Name: quantification of the abundance and charging levels of transfer rnas in escherichia coli
Uploaded: 2017-08-22T14:00:00.000+00:00
Duration: 10 min 34 s
Description: Watch this Scientific Journal Video about Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli at JoVE.com

作者感谢玛丽特拉 Warrer 出色的技术援助。这项工作得到丹麦议员支持独立研究中心 |自然科学 [1323年-00343B] 和丹麦国家研究基金会 [DNRF120]。

1.细菌培养制备。
<ol><li>成长所有文化在摇瓶培养/瓶体积比的顶多 1/6。在一个典型的实验设置，成长在 37.0 ° C 文化和摇晃在 160 rpm 在 morpholinepropanesulfonic 酸 （拖把） 最小中型 18 辅以首选的碳源，例如 0.2%的葡萄糖，为至少 10RNA 收获前的连续世代。</li>
	<li>RNA 收获的前一天稀释所需应变的拖把最小介质方式将所需的 OD 436 到达所需的时间第二天穿不了的文化。计算公式用于接种的穿不了区域性的体积: <img alt="Equation 1"src="/files/ftp_upload/56212/56212eq1.jpg"/> 注: 这里，OD 新 表示所需的 OD 436经过孵化，V 新 表示的稀释文化卷，OD 老 表示 OD 436 的穿不了的文化，从被稀释，G 表示的数代人的文化将会成长，从时间 (t 1) 稀释到的时间需要 (t 2) 的第二天，可以计算为: 时间差值 （min) t 1 和 t 2 除以生成时间 （分钟）。为确保稳态生长在采样时，应该是 G &#8805; 10;V 老 表示外种植文化应该转移到新鲜的培养基中的卷。这个方程是一个近似值，不考虑任何滞后期;经常观察后稀释外种植文化在新媒体中的一种现象。
	<ol><li>生长条件将取决于实验的目的但是限制细胞到细胞变异 tRNA 水平，提高重复性的结果，（推荐） 成长文化来稳定前采样状态 19.</li></ol></li></ol> 2。穗状花序在细胞制备 注: 表示编码 tRNA 全 异丙基 β-D-1-thiogalactopyranoside (IPTG) 控制下的 全 基因的 大肠杆菌 菌株 MAS1074，-诱导型启动子用作穗中应变。
<ol><li>摊薄 MAS1074 OD 436 的拖把 0.05 到穿不了文化最小培养基 100 微克/毫升氨苄西林和 0.2%的葡萄糖。成长在 37 ° C，震动在 160 rpm 文化。 
	注: 请参阅 讨论 用于替代参照应变。</li>
	<li>在 OD 436 ~ 0.1，添加 IPTG 至终浓度为 1 毫米，诱导表达的 tRNA 全。长出的文化对 OD 436 ~0.5 (~ 3 4 h 的增长) 和将培养瓶移到冰浴。保持在冰上的穗状花序在细胞培养，直到所有样本的 RNA 纯化都有都收获。</li>
</ol> 3。实验样品收获。
<ol><li>预暖 (37 ° C) 一个上限 100 毫升锥形瓶 （或类似） 包含 4 毫升 10%(w/v) 三氯乙酸 (TCA) 为每个样品放置在洗个温水澡。 
	注: 谨慎，TCA 后加热，分解生产包括氯化氢和氯仿的有毒和腐蚀性烟气。在水中的解决方案是一种强的酸;它与基地剧烈反应，是对许多金属有腐蚀性。使用这种液体时，应采取适当的预防措施。</li>
	<li>前夕细胞收获，测量和记录文化 OD 436。</li>
	<li>收获细胞，4 毫升的培养液转移到预热的烧瓶含有 TCA。</li>
	<li>混合样品通过用手旋转烧瓶，5 美国混合与 TCA 立即禁用所有酶的活性，从而保留转运的收费水平。 
	注意: 如果要收集多个样品，牢记收获的样品 TCA 冰上直到所有样本。</li>
</ol> 4。另外的穗状花序在细胞 注: 在步骤 2 中介绍的穗状花序在细胞的制备.
