蔗糖）成SW41超速离心管（14×89毫米）。 </li><li>使用5毫升注射器，加入5毫升50％的蔗糖溶液（400毫乙酸钾，25mM的钾的HEPES，pH值7.2，15mM的醋酸镁，200μM的放线菌酮，1mM的DTT，1mM的PMSF，40 U / ml的核糖核酸酶抑制剂，和50％蔗糖）使用长钢针15％的蔗糖的下面，然后取出针。 </li><lI&gt;用封口膜密封的梯度管的顶部，慢慢倾斜管倒向一侧，在稳定的位置，以防止滚动，因此它在桌面上水平地躺着。保持管在其一侧为至少2小时。在等待时，开始步6.5。 </li><li> 2小时后，缓慢地倾斜管背部直立和管转移到冰，允许它冷却至少15分钟前，样品加载（步骤14）。 </li><li>收集足够的疟原虫 -感染的血液阶段的文化产生100-500微克的总RNA，或大约100毫升的3-5％原虫异步文化。添加1/10含有10×（2毫摩尔）环己酰亚胺（CHX）培养基的该卷和在37℃下进行10分钟。 <ol><li>团培养离心500×g离心7分钟的离心分离机（制动器1），然后用室温1×含200μM的CHX PBS洗涤两次。重悬含200微米CHX寄生虫文化在1X PBS，并储存在冰。 </li></ol></l我&gt; <li>估计红细胞粒料的体积。添加裂解缓冲液（400毫乙酸钾，25mM的钾的HEPES，pH值7.2，15mM的醋酸镁，200μM的CHX，1mM的DTT，1mM的PMSF，40 U / ml的RNA酶抑制剂，1％（体积/体积）IGEPAL CA -630和0.5％（重量/体积）DOC）的红细胞沉淀到4.25 ml的终体积。孵育裂解液4 °下进行10分钟，同时旋转。 </li><li>溶胞产物转移到微量离心管，然后在16000×g离心和4离心样品中微量 °下进行10分钟。 </li><li>吹打1.25的冷的0.5M蔗糖缓冲液（400毫乙酸钾，25mM的钾的HEPES，pH值7.2，15mM的醋酸镁，200μM的放线菌酮，1mM的DTT，1mM的PMSF，40 U / ml的RNA酶毫升制备蔗糖垫抑制剂，和0.5M蔗糖）到SW55超速离心管（13×51毫米）。 </li><li>小心地用注射器和小（〜27）号针头的蔗糖垫层的顶层3.75 ml的裂解物上清液（来自步骤7）的。 STORE中的剩余裂解液（〜500微升）在-80°C至提取总RNA水平，如第1所示。 </li><li>加载样本到超速离心机转子，预冷却至4 °C，可容纳13.2毫升管。离心样品在366000×g离心和4℃146分钟。 </li><li>使用移液管，转移上清液成15mL的锥形管中。存储上清液在-80℃的游离（未结合的核糖体）的RNA的RNA分离。 </li><li>重悬核糖体沉淀在500μl核糖体再悬浮缓冲液（400毫乙酸钾，25mM的钾的HEPES，pH值7.2，15mM的醋酸镁，200μM的放线菌酮，1mM的DTT，1mM的PMSF，和40 U / ml的RNA酶抑制剂）的吹打5分钟。 </li><li>离心机在微量样品在16000×g离心和4 &#160; °下进行10分钟。 </li><li>在保持梯度步骤＃4冰上，除去封口膜，然后小心地层渐变USI顶部的核糖体悬浮液NG注射器和小（〜27）号针头。 </li><li>加载围成一个预冷超速离心机转子和离心加载梯度以200,000×g离心和4℃180分钟。当旋转完成后，储存于4离心梯度 °C，直到准备加载到分馏塔。 </li><li>加载一个空的超速离心管放入梯度精馏塔（这可以是在一个先前的运行中使用的管），然后用无RNA酶的水洗涤5分钟，在100×10的速度。 </li><li>期间在步骤6.16洗涤，置紫外吸收检测器的灵敏度。一个很好的出发灵敏度是0.2，但是这可能在稍后奔跑取决于阿254信号（其变化的基础上的寄生虫数等）来进行调节。 </li><li>一旦检测器灵敏度被设置，设置基线信号至零，同时水流过检测器。 </li><li>洗涤该馏分收集后，反向流体流动，直至线是空的，然后对物奥雅纳空超速离心管。 </li><li>运行通过精馏塔60％的蔗糖溶液，直到它离开针头装置。 </li><li>插入包含​​所述第一梯度的管中，在装载室的顶部，并拧紧封，注意不要过度拧紧。然后，刺穿与针管的底部。 </li><li>重置流体流动速度，以12.5×10（由泵前部的流动速度控制旋钮控制），然后设定自动级分收集器以收集18秒的级分（〜330微升/分）在微量离心管中。 </li><li>启动向前流的60％的蔗糖溶液中。 </li><li>当梯度液的首次下降落入离心管中，立即开始双方馏分收集和A 254信号的现场录音。 </li><li>一旦大幅下跌是在A 254的信号观察，对应50％和60％的蔗糖溶液之间的介面，停止流体流动和A 254两种</sUB&gt;录制。 </li><li>存储梯度分数在-80 &#160; °下以后使用。 </li><li>反向液流在100×10的速度，直到60％的蔗糖溶液清空出超速离心管。 </li><li>如果有附加梯度分馏，从装载室取出空超速离心管，并从步骤6.21重启。如果所有的样品都完成后，清洗设备，在步骤6.16和6.19进行。 </li></ol>

