Name: in vitro biochemical assays using biotin labels to study protein-nucleic acid interactions
Uploaded: 2019-07-17T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions at JoVE.com

我们感谢P.Jeremy Wang(宾夕法尼亚大学)所做的有益的编辑和讨论。我们还感谢西格丽德·埃卡德特的语言编辑。K. Z. 获得中国国家重点研发项目(2016YFA0500902,2018YFC1003500)和国家自然科学基金(31771653)的支持。L. Y. 得到了国家自然科学基金(81471502,31871503)和江苏省创新创业项目的支持。J.N.得到了浙江省医学科技项目(2019KY499)的支持。M.L.得到了国家自然科学基金(31771588)和"千名青年人才计划"的资助。

1. 蛋白质制备
<ol><li>MEIOB 和 MOV10 表达式构造<ol><li>生成 cDNA 表达式构造编码小鼠 MEIOB-A、C 和 E (图 1A) 和 MOV10。<ol>
<li>设置每个片段的聚合酶链反应 (PCR) 反应。在最终组合 1 μL 小鼠 cDNA(来自 C57BL/6 小鼠睾注)、1 μL dNTP、2 μL 10 μM 正向底漆、2 μL 10 μM 反向底漆、1 μL DNA 聚合酶、25 μL 2x PCR 缓冲液和 18 μL 双蒸馏 H2O (ddH2O)体积为 50 μL。 注:...

<ol>
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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=Hierarchical+roles+of+mitochondrial+Papi+and+Zucchini+in+Bombyx+germline+piRNA+biogenesis.">Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis.</a> Nature. 555 (7695), 260-264 (2018).</li><li>Anders, C., Jinek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+enzymology+of+Cas9.">In vitro enzymology of Cas9.</a> Methods in Enzymology. 546, 1-20 (2014).</li><li>Zhao, H., Zheng, J., Li, Q. 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研究蛋白质-核酸相互作用对于我们理解不同生物过程背后的分子机制至关重要。例如,MEIOB是哺乳动物25、26、27中梅氏症和生育力所必需的睾度特异性蛋白质。MEIOB包含一个OB域,它与单链DNA结合,并表现出3'至5'外源酶活性26,这与其在美质重组过程中的生理相关性直接相关。作为另一个例子,MOV10是一种具有无处不?...

蛋白质-核酸相互作用涉及许多重要的细胞过程,如DNA修复、复制、转录、RNA处理和翻译。蛋白质与染色质内特定DNA序列的相互作用是严格控制转录级别1的基因表达所必需的。对大量编码和非编码RNA的精确转录后调节需要任何蛋白质和RNA2之间广泛而复杂的相互作用。这些基因表达调控机制层包括一系列动态的分子间事件,通过转录/表观遗传因子或RNA结合蛋白与其核酸靶点的相互作用以及核酸靶点进行协调。蛋白质-蛋白质相互作用。为了解剖体内的蛋白质是否与核酸直接或间接相关,以及这种关联是如何发生和达到高潮的,进行体外生化测定,以检查相关蛋白质的结合亲和力或酶活性。设计DNA和/或RNA的基质。
已经开发了许多技术来检测和表征核酸-蛋白质复合物,包括电泳移动移位测定(EMSA),也称为凝胶缓动测定或带移检测3、4、5.EMSA 是一种多功能、灵敏的生化方法,广泛用于研究蛋白质与核酸的直接结合。EMSA依靠凝胶电泳带的变化,通常使用化学发光来检测生物素标签6,7,荧光素标签8,9的荧光,或通过放射性32P标签的自动辐射标签10,11。生化研究的其他用途是鉴定和表征蛋白质的核酸处理活性,如核酸反应,以评估核酸基质的裂解产物12,13,14和DNA/RNA结构展开测定,以评估赫利箱活性15,16,17。
在这种酶活性测定中,放射性同位素标记或荧光素标记的核酸由于其高灵敏度,经常被用作基质。对涉及32个P标记放射性追踪器的酶反应的放射图分析发现是敏感和可重复的18。然而,在世界上越来越多的实验室中,放射性同位素的使用受到限制甚至被禁止,因为与潜在接触相关的健康风险。除了生物安全问题外,其他缺点是使用放射性同位素工作所需的设备、所需的放射性许可证、32 P 的短半寿命(约 14天),以及探测器质量因无线电辐射。因此,开发了替代非同位素方法(即用荧光光对探针标记探头,通过荧光成像19进行检测)。但是,使用荧光标记的探头时,需要高分辨率成像系统。生物素是一种常用的标签,很容易与蛋白质和核酸等生物大分子结合。生物链球菌系统高效运行,提高了检测灵敏度,同时不增加非特异性背景20,21。除EMSA外,生物锡还广泛用于蛋白质纯化和RNA下拉,包括22、23、24。
该协议成功地使用生物素标记的核酸作为包括EMSA的体外生化检测的基质,此外还有未普遍使用的酶反应。MEIOB OB域被构造,两个突变体(截流和点突变)表示为GST融合蛋白25,26,27,以及小鼠MOV10重组FLAG融合蛋白16。本报告强调了这种用于蛋白质纯化和生物锡标记检测的合并方案的有效性,用于其他实验目的。