<ol><li>时所有样品 RNA 制备已收集，每个样品中添加 5%穗在单元格 （基于 OD）。 
	注: 穗在细胞培养所需的卷由公式计算: <img alt="Equation 2"src="/files/ftp_upload/56212/56212eq2.jpg"/> 凡 V 穗 表示卷的穗状花序在细胞培养需要 Vexp表示该卷收获实验细胞培养 （无 TCA)，OD exp 表示 OD 436 实验细胞培养成 TCA 收获时。OD 穗 表示 OD 436 的穗状花序在细胞培养。如果多个样品在不同耗氧物质收获，穗在细胞添加量应根据上面的公式，为每个样本调整。</li>
	<li>量化从穗在细胞贡献 RNA 物种感兴趣，准备只含有穗中细胞与 4 毫升 TCA 混合 4 毫升穗在细胞样品。准备 RNA 从这个示例中相同的方式与其他示例。</li>
	<li>（可选） 采取另一瓶含只给了实验样品，无穗在细胞，并将其量化实验样品给 tRNA 的贡献包括 全 水平; tRNA 全 在 中的内源性水平大肠杆菌 K-12 是可以忽略不计低。</li>
</ol> 5。RNA 制备 注: 此处所述的 RNA 制备协议基本上是由 Varshney 等人 描述 1;要避免在净化过程中的脱乙酰基的 aminoacylated 转运是重要的是保持样品在酸性 ph 值和在整个过程中纯化的 0 ° C。使用清洁无菌管/烧瓶来保存样品和解决方案，并准备所有解决方案与超纯水 （18.2 MΩ·cm 电阻率） 水.
<ol><li>每个样品到冰鲜离心管倒 8 毫升。离心样品 10 分钟在 9,500 x g 4 ° C.完全删除上清液，并在 0.3 毫升冷 0.3 M 醋酸钠，ph 值 4.5 和 10 毫米 EDTA。</li>
	<li>转移到冷 1.5 毫升离心管中每个样品和添加 0.3 毫升苯酚与相同的缓冲区平衡。 
	注: 谨慎，苯酚产生有毒气体和对皮肤高度腐蚀性。</li>
	<li>涡每个样品为 15 10 x s 与至少 1 min 之间每个旋涡。在涡流之间保持样品在 0 ° C.使用频繁的停顿，以确保样品保持冷。</li>
	<li>离心机样品 15 分钟在 20,000 x g 4 ° C.转让水阶段 （上部阶段） 到新的 1.5 mL 离心管。像以前一样添加 0.3 毫升冷苯酚和涡 4 x 15 s.</li>
	<li>离心机以离心 10 分钟 20,000 x g 4 ° C.转让水相向新、 冷 1.5 毫升的微量离心管含 96%乙醇 (~ 750 µ L) 量的 2.5 倍。沉淀 RNA 通过孵化管 1 h 在-20 ° C 或一夜之间在-80 ° C.</li>
	<li>离心机样品 30 分钟在 20,000 x g 4 ° C.弃上清，仔细而不会干扰 RNA 颗粒添加 1 毫升冷 70%乙醇。轻轻地反转一次洗用乙醇管内侧。 
	注: 颗粒很难看到在这一步。</li>
	<li>离心机以离心 10 分钟 20,000 x g 4 ° C.完全删除上清液和风干的颗粒，直到离开没有乙醇。通过大力旋涡中 20 微升冷 10 毫米钠醋酸 ph 值 4.5 和 1 毫米 EDTA 悬小球. 
	注意: 不过度干颗粒，它将变得困难，重悬。</li>
	<li>（可选） 评估测定 RNA 浓度测定吸光度在 260 nm。</li>
	<li>（可选） 通过琼脂糖凝胶电泳 20 评估质量的纯化 RNA。</li>
	<li>商店样品在-80 ° C. 
	注: 议定书 》 能够在这里暂停。</li>
</ol> 6。化学 deacylated 控制样品的制备 注: 区分的 aminoacylated 乐队从 deacylated 对应的北侧 tRNA 污点，化学 deacylated 分装备碱处理。大多数目的而言，这是足够到 deacylate 单一样本的每个北污点。
<ol><li>解冻的 RNA 样本在冰上。转移到新管中的 RNA 样品 4 µ L 分装。添加 46 µ L 三 HCl ph 值 9.0。孵育 2 h 在 37 ° C</li>
	<li>添加 15 微升 0.3 M 醋酸钠的 ph 值为 4.5，随后添加 125 µ L 的 96%的乙醇。沉淀 RNA，在-20 ° C 1 h.</li>