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Malaria Report. , (2014).</li><li>Livincstone, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+and+human+polymorphisms.">Malaria and human polymorphisms.</a> Annu Rev Genet. 5, 33-64 (1971).</li><li>Nagel, R. L., Roth, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+and+red+cell+genetic+defects.">Malaria and red cell genetic defects.</a> Blood. 74, 1213-1221 (1989).</li><li>Aidoo, M., 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=Protective+effects+of+the+sickle+cell+gene+against+malaria+morbidity+and+mortality.">Protective effects of the sickle cell gene against malaria morbidity and mortality.</a> Lancet. 359, 1311-1312 (2002).</li><li>Tiffert, T., 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=The+hydration+state+of+human+red+blood+cells+and+their+susceptibility+to+invasion+by+Plasmodium+falciparum.">The hydration state of human red blood cells and their susceptibility to invasion by Plasmodium falciparum.</a> Blood. 105, 4853-4860 (2005).</li><li>Fairhurst, R. M., 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=Abnormal+display+of+PfEMP-1+on+erythrocytes+carrying+haemoglobin+C+may+protect+against+malaria.">Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria.</a> Nature. 435, 1117-1121 (2005).</li><li>Cyrklaff, M., 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=Hemoglobins+S+and+C+interfere+with+actin+remodeling+in+Plasmodium+falciparum-infected+erythrocytes.">Hemoglobins S and C interfere with actin remodeling in Plasmodium falciparum-infected erythrocytes.</a> Science. 334, 1283-1286 (2011).</li><li>Friedman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocytic+mechanism+of+sickle+cell+resistance+to+malaria.">Erythrocytic mechanism of sickle cell resistance to malaria.</a> Proceedings of the National Academy of Sciences of the United States of America. 75, 1994-1997 (1978).</li><li>Pasvol, G., Weatherall, D. J., Wilson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanism+for+the+protective+effect+of+haemoglobin+S+against+P.+falciparum+malaria.">Cellular mechanism for the protective effect of haemoglobin S against P. falciparum malaria.</a> Nature. 274, 701-703 (1978).</li><li>Lecellier, C. H., 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+cellular+microRNA+mediates+antiviral+defense+in+human+cells.">A cellular microRNA mediates antiviral defense in human cells.</a> Science. 308, 557-560 (2005).</li><li>Niu, Q. W., 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=Expression+of+artificial+miroRNAs+in+transgenic+Arabidopsis+thaliana+confers+virus+resistance.">Expression of artificial miroRNAs in transgenic Arabidopsis thaliana confers virus resistance.</a> Nature biotechnology. 24, 1420-1428 (2006).</li><li>Zhao, G., Yu, D., Weiss, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+erythropoiesis.">MicroRNAs in erythropoiesis.</a> Curr Opin Hematol. 17, 155-162 (2010).</li><li>Lee, Y. T., 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=LIN28B-mediated+expression+of+fetal+hemoglobin+and+production+of+fetal-like+erythrocytes+from+adult+human+erythroblasts+ex+vivo.">LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo.</a> Blood. 122, 1034-1041 (2013).</li><li>Sangokoya, C., Doss, J. F., Chi, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-responsive+miR-485-3p+regulates+cellular+iron+homeostasis+by+targeting+ferroportin.">Iron-responsive miR-485-3p regulates cellular iron homeostasis by targeting ferroportin.</a> PLoS genetics. 9, e1003408 (2013).</li><li>Chen, S. Y., Wang, Y., Telen, M. J., Chi, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+analysis+of+erythrocyte+microRNA+expression+in+sickle+cell+diseases.">The genomic analysis of erythrocyte microRNA expression in sickle cell diseases.</a> PLoS ONE. 3, e2360 (2008).</li><li>Sangokoya, C., Telen, M. J., Chi, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA+miR-144+modulates+oxidative+stress+tolerance+and+associates+with+anemia+severity+in+sickle+cell+disease.">microRNA miR-144 modulates oxidative stress tolerance and associates with anemia severity in sickle cell disease.</a> Blood. 116, 4338-4348 (2010).</li><li>Deitsch, K., Driskill, C., Wellems, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+malaria+parasites+by+the+spontaneous+uptake+and+expression+of+DNA+from+human+erythrocytes.">Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes.</a> Nucleic acids research. 29, 850-853 (2001).</li><li>Liang, X. H., Haritan, A., Uliel, S., Michaeli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=trans+and+cis+splicing+in+trypanosomatids:+mechanism,+factors,+and+regulation.">trans and cis splicing in trypanosomatids: mechanism, factors, and regulation.</a> Eukaryotic cell. 2, 830-840 (2003).</li><li>Baum, J., 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=Molecular+genetics+and+comparative+genomics+reveal+RNAi+is+not+functional+in+malaria+parasites.">Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites.</a> Nucleic acids research. 37, 3788-3798 (2009).</li><li>Hall, N., 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+comprehensive+survey+of+the+Plasmodium+life+cycle+by+genomic,+transcriptomic,+and+proteomic+analyses.">A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.</a> Science. 307, 82-86 (2005).</li><li>Caro, F., Ahyong, V., Betegon, M., DeRisi, J. L. <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+regulatory+dynamics+of+translation+in+the+asexual+blood+stages.">Genome-wide regulatory dynamics of translation in the asexual blood stages.</a> eLife. 3, (2014).</li><li>Oehring, S. C., 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=Organellar+proteomics+reveals+hundreds+of+novel+nuclear+proteins+in+the+malaria+parasite+Plasmodium+falciparum.">Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum.</a> Genome biology. 13, R108 (2012).</li><li>Lasonder, E., 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=Analysis+of+the+Plasmodium+falciparum+proteome+by+high-accuracy+mass+spectrometry.">Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry.</a> Nature. 419, 537-542 (2002).</li><li>Bozdech, Z., 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=The+transcriptome+of+the+intraerythrocytic+developmental+cycle+of+Plasmodium+falciparum.">The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum.</a> PLoS biology. 1, E5 (2003).</li><li>Gardner, M. J., 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=Genome+sequence+of+the+human+malaria+parasite+Plasmodium+falciparum.">Genome sequence of the human malaria parasite Plasmodium falciparum.</a> Nature. 419, 498-511 (2002).</li><li>Zhang, M., Joyce, B. R., Sullivan, W. J., Nussenzweig, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+in+Plasmodium+and+toxoplasma+parasites.">Translational control in Plasmodium and toxoplasma parasites.</a> Eukaryotic cell. 12, 161-167 (2013).</li><li>Lacsina, J. R., LaMonte, G., Nicchitta, C. V., Chi, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+profiling+of+the+malaria+parasite+Plasmodium+falciparum.">Polysome profiling of the malaria parasite Plasmodium falciparum.</a> Molecular and biochemical parasitology. 179, 42-62 (2011).</li><li>Bunnik, E. M., 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=Polysome+profiling+reveals+translational+control+of+gene+expression+in+the+human+malaria+parasite+Plasmodium+falciparum.">Polysome profiling reveals translational control of gene expression in the human malaria parasite Plasmodium falciparum.</a> Genome biology. 14, R128 (2013).</li><li>LaMonte, G., 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=Translocation+of+sickle+cell+erythrocyte+microRNAs+into+Plasmodium+falciparum+inhibits+parasite+translation+and+contributes+to+malaria+resistance.">Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance.</a> Cell host &amp; microbe. 12, 187-199 (2012).</li><li>Lambros, C., Vanderberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Plasmodium+falciparum+erythrocytic+stages+in+culture.">Synchronization of Plasmodium falciparum erythrocytic stages in culture.</a> The Journal of parasitology. 65, 418-420 (1979).</li><li>Bourgeois, N., 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=Comparison+of+three+real-time+PCR+methods+with+blood+smears+and+rapid+diagnostic+test+in+Plasmodium+sp.+infection.">Comparison of three real-time PCR methods with blood smears and rapid diagnostic test in Plasmodium sp. infection.</a> Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 16, 1305-1311 (2010).</li><li>Abruzzo, L. V., 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=Validation+of+oligonucleotide+microarray+data+using+microfluidic+low-density+arrays:+a+new+statistical+method+to+normalize+real-time+RT-PCR+data.">Validation of oligonucleotide microarray data using microfluidic low-density arrays: a new statistical method to normalize real-time RT-PCR data.</a> Biotechniques. 38, 785-792 (2005).</li><li>Sangokoya, C., LaMonte, G., Chi, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+microRNAs+of+human+mature+erythrocytes.">Isolation and characterization of microRNAs of human mature erythrocytes.</a> Methods in molecular biology. 667, 193-203 (2010).</li><li>Rathjen, T., Nicol, C., McConkey, G., Dalmay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+short+RNAs+in+the+malaria+parasite+and+its+red+blood+cell+host.">Analysis of short RNAs in the malaria parasite and its red blood cell host.</a> FEBS Lett. 580, 5185-5188 (2006).</li><li>Xue, X., Zhang, Q., Huang, Y., Feng, L., Pan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+miRNA+were+found+in+Plasmodium+and+the+ones+identified+in+erythrocytes+could+not+be+correlated+with+infection.">No miRNA were found in Plasmodium and the ones identified in erythrocytes could not be correlated with infection.</a> Malar J. 7, 47 (2008).</li></ol>