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			<td height="21" style="height:21px;width:351px;">Equipment</td>
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			<td height="21" style="height:21px;width:351px;">Centrifuge</td>
			<td style="width:163px;">Eppendorf, Germany</td>
			<td style="width:104px;">5242R</td>
			<td style="width:176px;"></td>
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			<td height="21" style="height:21px;">Chemiluminescent Imaging System</td>
			<td>Tanon, China</td>
			<td>5200</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital sonifer</td>
			<td>Branson, USA</td>
			<td>BBV12081048A</td>
			<td style="width:176px;">450 Watts; 50/60 HZ</td>
		</tr>
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			<td height="21" style="height:21px;">Semi-dry electrophoretic blotter</td>
			<td style="width:163px;">Hoefer, USA</td>
			<td style="width:104px;">TE77XP</td>
			<td></td>
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			<td height="21" style="height:21px;width:351px;">Tube Revolver&#160;</td>
			<td style="width:163px;">Crystal, USA</td>
			<td style="width:104px;">3406051</td>
			<td></td>
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			<td height="21" style="height:21px;width:351px;">UV-light cross-linker</td>
			<td>UVP, USA</td>
			<td style="width:104px;">CL-1000</td>
			<td></td>
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			<td height="21" style="height:21px;width:351px;">Materials</td>
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			<td height="21" style="height:21px;">Amicon Ultra-4 Centrifugal Filter&#160;</td>
			<td>Milipore, USA</td>
			<td>UFC801096</td>
			<td>4 ml/10 K</td>
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			<td height="21" style="height:21px;width:351px;">Nylon membrane</td>
			<td>Thermo Scientific, USA</td>
			<td style="width:104px;">TG263940A</td>
			<td></td>
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			<td height="21" style="height:21px;width:351px;">TC-treated Culture Dish</td>
			<td>Corning, USA</td>
			<td style="width:104px;">430167</td>
			<td style="width:176px;">100 mm&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">TC-treated Culture Dish</td>
			<td>Corning, USA</td>
			<td style="width:104px;">430597</td>
			<td style="width:176px;">150 mm&#160;</td>
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			<td height="21" style="height:21px;width:351px;">Microtubes tubes</td>
			<td style="width:163px;">AXYGEN, USA</td>
			<td style="width:104px;">MCT-150-C</td>
			<td style="width:176px;">1.5 mL&#160;</td>
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			<td height="21" style="height:21px;width:351px;">Tubes</td>
			<td style="width:163px;">Corning, USA</td>
			<td style="width:104px;">430791</td>
			<td style="width:176px;">15 mL</td>
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			<td height="21" style="height:21px;width:351px;">Reagents&#160;</td>
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			<td height="21" style="height:21px;">Ampicillin</td>
			<td>SunShine Bio, China</td>
			<td>8h288h28</td>
			<td></td>
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			<td height="21" style="height:21px;width:351px;">Anti-FLAG M2 magnetic beads</td>
			<td style="width:163px;">Sigma, USA</td>
			<td style="width:104px;">M8823</td>
			<td></td>
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			<td height="21" style="height:22px;">ATP</td>
			<td>Thermo Scientific, USA</td>
			<td>591136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BCIP/NBT Alkaline Phosphatase Color Development Kit</td>
			<td>Beyotime, China</td>
			<td>C3206</td>
			<td></td>
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			<td height="24" style="height:24px;">CelLyticTM M Cell Lysis Reagent&#160;</td>
			<td>Sigma, USA</td>
			<td>107M4071V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ClonExpress II one step cloning kit&#160;</td>