	<li>离心机以离心 30 分钟 20,000 x g 4 ° C.完全除去上清，悬在 4 µ L 冷 10 毫米钠醋酸 ph 值 4.5 和 1 毫米 EDTA。 
	注: 议定书 》 可以暂停在这里，如果样品被存放在-80 ° C.</li>
</ol> 7。凝胶电泳和北弄脏 <ol><li>混合 4 µ L 样品 (未经处理或化学 deacylated) 与 6 µ L 加载缓冲区 （0.1 M 醋酸钠 (pH 5.0)、 8m 尿素、 0.05%溴酚蓝和 0.05%二甲苯蓝）。 
	注: 请记住，包括样品只有穗中的单元格，化学 deacylated 样品 （和无穗在细胞，如果一个人准备样品）。</li>
	<li>装载到 0.4 m m 厚 6.5%聚丙烯酰胺凝胶 (19:1 acrylam-ide:bisacrylamide) 包含在 0.1 M 醋酸钠 (pH 5.0) 8m 尿素样品。60 厘米长凝胶用于适当分离的 tRNA 物种。</li>
	<li>在 4 ° c，10 V/cm 凝胶电泳法分离 RNA 直到溴酚蓝到达的凝胶 (~ 20 h) 底部。</li>
	<li>地区从二甲苯蓝染料和 20 厘米下来对溴酚蓝染料用于印迹 （两种染料之间的距离应该是 25-30 厘米）。仔细单独两个玻璃板材，以便这种凝胶仍然是其中之一。一块薄薄的滤纸现降至所需的吸墨纸区域的大小，放在将用于吸水凝胶的一部分。凝胶中未涵盖的过滤纸的部分是切断和丢弃。 使用过滤纸小心地提起凝胶这块远离玻璃板。 
	注: 大凝胶是很脆弱所以小心轻放。</li>
	<li>正电荷的尼龙膜 （例如中 N + 膜） 放在凝胶上，是在使用 40 毫米醋酸 (pH 8.1)，2 毫米 EDTA 作为传输缓冲区的 90 分钟 20 V 裁。</li>
	<li>交联 RNA 对使用 UV 光膜 (0.12 J/厘米 2). 
	注: 膜现在可以存储在室温和议定书 》 能够在这里暂停。</li>
</ol> 8。探针的设计与验证 注: 小心北污点探针的设计与验证至关重要获得可靠的结果，避免与其他 RNA 种类不需要的交叉杂交。
<ol><li>使用短合成寡核苷酸探针。设计中一种方式，它是相辅相成的感兴趣的 tRNA 序列最独特的地方 — — 这往往是反密码子环的探针序列。 
	注: 从各种生物的预测 tRNA 序列的列表可以找到在基因组的 tRNA 数据库 (GtRNAdb) 21.</li>
	<li>要获得预测的序列的长度调整熔化温度 55-65 ° c。</li>
	<li>爆炸 （基本局部比对搜索工具） 所选序列对基因组序列的菌株在实验中，用来验证序列是唯一选定的 tRNA， 22 中所述。 
	注: 表 1 列出了建议的探针序列，为几个不同的 Trna 的 大肠杆菌 K12。如果探针是特定为感兴趣的 tRNA，只有两个波段上均可见北污点 (带电和不带电的 tRNA) 和只有一个乐队是在车道与 deacylated 样本 (不带电 tRNA) 中可见。如果两个以上乐队目前或如果需要进一步验证的特异性探针，见讨论。</li>
</ol> 9。北部的印迹杂交 注: 在以下步骤中，膜按顺序杂交探针补充所需的特定转运，包括探头特定参考 tRNA 全。
<ol><li>膜置于杂交管中，加入 6 毫升杂交溶液 (0.9 M 氯化钠，0.05 M NaH 2 PO 4 (pH 7.7)，5 毫米 EDTA，0.5%(w/v) SDS，100 毫克/毫升的剪切和变性鲱鱼精子 DNA 和 5 x Denhardt &#39; s 解决方案 (100 xDenhardt &#39; s 解决方案 = 2%牛血清白蛋白、 2%吡咯烷酮 40 和 2%蔗糖))。</li>
	<li>预杂交膜中的缓冲区的旋转在 42 ° C.添加 30 皮摩尔放射性寡聚 DNA 探针 5 1 h &#39; 端-带有 32 P （编写由山姆布鲁克 等人 所述 23) 注: 警告，高能 β 排放量 32 P 可以呈现大量的皮肤和眼睛剂量危害。当使用 32 页放射性废物应按处理适当的体制安全协议时应采取适当的预防措施。</li>
	<li>孵育探针与膜隔夜旋转在 42 ° C.删除探针一次性 10 毫升吸管。 