The authors have no competing financial interests to declare.

哈佛商学院在疟疾流行地区最常见的异常血红蛋白之一，主要是因为它提供保护，防止造成P.重症疟疾疟原虫 。该技术需要在P的基因调控的人类微小RNA的作用表征恶性详述整个手稿。通过提取总RNA以这样的方式，以包括所有的小RNA，并通过执行一个相对简单的寄生虫溶胞步骤，这些融合的RNA能够通过各种独立技术来鉴定。 这些步骤都比较简单，但有一些问题，如果不遵守正确的污染敏感。在寄生虫提取，关键是要执行皂素裂解以除去污染性宿主红细胞。这是在RNA分离的皂苷裂解步骤，尤其如此。宿主红细胞所述miRNA组分的大小克几个数量比该寄生虫，部分由于大多数红细胞reater是未感染的，所以至关重要的是要除去尽可能多红细胞污染成为可能。同样重要的是要保持在一个健康的和适当的阶段疟疾培养（如需要在感染周期中的特定点），以获得准确的miRNA谱。类似地，酚 - 氯仿提取期间，这是非常重要的，以确保水相都清晰，如果没有，重复酚 - 氯仿提取，以避免任何剩余污染。 另外，在脱硫生物素捕获实验，关键是要洗脱用过量的生物素，并使用最小的链亲和素珠，以确保适当的特定溶出。结合RNA的通过与生物素竞争的珠子，而不是完全变性自己的溶出，有助于显着降低背景RNA富集。最后，经过缺失捕获试验突出了s至演示如何这些miRNA捕获，但几个其它技术也用在先前的研究，以证明这些融合的RNA，如northern印迹和核糖核酸酶保护测定29的重要途径之一。利用转染微RNA检索修饰转录可以具有更广泛的应用，如相关蛋白复合物可以介导转染的miRNA的运输和处理的纯化的这样的方法。 虽然各种全基因组的方法已被用于研究疟疾的蛋白质组和转录组22-25，也有一些方法在全球范围内研究翻译调控的P.恶性 21,26。这种方法的限制排除了翻译调控中P的全球分析疟原虫 。其中最常见的实验方法来研究转录调控是多核糖体分析，评估其整体平移根据加载到感兴趣的特定mRNA的核糖体人的活动。具体在这个手稿是一个优化的多核糖体分析方法在疟疾寄生虫裂解无论是感染红细胞和内寄生虫使用。以前的方法也没有收复大部分多聚核糖体，并使它看起来就像疟原虫没有多聚核糖体的大量人群。有高浓度的乙酸钾和镁使用裂解缓冲液使得有可能以溶解两个红细胞和寄生虫而增溶膜结合核糖体，以允许完整多核糖的捕获。 这种高性价比的核糖体纯化和分析的方法，将有助于弥合疟疾转录组和蛋白质组之间的现有知识的差距并启用P的平移状态下的全基因组分析疟原虫 。这种方法已被用于比较的早期和晚期阶段b的平移状态lood阶段寄生虫28，这已紧密协调的基因表达。上转录的核糖体加载数据可以容易地获得和分析，以确定在特定的mRNA核糖体的密度。此外，当山梨糖醇的同步组合，改变核糖体密度可以被用来确定如何翻译贯穿于寄生虫的生命周期管理。使用这种方法的核糖体隔离需要大量培养的，不幸的是限制了在其中可以进行疟疾生命周期的阶段。大量输入材料也将限制它的使用既在体外实验恶性疟原虫株，并可能限制其适用于其他传染性生物体。任何人使用此过程也将能够以筛选基因，显示异常翻译型材，其中不与核糖体相关联的，反映了基因调控的可选模式特定转录。最后，这种做法将使我们能够评估新的抗疟疾药物是如何影响蛋白质的翻译，并确定耐药性的翻译为基础的机制。这条管线在识别和确定的转录后调控在许多实验系统极大的效用。