			<td>Vazyme, China</td>
			<td>C112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemiluminescent Nucleic Acid Detection Kit</td>
			<td>Thermo Scientific, USA</td>
			<td>T1269950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP</td>
			<td>Sigma-Aldrich, USA</td>
			<td>DNTP100-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DMEM</td>
			<td style="width:163px;">Gibco, USA</td>
			<td style="width:104px;">10569044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DPBS buffer</td>
			<td style="width:163px;">Gibco, USA</td>
			<td style="width:104px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EDTA</td>
			<td>Invitrogen, USA</td>
			<td style="width:104px;">AM9260G</td>
			<td>0.5 M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EDTA free protease inhibitor cocktail</td>
			<td style="width:163px;">Roche, USA</td>
			<td style="width:104px;">04693132001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EndoFree Maxi Plasmid Kit&#160;</td>
			<td>Vazyme, China</td>
			<td>&#160; 
			DC202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPure Gel DNA Extraction Mini Kit</td>
			<td>Vazyme, China</td>
			<td>DC301-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">FBS</td>
			<td style="width:163px;">Gibco, USA</td>
			<td style="width:104px;">10437028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">FLAG peptide</td>
			<td style="width:163px;">Sigma, USA</td>
			<td style="width:104px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Glycerol</td>
			<td style="width:163px;">Sigma, USA</td>
			<td style="width:104px;">SHBK3676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GST Bulk Kit</td>
			<td style="width:163px;">GE Healthcare, USA</td>
			<td style="width:104px;">27-4570-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffer</td>
			<td>Sigma, USA</td>
			<td>SLBZ2837</td>
			<td>1 M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG</td>
			<td>Thermo Scientific, USA</td>
			<td>34060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">KoAc</td>
			<td style="width:163px;">Sangon Biotech, China</td>
			<td style="width:104px;">127-08-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamin 3000 Transfection Reagent</td>
			<td>Thermo Scientific, USA</td>
			<td style="width:104px;">L3000001</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">MgCl2</td>
			<td>Invitrogen, USA</td>
			<td style="width:104px;">AM9530G</td>
			<td>1 M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Invitrogen, USA</td>
			<td>AM9759 
			&#160;</td>
			<td>5 M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NP-40</td>
			<td style="width:163px;">Amresco, USA</td>
			<td style="width:104px;">M158-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM medium</td>
			<td>Gibco, USA</td>
			<td style="width:104px;">31985062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Gibco, USA</td>
			<td>10010023</td>
			<td>PH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">RNase Inhibitor</td>
			<td style="width:163px;">Promega, USA</td>
			<td style="width:104px;">N251B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Streptavidin alkaline phosphatase</td>
			<td style="width:163px;">Promega, USA</td>
			<td style="width:104px;">V5591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TBE</td>
			<td style="width:163px;">Invitrogen, USA</td>
			<td style="width:104px;">15581044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCI Buffer&#160;</td>
			<td>Invitrogen, USA</td>
			<td>15567027</td>
			<td>1 M, PH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCI Buffer&#160;</td>
			<td>Invitrogen, USA</td>
			<td>15568025</td>
			<td>1 M, PH 8.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tween-20</td>
			<td style="width:163px;">Sangon Biotech, China</td>
			<td style="width:104px;">A600560</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro biochemical assays using biotin labels to study protein-nucleic acid interactions