	注意: 如果储存在冰箱的探针可重复使用。</li>
	<li>在杂交管中，洗膜不久在 50 毫升的 0.3 M 氯化钠，柠檬酸钠 30 毫米，0.1%(w/v) SDS 在室温。 
	注意: 这第一次洗包含多绑定的探针和必须作为高度放射性处理。</li>
	<li>重复一次，一步 9.4，然后将膜移动到平塑料集装箱或托盘包括盖子。覆盖膜与洗涤液 （0.3 M 氯化钠，柠檬酸钠 30 毫米，0.1%(w/v) SDS） 和孵育 30min 室温。</li>
	<li>改变洗涤液和持续冲洗，直到令人满意的信号的信噪比得到了 （通常 3-4 更改的洗涤液）。使用盖革-米勒管来检测噪声对信号比通过膜的从一个空的区域辐射测量的更改和比作的地区地方探头具有杂交特指 tRNA。</li>
	为了能够带从膜探针接触之后，<li>保证，（重要） 膜也不干溶出过程之前。将膜放在薄薄的塑料袋或包裹它紧紧地使用塑料包装和密封就是气密的磁带或焊接。在 phosphorimaging 屏幕上放置密封的膜。 
	注: phosphorimaging 屏幕上所需的曝光时间测定探针的具体活动的 RNA 量探讨佛r 和探针，和可以杂交效率相差很大;从几分钟到几天。与经验，在盖革-米勒管膜上检测到的辐射强度给需要的曝光时间很好的说明。为可靠地进行量化，很重要的屏幕不过度曝光。</li>
	<li>Phosphorimaging 屏幕使用激光扫描仪扫描和保存在文件 &#34;.gel &#34; 格式。 
	注: 高信号的信噪比是必要的可靠的量化。探测的 tRNA 应导致在两个频段;一个包含 aminoacylated tRNA （慢迁移） 和一个包含 deacylated tRNA （快迁移）。请参见 图 2。</li>
</ol> 10。剥膜 注: 膜可探测的另一个 tRNA 之前，先前的探头必须先删除由剥膜。
<ol><li>热足够剥缓冲区 （15 毫米氯化钠，柠檬酸钠 1.5 毫米，0.1%(w/v) SDS） 覆盖膜和倒膜。孵育 15 分钟在室温。</li>
	<li>可以用盖革-米勒管检测重复步骤 10.1 直到无辐射; 膜现在可以重新杂交后第九条中所述的步骤。</li>
</ol> 11。量化 tRNA 和收费水平 <ol><li>负载.gel 文件到成像软件 （见 表的材料）。画一条垂直线沿着每个样品车道方式它横跨了大部份的车道的宽度，但不包括乐队的边缘其中信号可以是不平衡的如 图 3A 所示。</li>
	<li>可视化计数从每个车道通过单击 &#34; 分析 &#34;-&#62; &#34; 创建图 &#34;。手动定义表示带电和不带电的 tRNA 从每个车道，两座山峰，如 图 3B 所示。</li>
	<li>从每个峰获得计数，通过单击 &#34; 分析 &#34;-&#62; &#34; 地区报告 &#34;，并记录下每个两个峰的面积。</li>
	<li>当 tRNA 全 和至少一个 tRNA 的兴趣，探讨了污点正常化的 tRNA tRNA 全 感兴趣的水平。
	<ol><li>计算的 tRNA 源自每个车道的穗状花序在细胞比例和从该行车线行驶，利用方程的总信号中减去: <img alt="Equation 3"src="/files/ftp_upload/56212/56212eq3.jpg"/> 注: 在这里，样品 tRNA = 从单线的探测所需的 tRNA; 膜得到的信号样品 全 = 从同一行车线探测 tRNA 全; 对同一种膜得到的信号Ref tRNA = 从车道只有穗在细胞 rna 的同一种膜有人探测其是否需 tRNA; 得到的信号Ref 全 = 从车道只有穗在细胞 rna 的同一种膜有人探测其是否 tRNA 全 得到的信号。</li>
		<li>实现正常化，来自每个车道的更正的 tRNA 值除以来自同一行车线的 tRNA 全 信号。 
		注: 从采样细胞 tRNA 全 信号的贡献是可以忽略不计 (见 图 2，车道标记 &#34;-全 &#34;)。</li>
	</ol></li> <li>计算收费水平每条车道除以信号的峰值代表 tRNA 与信号总和从这两个峰的 tRNA （带电 + 不带电荷）。</li></ol>