疟疾，造成疟原虫属顶复门寄生虫，是人类最常见的寄生虫病，感染全球大约200万人，每年造成周围60万人死亡1。感染人类的五个疟原虫物种的，最相关的人类疾病是P.疟原虫和P.间日疟原虫 ，由于严重疟疾并发症其广泛分布和潜力。疟原虫的生命周期既需要蚊子和人类的感染。当受感染的蚊子叮咬人类，寄生虫通过血液循环到达肝脏，在首轮的复制发生移动。后从主机肝裂殖子破裂，它们感染附近的红血细胞，发起或者无性或有性复制。复制的无性阶段，持续48小时在体育疟原虫，是本研究的焦点，因为它是最发作的同时在源咏叹调 ​​症状和容易概括体外 。 虽然一些公共卫生措施，包括改善抗疟疾疗法，已经有所减少全球疟疾负担，抗药性疟原虫的持续出现，呈现为疟疾防治工作中的问题。这可能意味着新的治疗方法的一个领域是研究遗传变异如何不同赋予抵抗疟疾。在疟疾流行的地区，各种红细胞多态性的现象相当普遍2,3。这些突变，镰状细胞是也许是最突出，通常与对有症状疟疾感染4发病实质性抗性相关。由它们引起红细胞抵抗疟疾感染的基本机制是不完全的了解。寄生红细胞与血红蛋白突变，受增强吞噬功能，通过增强细胞的刚性和脱水，这与减少入侵体育相关疟原虫 5。 HBC的等位基因也影响蛋白质表达在红细胞表面和与细胞骨架的重塑，进一步抑制寄生虫发育6,7。最后，P.疟原虫生长不佳内纯合镰刀（HBSS）红细胞8,9 体外 ，提示疟疾电阻的固有红细胞因素。然而，虽然所有这些机制似乎发挥作用，但它们不能完全解释后面疟疾镰状细胞抗性的机制。 一个潜在的一套红细胞因素仍然知之甚少是成熟红细胞中的miRNA存在的大池。微RNA是小的非编码RNA，19-25个核苷酸的大小，其中由3&#39;UTR内的碱基配对介导的翻译和/或靶mRNA的稳定性。他们有牵连的哺乳动物的免疫反应的控制，包括抑制Ò˚F病毒复制10，并分别显示出赋予对病毒在植物中它们也已显示出调节一些红细胞过程，包括红细胞生成11,12和铁代谢13。以前的研究确定了红细胞的miRNA，其表达显着改变的HBSS红细胞14,15的丰富和多样化的人口。由于成熟的红细胞缺乏积极的转录和翻译，这些红细胞miRNA的功能作用尚不清楚。由于显著物质交换的宿主细胞和P之间发生的红细胞内发育周期（IDC）16中疟原虫，有人推测的HbS红细胞内改变的miRNA谱可以直接有助于细胞内在疟疾电阻。 这些研究最终导致管道的发展分离，鉴定和功能上的米内研究人类miRNA的作用alaria寄生虫P.疟原虫，这表明这些宿主/人miRNA的最终共价保险丝然后平移地抑制寄生虫mRNA转录物17。这提供了通过转拼形成的第一跨物种的嵌合转录物的示例，并且暗示，这种miRNA的体mRNA融合物可以在其它物种中，包括其他寄生虫可以发生。所有锥虫的mRNA为反式剪接与拼接领袖（SL）来调节多顺反子转录物18的分离。由于P.疟原虫缺乏直系同源对的Dicer /阿戈19,20，它是可能的红细胞的miRNA劫持在巴斯德类似SL机械恶性融入靶基因。最近在体育研究疟原虫有实际上表示的5&#39;剪接前导序列21的存在。这项研究详细介绍了导致人类寄生虫的miRNA基因融合基因的发现，这些方法包括转录和translational调节技术。这些方法的总体目标是研究的小RNA在P的基因调控，表型和翻译潜在的影响疟原虫成绩单。 人类寄生虫嵌合成绩单初步查明依赖的RNA分析技术，如实时PCR，转录组测序和EST库的采集，其中包括总额和小RNA，而不是用技术只孤立的小RNA使用。分离所有的RNA一起在一个大的池，而不是分开，允许两个易位人类小RNA在寄生虫的标识以及这些小RNA序列的存在作为较大序列的一部分。然后，这需要这些融合mRNA的翻译状态的分析，以确定这些融合的功能性后果。 而在寄生虫的基因组的表征广泛的努力ð转录纷纷加入到寄生虫的生物学22-25的了解，远不如所了解的mRNA的转录组跨越P的生命周期中的翻译调控疟原虫 26。寄生虫的蛋白质组的这种有限的理解已经阻碍了双方的了解寄生虫的生物学，并确定为下一代抗疟疾治疗的新目标的能力。寄生虫的细胞生物学的理解，这仍存在差距主要是由于缺乏足够的技术来研究翻译调控的P.疟原虫 。最近的一篇论文描述了使用P的核糖体足迹的恶性确定全球翻译状态21。一个公认的转录的平移电位测定是由多核糖仿形测定相关的核糖体的数目。然而，当这种技术应用到P.疟原虫&lt;/ em&gt;的，它是无法收回大部分多核糖体和捕获主要monosomes。近日，几组27,28优化了P.疟原虫多核糖体的技术，通过同时裂解红细胞和寄生虫保存多核糖体和它们在宿主红细胞28无性发育过程中表征这些疟原虫的核糖体占用和翻译潜能。 总的来说，这些方法证明人类miRNA与寄生虫mRNAs的所观察到的融合调制那些融合的mRNA，其使用先前报道的方法27表明的寄生虫蛋白质翻译，并且是在疟疾的HBA阻力的主要决定因素和HBSS红细胞17。这些方法将在任何系统，查找以确定和功能上探索RNA剪接事件是有用的，这些融合的RNA是否在第疟原虫或其他真核系统。