蛋白质-核酸相互作用在生物过程(如转录、重组和RNA代谢)中起着重要作用。研究蛋白质-核酸相互作用的实验方法需要使用荧光标记、放射性同位素或其他标签来检测和分析特定的目标分子。Biotin 是一种非放射性核酸标签,通常用于电泳移动转移测定 (EMSA),但一直没有定期用于监测核酸过程中的蛋白质活性。该协议说明了生物锡标签在体外酶反应过程中的效用,证明该标签与一系列不同的生化测定有效。具体来说,根据之前使用放射性同位素32P标记基质的发现,通过生物素标记EMSA确认MEIOB(一种专门参与美智重组的蛋白质)是一种DNA结合蛋白,即MOV10(一种RNA合壳)可解析生物素标记的RNA双工结构,而MEIOB可使生物素标记的单链DNA。研究表明,生物素能够在体外各种核酸相关生化检测中替代32P。

MEIOB的蛋白质结构和本研究中使用的表达结构如图1A所示。 MEIOB 中的 OB 折叠是紧凑的桶状结构,可以识别和与单链核酸相互作用。其中一个OB域(aa 136-307,构造A)结合单绞合DNA(sSDNA),截断蛋白(aa 136-178截断,构造C)和点突变形式(R235A突变,构造E)的MEIOB没有DNA结合活性26。GST-MEIOB融合蛋白在BL21细菌中被过度表达,随后的分离步骤导致通过Coomassie蓝色染色和?...

Watch this Scientific Journal Video about In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions at JoVE.com