<ol>
	<li>Varshney, U., Lee, C. -. P., RajBhandary, U. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+aminoacylation+levels+of+tRNAs+in+vivo.+Application+to+studying+recognition+of+Escherichia+coli+initiator+tRNA+mutants+by+glutaminyl-tRNA+synthetase.">Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase.</a> J Biol Chem. 266 (36), 24712-24718 (1991).</li><li>Sorensen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charging+levels+of+four+tRNA+species+in+Escherichia+coli+Rel(+)+and+Rel(-)+strains+during+amino+acid+starvation:+a+simple+model+for+the+effect+of+ppGpp+on+translational+accuracy.">Charging levels of four tRNA species in Escherichia coli Rel(+) and Rel(-) strains during amino acid starvation: a simple model for the effect of ppGpp on translational accuracy.</a> J Mol Biol. 307 (3), 785-798 (2001).</li><li>Kr&#252;ger, M. K., S&#248;rensen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminoacylation+of+hypomodified+tRNAGlu+in+vivo.">Aminoacylation of hypomodified tRNAGlu in vivo.</a> J. Mol. Biol. 284 (3), 609-620 (1998).</li><li>Svenningsen, S. L., Kongstad, M., Stenum, T. S., Mu&#241;oz-G&#243;mez, A. J., S&#248;rensen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+RNA+is+highly+unstable+during+early+amino+acid+starvation+in+Escherichia+coli.">Transfer RNA is highly unstable during early amino acid starvation in Escherichia coli.</a> Nucleic Acids Res. 45 (2), 793-804 (2017).</li><li>Enriquez, J. A., Attardi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+aminoacylation-induced+conformational+changes+in+human+mitochondrial+tRNAs.">Evidence for aminoacylation-induced conformational changes in human mitochondrial tRNAs.</a> Proc Natl Acad Sci U S A. 93 (16), 8300-8305 (1996).</li><li>Parker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Errors+and+alternatives+in+reading+the+universal+genetic+code.">Errors and alternatives in reading the universal genetic code.</a> Microbiol Rev. 53 (3), 273-298 (1989).</li><li>Traxler, M. F., 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+global,+ppGpp-mediated+stringent+response+to+amino+acid+starvation+in+Escherichia+coli.">The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli.</a> Mol Microbiol. 68 (5), 1128-1148 (2008).</li><li>Zhong, 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=Transfer+RNAs+Mediate+the+Rapid+Adaptation+of+Escherichia+coli+to+Oxidative+Stress.">Transfer RNAs Mediate the Rapid Adaptation of Escherichia coli to Oxidative Stress.</a> PLoS Genet. 11 (6), e1005302 (2015).</li><li>Kane, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+rare+codon+clusters+on+high-level+expression+of+heterologous+proteins+in+Escherichia+coli.">Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli.</a> Curr Opin Biotechnol. 6 (5), 494-500 (1995).</li><li>Baca, A. M., Hol, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+codon+bias:+a+method+for+high-level+overexpression+of+Plasmodium+and+other+AT-rich+parasite+genes+in+Escherichia+coli.">Overcoming codon bias: a method for high-level overexpression of Plasmodium and other AT-rich parasite genes in Escherichia coli.</a> Int J Parasitol. 30 (2), 113-118 (2000).</li><li>Li, S., Pelka, H., Schulman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anticodon+and+discriminator+base+are+important+for+aminoacylation+of+Escherichia+coli+tRNA+(Asn).">The anticodon and discriminator base are important for aminoacylation of Escherichia coli tRNA (Asn).</a> J Biol Chem. 268 (24), 18335-18339 (1993).</li><li>Rizzino, A. A., Freundlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+in+vivo+aminoacylation+by+periodate+oxidation:+tRNA+alterations+and+iodate+inhibition.">Estimation of in vivo aminoacylation by periodate oxidation: tRNA alterations and iodate inhibition.</a> Anal Biochem. 66 (2), 446-449 (1975).</li><li>Dittmar, K. A., Sorensen, M. A., Elf, J., Ehrenberg, M., Pan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+charging+of+tRNA+isoacceptors+induced+by+amino-acid+starvation.">Selective charging of tRNA isoacceptors induced by amino-acid starvation.</a> EMBO Rep. 6 (2), 151-157 (2005).</li><li>Zhou, K., 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=Novel+reference+genes+for+quantifying+transcriptional+responses+of+Escherichia+coli+to+protein+overexpression+by+quantitative+PCR.">Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR.</a> BMC Mol Biol. 12 (1), 18 (2011).</li><li>Jacobson, A., Gillespie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+events+occurring+during+recovery+from+prolonged+glucose+starvation+in+Escherichia+coli.">Metabolic events occurring during recovery from prolonged glucose starvation in Escherichia coli.</a> J Bacteriol. 95 (3), 1030-1039 (1968).</li><li>McCarthy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+magnesium+starvation+on+the+ribosome+content+of+Escherichia+coli.">The effects of magnesium starvation on the ribosome content of Escherichia coli.</a> Biochim Biophys Acta (BBA)-Specialized Section on Nucleic Acids and Related Subjects. 55 (6), 880-889 (1962).</li><li>Zundel, M. A., Basturea, G. N., Deutscher, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+ribosome+degradation+during+starvation+in+Escherichia+coli.">Initiation of ribosome degradation during starvation in Escherichia coli.</a> Rna. 15 (5), 977-983 (2009).</li><li>Piir, K., Paier, A., Liiv, A., Tenson, T., Maivali, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+degradation+in+growing+bacteria.">Ribosome degradation in growing bacteria.</a> EMBO Rep. 12 (5), 458-462 (2011).</li><li>Schaechter, M., Maaloe, O., Kjeldgaard, N. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependency+on+medium+and+temperature+of+cell+size+and+chemical+composition+during+balanced+grown+of+Salmonella+typhimurium.">Dependency on medium and temperature of cell size and chemical composition during balanced grown of Salmonella typhimurium.</a> J Gen Microbiol. 19 (3), 592-606 (1958).</li><li>Neidhardt, F. C., Bloch, P. L., Smith, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+medium+for+enterobacteria.">Culture medium for enterobacteria.</a> J Bacteriol. 119 (3), 736-747 (1974).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (1989).</li><li>Sorensen, M. A., Jensen, K. F., Pedersen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+concentrations+of+ppGpp+decrease+the+RNA+chain+growth+rate.+Implications+for+protein+synthesis+and+translational+fidelity+during+amino+acid+starvation+in+Escherichia+coli.">High concentrations of ppGpp decrease the RNA chain growth rate. Implications for protein synthesis and translational fidelity during amino acid starvation in Escherichia coli.</a> J Mol Biol. 236 (2), 441-454 (1994).</li><li>Tian, 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=Rapid+Curtailing+of+the+Stringent+Response+by+Toxin-Antitoxin+Module-Encoded+mRNases.">Rapid Curtailing of the Stringent Response by Toxin-Antitoxin Module-Encoded mRNases.</a> J Bacteriol. 198 (14), 1918-1926 (2016).</li></ol>