<table><tbody><tr><td>&lt;strong&gt;REAGENTS-All reagents must be RNAse-free.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Diethyl pyrocarbonate</td><td>Sigma</td><td>D5758</td><td>DEPC</td></tr><tr><td>DEPC-treated water (see REAGENT SETUP)</td><td></td><td></td><td></td></tr><tr><td>Yoyo-1 DNA fluorescent dye</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Gene Pulser II electroporator</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>0.2 cm Electroporation cuvettes</td><td></td><td></td><td></td></tr><tr><td>4 M potassium acetate</td><td>Mallinckrodt</td><td>6700</td><td></td></tr><tr><td>2 M potassium HEPES</td><td>Sigma</td><td>H0527</td><td>pH 7.2</td></tr><tr><td>1 M magnesium acetate</td><td>Sigma</td><td>M-2545</td><td></td></tr><tr><td>1 M dithiothreitol</td><td>Research Products International</td><td>D11000</td><td>DTT</td></tr><tr><td>100 mM phenylmethylsulfonate fluoride</td><td>Sigma</td><td>P7626</td><td>PMSF, dissolved in isopropanol</td></tr><tr><td>10% (v/v) Igepal CA-630</td><td>Sigma-Aldrich</td><td>I3021</td><td></td></tr><tr><td>10% (w/v) sodium deoxycholate</td><td>Sigma</td><td>D-6750</td><td>DOC</td></tr><tr><td>Cycloheximide</td><td>Sigma</td><td>C-7698</td><td>CHX</td></tr><tr><td>Sucrose</td><td>Mallinckrodt</td><td>8360-04</td><td></td></tr><tr><td>RNAseOUT (Invitrogen, cat. no. 100000840)</td><td>Invitrogen</td><td>100000840</td><td></td></tr><tr><td>RPMI-1640 w/ L-glutamine</td><td>Cellgro</td><td>10-040-CV</td><td></td></tr><tr><td>10% AlbuMAX I</td><td>Invitrogen</td><td>11020-039</td><td>w/v in sterile water</td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15750-060</td><td></td></tr><tr><td>HT supplement (100x) (Invitrogen, cat. no. 11067030)</td><td></td><td></td><td></td></tr><tr><td>45% (w/v) sucrose (Sigma, cat no. G8769)</td><td></td><td></td><td></td></tr><tr><td>1 M HEPES buffer (Gibco, cat. no. 15630-080)</td><td></td><td></td><td></td></tr><tr><td>1x phosphate buffered saline (PBS; Cellgro, cat. no. 2109310CV)</td><td></td><td></td><td></td></tr><tr><td>Corning 500 ml vacuum filter flask (VWR, cat. no. 430769)</td><td></td><td></td><td></td></tr><tr><td>Glass slides (VWR, cat. no. 48311-702)</td><td></td><td></td><td></td></tr><tr><td>Giemsa stain (50X) (VWR, cat no. m708-01)</td><td></td><td></td><td></td></tr><tr><td>mirVana miRNA isolation kit (Ambion, ThermoFisher Scientific).</td><td></td><td></td><td></td></tr><tr><td>5&#039; Biotin conjugated mature miRNA (sequence varies by miRNA of interest) (Dharmacon)</td><td></td><td></td><td></td></tr><tr><td>Biotin powder (Sigma-Aldrich)</td><td></td><td></td><td></td></tr><tr><td>1 M Potassium Chloride</td><td></td><td></td><td></td></tr><tr><td>Streptavidin Sepharose High Performance Beads (GE Healthcare)</td><td></td><td></td><td></td></tr><tr><td>Ribosome resuspension buffer (see REAGENT SETUP)</td><td></td><td></td><td></td></tr><tr><td>Lysis buffer (see REAGENT SETUP)</td><td></td><td></td><td></td></tr><tr><td>15% (w/v) sucrose gradient solution (see REAGENT SETUP)</td><td></td><td></td><td></td></tr><tr><td>50% (w/v) sucrose gradient solution (see REAGENT SETUP)</td><td></td><td></td><td></td></tr><tr><td>0.5 M sucrose cushion (see REAGENT SETUP)</td><td></td><td></td><td></td></tr><tr><td>60% (w/v) sucrose chase solution (see REAGENT SETUP)</td><td></td><td></td><td></td></tr><tr><td>Plasmodium cell culture media (see REAGENT SETUP)</td><td></td><td></td><td></td></tr><tr><td>RNA capture Wash Buffer (see REAGENT SETUP)</td><td></td><td></td><td></td></tr><tr><td>RNA Elution Buffer (see REAGENT SETUP)</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;REAGENT SETUP&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Plasmodium cell culture media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>500 ml RPMI-1640 w/ L-glutamine</td><td></td><td></td><td></td></tr><tr><td>0.5 ml gentamicin</td><td></td><td></td><td></td></tr><tr><td>5 ml HT supplement</td><td></td><td></td><td></td></tr><tr><td>2.5 ml 45% glucose</td><td></td><td></td><td></td></tr><tr><td>25 ml 10% AlbuMAX I</td><td></td><td></td><td></td></tr><tr><td>12.5 ml 1 M HEPES</td><td></td><td></td><td></td></tr><tr><td>Sterile filter with 0.2 mm vacuum filter flask.