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

这里介绍的是使用生物素标签的体外生化测定方案,可广泛用于研究蛋白质-核酸相互作用。

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

该协议为研究蛋白质-核酸相互作用提供了一个生物素标签平台，证明其稳健、可靠、高效且价格实惠。同一批生物素标记的核酸可以长期使用，保持实验的可重复性。最后，此处描述的所有生物素标记检测都可以在一天内进行，不需要特殊设备。演示这个程序的将是来自我们实验室的研究生LinaYu。准备 MEIOB 和 MOV10 构造，并将其转换为适当的细菌。要提取 MEIOB，通过离心将细胞颗粒，然后重新在 20 毫升的冰冷的杜尔贝科磷酸盐缓冲盐水（DPBS）缓冲液中重新填充。声波的细菌悬浮液在冰上。然后，将12，000倍g和4摄氏度的利酸盐离心15分钟，然后将上分液转移到一个新鲜的管中。预洗谷胱甘肽-塞法罗斯珠，并在四摄氏度下用谷氨酸孵育两小时。将混合物在750倍g和4摄氏度下离心一分钟，以分粒珠子。用10毫升的冰冷PBS冲洗珠子8次。然后，加入一毫升的洗脱缓冲液，在4摄氏度下孵育珠子10分钟，然后通过离心将珠子孵育。重复洗脱六次，将六个分数汇集在一起。将洗出的蛋白质转移到离心过滤器中，以7，500倍g的离心浓缩，最终体积为100至200微升。要提取MOV10，在 HEK293T 细胞中表达蛋白质，然后根据手稿方向对细胞进行颗粒。将颗粒重新吸收在三毫升细胞解液缓冲液中，并服用完整的无 EDTA 蛋白酶抑制剂鸡尾酒，并在冰上孵育 30 分钟。然后，在摄氏四度时将酸盐离心。使用 K150 缓冲液清洗两次防旗磁珠，准备防旗磁珠。洗涤后，将珠子重新在一毫升冰冷的K150缓冲液中，在4摄氏度下温和旋转孵育两分钟。收集磁铁上的珠子，取出上一液。将珠子加入细胞酸盐，在四摄氏度下孵育两小时。根据手稿方向用 K150 缓冲液清洗珠子，并在 FLAG 洗脱缓冲液的 300 微升中重新填充珠子。将珠子放在磁铁上，用MOV10蛋白收集上清液，并确定蛋白质浓度。准备RNA寡核苷酸，将每个寡糖稀释至20微摩尔，然后根据手稿指示将寡核苷酸退火，形成RNA双面体。对于 MEIOB 电泳移动性移位测定（EMSA），请根据手稿指示混合试剂。加入水，最终反应量为20微升。在室温下孵育反应30分钟，然后加入5倍止损缓冲液。对于MOV10直升机体活性测定，根据手稿说明混合试剂，并加入水，最终反应量为20微升。在37摄氏度下孵育反应10分钟、30分钟和60分钟，然后加入5倍停止缓冲液以阻止反应。然后，制备20%原生聚丙烯酰胺凝胶，将每个样品的20至25微升装入每个井中。在冰浴上以 100 伏的电压运行凝胶，直到布罗莫酚蓝色标记迁移到凝胶的底部。丙烯酰胺是有害和有毒的。使用适当的个人防护设备进行处理非常重要。拆解凝胶板，通过去除装载井和未使用的通道来修剪凝胶。将凝胶放在0.5x TBE缓冲液中。将滤纸和尼龙膜切割成凝胶大小，预先湿润纸张和膜，并组装堆栈进行转移。将样品在90毫安的半干电泳装置中从凝胶转移到膜上20分钟。然后，将样品以每厘米120毫焦耳的平方照射，将样品交联45至60秒。对于化学发光检测，首先在膜中加入20毫升的阻尼缓冲液，并在轻轻摇动时孵育15至30分钟。小心地删除阻塞缓冲区，并将其替换为偶联/阻塞缓冲区。再次孵育膜15分钟。每次洗涤时摇动五分钟，清洗膜四次。然后，在膜中加入30毫升基板平衡缓冲液，在20至25转/时摇动时孵育5分钟。用基板工作溶液覆盖膜的整个表面，孵育五分钟。孵育后，在化学发光成像系统中扫描膜一至三秒钟。通过库马西蓝色染色和西印迹分析，可以验证 MEIOB 蛋白 A、C 和 E 的纯化。红色箭头表示纯化的 MEIOB 蛋白的位置。MEIOB 和单链 DNA 的相互作用可以使用 EMSA 进行可视化。观察到浓度依赖结合和裂解。不出所料，只有野生型 MEIOB-A 与 DNA 相互作用，而突变 MEIOB-E 和 MEIOB-C 则不与 DNA 相互作用。也可以观察到 MEIOB 和单链RNA的相互作用。野生型 MEIOB 显示单链RNA上的浓度依赖结合和外核酶活性。然而，定量比较表明，MEIOB结合DNA的效率高于RNA。MOV10的纯化用库马西蓝色染色进行验证，在RNA双面体上测量了该蛋白的血体活性，其悬垂为5个。随着反应时间的增加，MOV10 逐渐解除双链RNA。为了降低检测成本，化学致发光检测可以通过检测试剂试剂的两倍稀释或自制试剂进行。此处描述的协议成功地将生物素标记的核酸用作体外生化测定（如 EMSA）和未常用生物素的酶反应的基质。

Research

JoVE Journal

Genetics

In Vitro Transcription Assays and Their Application in Drug Discovery

In Vitro Transcription Assays and Their Application in Drug Discovery

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

科研

遗传学

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

可再生生物燃料及生化生产的非常规酵母基因工程

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...