作者没有透露。

此协议说明如何同时测量特定大肠杆菌的收费水平转运和比较不同样品中转运的相对水平。议定书 》 的关键点是 1） 处理方式保留细胞 tRNA 的收费水平，2） 正常化 tRNA 量方式，可以可靠地比较不同样品中的相对 tRNA 水平，和 3） 确保 sp 样品ecificity 的选定的探头，用于转运的兴趣。这些点在下面分别讨论。
tRNA 净化
转运是纯化大肠杆菌在温?...

在下文中，我们目前的量化具体转运方法和测量他们的收费水平由北弄脏。该方法基于第一次由 Varshney等人的技术1.由成三氯乙酸 (TCA) 收获细胞和保持在 0 ° C，整个 RNA 纯化的样品，tRNA 与氨基酸之间的酯键是保守的2，，34。Aminoacylated 转运可以区分从 nonacylated 同行通过凝胶电泳和北弄脏，适当降低流动性 aminoacylated tRNA 凝胶，引起共价键绑定的氨基酸5中。此外，我们目前的协议规范 tRNA 数量由加法的穗状花序在细胞特异性表达很少使用的 tRNA全 4。
转运是一些最丰富的分子在细菌细胞和翻译机械绝对重要部分。转运绑定氨基酸并将它们传输到翻译的核糖体。氨基酸对转运 （氨或充电） 的绑定被通过氨酰 tRNA 合成酶。不同带电的 Trna 的相对丰度是合成的重要为确保保真度的蛋白质，因为同源任职被控为给定的信使 RNA (mRNA) 密码子 tRNA 增加附近同源 trna 上将的可能性错误地将其氨基酸交付越来越多肽链在核糖体6。TRNA 的重要性反映在大肠杆菌细胞广泛响应 tRNA; 收费水平严重下降严格的响应。严格的反应中转运、 核糖体 Rna 和大多数基因的合成降低有利于与氨基酸生物合成和应力生存相关的特定基因转录和细胞的增长速度大大降低7.此外，最近由我们和其他人的工作表明，大肠杆菌积极降低其 tRNA 翻译4，8的极限应力是响应表明 tRNA 水平调整可能绝大多数为应付这样的压力是重要的。因此，可靠的测量 tRNA 丰度和收费水平将充分了解细菌应激反应的重要工具。
大肠杆菌常用表达重组蛋白和由于密码子用法的物种之间存在差异，次优的表达是一个经常遇到的问题9。这可以由表达式的翻译重组 mRNA10所需的额外转运绕过。收费水平在这种菌株的 tRNA 的测量可以引导疑难解答工作及帮助优化蛋白表达。
此方法还使检测的"mischarging";与一个非同源的氨基酸的 tRNA 分子 aminoacylated。由不同的氨基酸酰化可能会导致 tRNA 要迁移与略有不同的速度通过聚丙烯酰胺凝胶1，11。在某些情况下，该方法也可用于区分不同的修饰模式否则为相同转运3日。
另一种建立生化过程的 tRNA 收费水平调查是高碘酸氧化。这种方法依赖于观察 aminoacylated tRNA 保护从高碘酸钾氧化和不带电的 tRNA 不是。后高碘酸氧化处理充电 tRNA 用于估计收获 RNA 的收费水平。然而，充电几个转运已被证明受治疗从而提供一些不准确12。这里介绍的方法从纯化 RNA 直接充电，因此从化学或酶促反应中排除任何偏见的措施。此方法的一个局限是只有一个 tRNA 种类检测一次，所以虽然同样北污点可以剥离和 reprobed 为多个转运，既费时又有点费力在许多转运上收集数据。
规范样品到彼此的可靠方法是十分重要的研究，目标是跨不同的样品比较分子的相对水平。在这里我们介绍正常化过程为大肠杆菌细胞特异性表达稀有 tRNA全小分装为穗状花序在添加到 RNA 纯化前的所有实验样品的 RNA。此方法很有用不仅审查转运时但相对定量的任何种类的 RNA 时的实验装置是这样没有内源性 RNA 可以信任要出席所有样品中相同的细胞浓度。例如，"管家"RNA 样核糖体 RNA 水平经常用作内源性的参考比较另一个 RNA 之间不同的相对数量样品13。但这是没有多大用处如果参考 RNA 细胞浓度变化之间的样品，可以出现这种情况为核糖体 RNA，如果采样发生在应力响应15，，1617期间或进入固定相17。增加了穗在细胞 RNA 纯化前对实验样品提供规范化的采样设置独立固体和准确的方式。标准化的另一种是另外的一个或多个穗状花序在 Rna 的实验样品 RNA 纯化后。然而，这种方法不占任何在 RNA 恢复样品之间的差异。
利用大肠杆菌细胞 RNA 参考为穗状花序在单元格 （见图 1） 在实验中对大肠杆菌有它结果的缺点，overexpress 另外少量的外源性大肠杆菌的总 RNA样品。我们通过分析示例包含只有穗在细胞中平行实验的样本 （见图 2，车道标记为"全"蛋白免疫印迹北） 更正此添加。议定书 》，提出了一种针对大肠杆菌K-12 但可能是适用于大多数的细菌种类。