</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Plasmodium cell culture media containing 10x CHX&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Same recipe as Plasmodium cell culture media above, with the addition of:</td><td></td><td></td><td></td></tr><tr><td>2 mM cycloheximide</td><td></td><td></td><td></td></tr><tr><td>Media is prepared as normal, then add CHX to a final concentration of 2 mM and filter.&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;1x PBS containing cycloheximide&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cycloheximide is added fresh to 1x PBS to a final concentration of 200 mM, and kept at 4 &lt;sup&gt;O&lt;/sup&gt;C until use.</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Ribosome resuspension buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>400 mM potassium acetate</td><td></td><td></td><td></td></tr><tr><td>25 mM potassium HEPES, pH 7.2</td><td></td><td></td><td></td></tr><tr><td>15 mM magnesium acetate</td><td></td><td></td><td></td></tr><tr><td>200 mM cycloheximide (add fresh)</td><td></td><td></td><td></td></tr><tr><td>1 mM DTT (add fresh)</td><td></td><td></td><td></td></tr><tr><td>1 mM PMSF (add fresh)</td><td></td><td></td><td></td></tr><tr><td>40 U/ml RNaseOUT (add fresh)</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Lysis buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Same recipe as ribosome resuspension buffer above, with the addition of:</td><td></td><td></td><td></td></tr><tr><td>1% (v/v) Igepal CA-630</td><td></td><td></td><td></td></tr><tr><td>0.5% (w/v) DOC</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Sucrose solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Same recipe as ribosome resuspension buffer above, with the addition of varying amounts of sucrose:</td><td></td><td></td><td></td></tr><tr><td>15% (w/v) sucrose to make 15% sucrose gradient solution</td><td></td><td></td><td></td></tr><tr><td>50% (w/v) sucrose to make 50% sucrose gradient solution</td><td></td><td></td><td></td></tr><tr><td>0.5 M sucrose to make 0.5 M sucrose cushion</td><td></td><td></td><td></td></tr><tr><td>As above, cycloheximide, DTT, PMSF, and RNaseOUT must be added fresh.</td><td></td><td></td><td></td></tr><tr><td>The 60% sucrose chase solution requires only sucrose and water, no other components</td><td></td><td></td><td></td></tr><tr><td>60% (w/v) sucrose in DEPC-treated water to make 60% sucrose chase solution</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;RNA Capture Wash Buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>20 mM&amp;nbsp; KCl</td><td></td><td></td><td></td></tr><tr><td>5 unit/ml Rnase Out (Invitrogen)</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;RNA Capture Elution Buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Same recipe as RNA Capture Wash Buffer, with the addition of:</td><td></td><td></td><td></td></tr><tr><td>2 mM Biotin</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SW55 Ti ultracentrifuge rotor (Beckman, cat. no. 342194)</td><td></td><td></td><td></td></tr><tr><td>SW41 Ti ultracentrifuge rotor (Beckman, cat. no. 331362)</td><td></td><td></td><td></td></tr><tr><td>Ultra-Clear&lt;sup&gt;TM&lt;/sup&gt; &amp;frac12; x 2 in (13 x 51 mm) ultracentrifuge tubes for the SW55 (Beckman, cat. no. 344057)</td><td></td><td></td><td></td></tr><tr><td>Polyallomer 9/16 x 3 &amp;frac12; in (14 x 89 mm) ultracentrifuge tubes for the SW41 (Beckman, cat. no. 331372)</td><td></td><td></td><td></td></tr><tr><td>L8-80M ultracentrifuge (Beckman)</td><td></td><td></td><td></td></tr><tr><td>Steel blunt syringe needle, 4 inch, 16 G (Aldrich, cat. no. Z261378)</td><td></td><td></td><td></td></tr><tr><td>1 ml syringe with 27 G&amp;frac12; needle (Becton Dickinson, cat no. 309623)</td><td></td><td></td><td></td></tr><tr><td>5 ml syringe (Becton Dickinson, cat. no. 309646)</td><td></td><td></td><td></td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>Tube rotator, end-over-end</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge, refrigerated</td><td></td><td></td><td></td></tr><tr><td>Density gradient fractionation system (Teledyne Isco, cat. no. 69-3873-179)</td><td></td><td></td><td></td></tr><tr><td>TracerDAQ (Measurement Computing)</td><td></td><td></td><td></td></tr><tr><td>Eppendorf 5810R centrifuge</td><td></td><td></td><td></td></tr><tr><td>Eppendorf A-4-81 rotor (Eppendorf, cat. no. 022638807)</td><td></td><td></td><td></td></tr></tbody></table>

methods to investigate the regulatory role of small rnas and ribosomal occupancy of plasmodium falciparum

遗传变异负责镰状细胞等位基因（HBS）使红细胞的疟原虫，P.抗感染疟原虫 。这个阻力，这是众所周知的是多因素的分子基础，仍然不完全理解。最近的研究发现，红细胞微RNA，一旦转移进入疟原虫的差异表达，同时影响基因调控和寄生虫生长。这些miRNA后来所示，通过5&#39;RNA融合寄生虫mRNA的谨慎的子集形成了嵌合体RNA转录抑制mRNA的翻译。在这里，中使用的技术来研究基因调控和P的平移潜在的功能作用和假定机制底层红细胞的microRNA 疟原虫，包括改性合成微小RNA转染到宿主红细胞，进行详细说明。最后，一​​个多核糖梯度法是用来确定TRA的程度nslation这些成绩单。一起，这些技术使我们能够证明红细胞微RNA的失调水平有助于镰状红细胞的细胞内在疟疾电阻。

人类的microRNA在P的全球分析疟原虫 这里介绍的技术被用来提取寄生虫微小RNA在多种条件。有一点要注意的是，在32，这往往充当了两个本文以及原研29提出的微小RNA数据的参考点指示进行RNA提取的未感染的红细胞。这些以往的研究也表明，HBSS红细胞具有一定的miRNA中，miR-451特别是更大的水平。上面所指出的技术足以证明在哈佛商学院（HBA卡或HBSS）感染寄生虫的变化，miRNA的丰度。 RNA样品提取为上述和归一化表示对18S rRNA基因。从HBSS红细胞寄生虫的样品，例如，不显示在几种微小RNA，包括的miR-451（图1）的水平一显著增加。的miRNA来自主机erythr摄取ocyte也与以往的研究34,35一致。 HBAA红细胞用miR-451的转染增加HBAA的miR-451类似于HBSS红细胞水平 （图1）。流式细胞术，然后用来测量寄生虫感染，以评估miRNA的易位时寄生虫生长的效果。根据是，转染的miR-451中，miR-223和miR-181a的HBAA红细胞，以及一个单链DNA寡，进行了检查的微小RNA在寄生虫生长的影响。的ssDNA或miR-181a的转染过在寄生虫血症没有影响（ 图2A - D）的同时，转染用miR-451明显减少寄生虫血症，这只能由被减轻共转的反义的miR-451寡。 通过2&#39;-O-ME反义​​微RNA转染阻断的microRNA表现出相互影响。 HBSS红细胞，与自然较高的miR-451水平，分别转<html>反义的miR-451或2&#39;-O-甲基寡核苷酸（ 图3）。的miR-451的抑制在这两个HBSS红细胞（ - F 图3C）增加寄生虫生长。寄生虫百分数计算从至少3个独立转染，然后平均，并作为相对于HBSS 生长 （图3F）的倍数变化。 中的miRNA基因融合RNA的生物素捕获法捕获 5&#39;-脱硫生物素 - 的miR-451转染后（5&#39;Db-的miR-451）和5&#39;Db-的miR-181，如在方法部分5所示，已确定，所观察到的miRNA序列源自与人微RNA。定量RT-PCR分析表明，5&#39;Db-的miR-451的转染导致了PKA-R融合转录富集（图4）。 多核糖体分离，以确定P的核糖体占用疟原虫 帐篷"&gt;典型的甲254跟踪示于图5A（与图5B的rRNA含量的北部确认），与由左到右的级分密度的增加在图上（即较轻馏分到左边），初始阿254是相当高，由于吸收由血红蛋白在早期的级分，但很快下降到基线。第一小峰对应于核糖体小亚基（40S），紧接着第二个峰，这是稍大并对应于大核糖体亚基（60S），第三个峰，这是一般最大为恶性疟原虫 ，对应于monosome峰（80S），由于它的高度，80S应作为一个指南调整UV检测器的灵敏度对于后续运行（步骤6.17）。此外，由于上世纪80年代高峰期的大小，通常为60S信号模糊就像一个"肩膀"到monosome高峰期的开始，如果这两个60S和80S信号都很大。 继monosome峰是对应于多聚核糖体的峰值。每个连续的峰代表代表核糖体的逐渐变大的数字的多核糖体级分（2，3，4，等等）。每个多核糖峰是比以前的峰也较小，减少的高度由2-3倍随着每次连续多核糖体数目。典型的运行将允许多核糖5-7核糖体组成的分辨率，虽然分辨率高达一个九聚体的多核糖已观察在一些运行与大量原料。高阶多核糖构成的微量的剩余部分，​​并且不能这些梯度条件下解决。 <img alt="图1" src="/files/ftp_upload/53214/53214fig1.jpg" /> 图1:MIR-451升高于HBSS红细胞 Intraparasitic的miR-451的水平，由实时PCR和所确定的归一化对18S rRNA基因，来自寄生虫从指定的隔离 。红细胞类型。值是平均值±SE。 <img alt="图2" src="/files/ftp_upload/53214/53214fig2.jpg" /> 图2:的miR-451的过表达在正常的红细胞抑制寄生虫生长（A - C）的指示的感染率， ​​在总红细胞的百分比，由M1表示，为示出红血球类型代表FACS图。 （D）来自FACS和标准化对未转染HBAA红细胞复合感染率。在面板的AC值是代表FACS图，而D是平均值±SE。 <img alt="图3" src="/files/ftp_upload/53214/53214fig3.jpg" /> 图3:的miR-451的镰状细胞红细胞抑制升高寄生虫生长（A - E）的代表性FACS图，指示感染率， ​​在总红细胞的百分比，由M1表示，为示器ythrocyte类型。 （F）来源于FACS并绘制对未转染HBAA红细胞的标准值复合感染率。在面板曝光值是代表FACS图，F是指±SE。 <img alt="图4" src="/files/ftp_upload/53214/53214fig4.jpg" /> 图4:的miR-451的转丰富的PKA-R融合基因，并表明，miRNA的融合红细胞是在原籍已被抓获链霉指示脱硫生物共轭的miRNA基因融合基因的富集。值是使用实时PCR测量并绘制为相对值相对于未转染的寄生虫。值是平均值±SE。 <img alt="图5" src="/files/ftp_upload/53214/53214fig5.jpg" /> 图5:核糖体寄生虫可通过蔗糖梯度隔离（A - B）Examp勒的痕迹，从恶性疟原虫中提取的多聚核糖体轮廓。一个核糖体轮廓恶性疟原虫提取，与40S，60S，80S和核糖体峰（A），跟踪（＃分别与指示相关核糖体的数量）显示。 （B）的18S和28S rRNA的整个核糖体部分RNA印迹。值是平均值±SE。

Watch this Scientific Journal Video about Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum at JoVE.com

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

methods-to-investigate-regulatory-role-small-rnas-ribosomal-occupancy

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

8.8K 次观看.  Duke University School of Medicine. Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts. 

视频: Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

科研

遗传学

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

生物化学

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

MS2亲和力纯化与革兰阳性细菌中的RNA测序相结合

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

小核糖核酸病毒取向的微小RNA基于调控

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

免疫与感染

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

生物学

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336&#916;micC by the &#955; Red-based recombination system and the complemented mutant 50336&#916;micC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336&#916;micC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336&#916;micC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336&#916;micC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336&#916;micC and 50336&#916;micC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An &#955;-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

非编码小RNA MicC有助于肠炎 沙门氏菌 外膜蛋白的毒力

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

增强Northern印迹检测小RNA种类的<em&gt;果蝇</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

方法调查小RNA和核糖体占用的调控作用

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的快速方法新颖的RNA结合蛋白隔离

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

MS2亲和力纯化与革兰阳性细菌中的RNA测序相结合

小核糖核酸病毒取向的微小RNA基于调控

单细胞基因转录的RNA荧光分析

非编码小RNA MicC有助于肠炎沙门氏菌外膜蛋白的毒力

增强Northern印迹检测小RNA种类的

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

方法调查小RNA和核糖体占用的调控作用

摘要

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此视频中的章节

的快速方法新颖的RNA结合蛋白隔离

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

MS2亲和力纯化与革兰阳性细菌中的RNA测序相结合

小核糖核酸病毒取向的微小RNA基于调控

单细胞基因转录的RNA荧光分析

非编码小RNA MicC有助于肠炎 沙门氏菌 外膜蛋白的毒力

增强Northern印迹检测小RNA种类的

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

非编码小RNA MicC有助于肠炎沙门氏菌外膜蛋白的毒力