<table><tbody><tr><td>Urea</td><td>Merck</td><td>57-13-6</td><td>Purity &gt;99%</td></tr><tr><td>Polyacrylamide</td><td>Serva</td><td>79-06-7</td><td></td></tr><tr><td>Bis (N,N&#039;-Methylene-bis-acrylamide)</td><td>BIO-RAD</td><td>161-0201</td><td></td></tr><tr><td>Trichloroacetic acid (TCA)</td><td>Sigma</td><td>76-03-9</td><td></td></tr><tr><td>Phenol</td><td>Merck</td><td>108-95-2</td><td></td></tr><tr><td>IPTG</td><td></td><td></td><td></td></tr><tr><td>Glucose</td><td></td><td></td><td></td></tr><tr><td>Sodium Acetate</td><td></td><td></td><td></td></tr><tr><td>EDTA</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>Tris</td><td></td><td></td><td></td></tr><tr><td>Hydrochloric acid</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td></td><td></td><td></td></tr><tr><td>NaH2PO4</td><td></td><td></td><td></td></tr><tr><td>Sodium Citrate</td><td></td><td></td><td></td></tr><tr><td>SDS</td><td></td><td></td><td></td></tr><tr><td>Herring Sperm DNA</td><td>Sigma</td><td>100403-24-5</td><td></td></tr><tr><td>BSA</td><td></td><td></td><td></td></tr><tr><td>Polivinylpyrrolidone</td><td></td><td></td><td></td></tr><tr><td>Ficoll</td><td></td><td></td><td></td></tr><tr><td>P32 &amp;gamma;ATP</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>DNA oligos (probes)</td><td>TAG Copenhagen</td><td></td><td></td></tr><tr><td>Hybond N&lt;sup&gt;+&lt;/sup&gt; membrane</td><td>GE Healthcare</td><td>RPN203B</td><td></td></tr><tr><td>Crosslinker</td><td></td><td></td><td></td></tr><tr><td>Electroblotter</td><td>BIO-RAD</td><td></td><td></td></tr><tr><td>Typhoon FLA 7000 Scanner</td><td>GE Healthcare</td><td>28955809</td><td></td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td></td></tr><tr><td>Hydridization oven</td><td></td><td></td><td></td></tr><tr><td>Geiger-M&amp;uuml;ller tube</td><td></td><td></td><td></td></tr><tr><td>Phosphor imager screen</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Hybridization tube</td><td></td><td></td><td></td></tr><tr><td>Culture Flasks</td><td></td><td></td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>20 ml centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ImageQuant</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

quantification of the abundance and charging levels of transfer rnas in escherichia coli

转运 RNA (tRNA) 是任何一种生物的平移机构的重要组成部分。转运绑定并将氨基酸转移到翻译的核糖体。不同的 Trna 的相对水平和 aminoacylated 比 tRNA 到总 tRNA，称为收费水平，是在确定的精度和速度的翻译的重要因素。因此，丰度和收费水平的转运是重要的变量来衡量学习蛋白质合成，例如在不同应力条件下的时候。在这里，我们描述为收获 tRNA 和直接测量的相对丰度和特定 tRNA 物种在大肠杆菌中的绝对收费水平的方法。TRNA tRNA 和其氨基酸之间不稳定的纽带保留的一种收获。RNA 然后遭受凝胶电泳和北弄脏，导致分离带电和不带电的转运。具体转运不同样品中的水平可以相比的穗状花序在细胞正常化添加。在 RNA 纯化之前, 我们添加 5%的过量罕见的 tRNA全对每个样本的大肠杆菌细胞。样品中感兴趣的 tRNA 物种的数量然后归的 tRNA全在相同的样本量。穗状花序在细胞 RNA 纯化前的加法有另外它也占任何细胞裂解效率差异样品的纯化穗在 Rna 的优势。

使用过程形容这里，丰度和收费水平的三个转运测定大肠杆菌K-12 之前和期间氨基酸饥饿。
精氨酸营养缺陷型菌株 NF9154 (thr 列伊他啊 thi mtl supE44)了种植至少十代拖把辅以 0.4%的甘油、 50 微克/毫升苏氨酸、 亮氨酸、 精氨酸、 和 5 微克/毫升组氨酸在摇晃在 37 ° C 的最小介质中160 转/分。精氨酸饥...

Watch this Scientific Journal Video about Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli at JoVE.com

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

丰度和收费水平的转移 Rna 在大肠杆菌中的量化

在这里，我们提出一种直接测量转移 RNA 从纯化的大肠杆菌RNA 以及方式比较的转运 RNA 相对水平或任何其他短的 RNA，跨越不同的样本基础，增设的穗状花序在收费水平方法单元格引用基因的表达。

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

quantification-abundance-charging-levels-transfer-rnas-escherichia

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

9.2K 次观看.  University of Copenhagen. 在这里，我们提出一种直接测量转移 RNA 从纯化的大肠杆菌RNA 以及方式比较的转运 RNA 相对水平或任何其他短的 RNA，跨越不同的样本基础，增设的穗状花序在收费水平方法单元格引用基因的表达。

视频: Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

科研

遗传学

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

定量细菌中蛋白质-RNA结合的测定

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

使用 AQRNA-seq 分析 tRNA 动力学

AQRNA-seq for Quantifying Small RNAs

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

作者聚焦:AQRNA-seq 在绘制小 RNA 和解开蛋白质翻译机制中的作用

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

用于定量小 RNA 的 AQRNA-seq

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

细菌中的RIBO-seq:NGS测序样本收集和库准备协议

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

生物化学

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

无需梯度测定器或分馏系统的多聚体分析

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

侵染微生物利用数字条码平台的基因表达谱

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

免疫与感染

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

（充电）酰化tRNA的使用微阵列技术水平的全基因组分析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

丰度和收费水平的转移 Rna 在大肠杆菌中的量化

摘要

探索更多视频

此视频中的章节

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

定量细菌中蛋白质-RNA结合的测定

用于定量小 RNA 的 AQRNA-seq

用于定量小 RNA 的 AQRNA-seq

用于定量小 RNA 的 AQRNA-seq

细菌中的RIBO-seq:NGS测序样本收集和库准备协议

无需梯度测定器或分馏系统的多聚体分析

侵染微生物利用数字条码平台的基因表达谱

（充电）酰化tRNA的使用微阵列技术水平的全基因组分析