Name: fluorescent visualization of mango-tagged rna in polyacrylamide gels via a poststaining method
Uploaded: 2019-06-21T12:30:00
Description: Watch this Scientific Journal Video about Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method at JoVE.com

作者感谢拉兹万·科约卡鲁和阿米尔·阿卜杜拉赫扎德的技术援助,感谢莱娜·多尔戈舍纳对手稿进行校对。加拿大自然科学和工程研究理事会(NSERC)向P.J.U提供的业务赠款为该项目提供了资金。

1. 试剂的制备
<ol><li>
TO1-生物染色后溶液(凝胶染色溶液)
	<ol>
<li>在 25°C 下,在 pH 7.2 下,通过加入 342 mL 的 Na2HPO4和 158 mL 的 1 M NaH2PO4,在 pH 7.2 下制造 1 L 的 1 m 磷酸盐缓冲液。通过添加适当的磷酸盐溶液,在 50 mM 时将 pH 调节到 7.2。使用 0.2 μm 过滤器进行无菌过滤器,并在室温下将溶液储存在塑料制品中。</li>
		<li>准备5x凝胶染色溶液(不含TO1-生物素),如下?...

<ol>
	<li>Dolgosheina, E. 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=RNA+Mango+Aptamer-Fluorophore:+A+Bright,+High-Affinity+Complex+for+RNA+Labeling+and+Tracking.">RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking.</a> ACS Chemical Biology. , (2014).</li><li>Autour, A., 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=Fluorogenic+RNA+Mango+aptamers+for+imaging+small+non-coding+RNAs+in+mammalian+cells.">Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.</a> Nature Communications. 9, 656 (2018).</li><li>Dolgosheina, E. V., Unrau, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorophore-binding+RNA+aptamers+and+their+applications:+Fluorophore-binding+RNA+aptamers.">Fluorophore-binding RNA aptamers and their applications: Fluorophore-binding RNA aptamers.</a> Wiley Interdisciplinary Reviews: RNA. , (2016).</li><li>Panchapakesan, S. S., 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=Ribonucleoprotein+Purification+and+Characterization+using+RNA+Mango.">Ribonucleoprotein Purification and Characterization using RNA Mango.</a> RNA. , 1592-1599 (2017).</li><li>Trachman, R. 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=Structural+basis+for+high-affinity+fluorophore+binding+and+activation+by+RNA+Mango.">Structural basis for high-affinity fluorophore binding and activation by RNA Mango.</a> Nature Chemical Biology. 13, 807 (2017).</li><li>Trachman, R. 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=Crystal+Structures+of+the+Mango-II+RNA+Aptamer+Reveal+Heterogeneous+Fluorophore+Binding+and+Guide+Engineering+of+Variants+with+Improved+Selectivity+and+Brightness.">Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness.</a> Biochemistry. 57, 3544-3548 (2018).</li><li>Trachman, R., 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=Mango-III+is+a+compact+fluorogenic+RNA+aptamer+of+unusual+structural+complexity.">Mango-III is a compact fluorogenic RNA aptamer of unusual structural complexity.</a> Nature Chemical Biology. , (2018).</li><li>Panchapakesan, S. S. S., Jeng, S. C. Y., Unrau, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+complex+purification+using+high-affinity+fluorescent+RNA+aptamer+tags.">RNA complex purification using high-affinity fluorescent RNA aptamer tags.</a> Annals of the New York Academy of Sciences. , (2015).</li><li>Filonov, G. S., Kam, C. W., Song, W., Jaffrey, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-gel+imaging+of+RNA+processing+using+Broccoli+reveals+optimal+aptamer+expression+strategies.">In-gel imaging of RNA processing using Broccoli reveals optimal aptamer expression strategies.</a> Chemistry &amp; Biology. 22, 649-660 (2015).</li><li>Milligan, J. F., Groebe, D. R., Witherell, G. W., Uhlenbeck, O. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Nucleic+Acids+by+Extraction+with+Phenol:Chloroform.">Purification of Nucleic Acids by Extraction with Phenol:Chloroform.</a> Cold Spring Harbor Protocols. 2006 (1), (2006).</li><li>Tuma, R. S., 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=Characterization+of+SYBR+Gold+Nucleic+Acid+Gel+Stain:+A+Dye+Optimized+for+Use+with+300-nm+Ultraviolet+Transilluminators.">Characterization of SYBR Gold Nucleic Acid Gel Stain: A Dye Optimized for Use with 300-nm Ultraviolet Transilluminators.</a> Analytical Biochemistry. 268, 278-288 (1999).</li><li>Streit, S., Michalski, C. 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芒果荧光标签的一个显著优点是单个标签可以多种方式使用。这些贴合剂的高亮度和亲和力使它们不仅可用于细胞可视化2,而且可用于体外RNA或RNP纯化4。因此,凝胶成像以简单明了的方式扩展了芒果标签的多功能性。芒果凝胶成像灵敏度略低于北方印贝14,但可以轻松检测60~120 fmol的RNA样品,无需冗长而繁琐的膜转移和探测步骤。这与之前在<sup clas...

芒果是一个RNA标记系统,由一组四个小型荧光RNA贴合剂组成,与硫亚酮橙的简单衍生物(TO1-Biotin,图1A)1、2、3紧密结合( 纳米摩尔结合).结合后,这种配体的荧光会根据特定的贴合体增加1,000至4,000倍。芒果系统的高亮度,超过增强的绿色荧光蛋白(eGFP),结合RNA芒果贴合剂的纳米结合亲和力,使其可用于成像和RNA的纯化复合体 2,4。
芒果一号、第五号、二号、三号7的X射线结构已经确定为高分辨率,所有三个贴合体都利用RNA四联体结合TO1-Biotin(图1B+D)。所有三个贴实器的紧凑核心通过紧凑的适配器图案从外部RNA序列中分离出来。芒果I和II都利用灵活的GNRA环适配器将芒果芯连接到任意RNA双工(图1B,C)。相比之下,芒果III使用刚性三重体图案将其核心连接到任意RNA螺旋(图1D,紫色残留物),而芒果IV的结构目前尚不清楚。由于这些贴合剂的配体结合核心通过这些螺旋适配器从外部RNA序列中分离出来,因此它们似乎都可以简单地被合并到各种RNA中。细菌6S调节RNA(芒果I),酵母孢子菌(芒果I)的成分,以及人类5SRNA,U6RNA和C/D scaRNA(芒果II和IV)都成功地标记在这种方式2,8,这表明许多生物RNA可以使用RNA芒果贴切器系统进行标记。
变性和原生凝胶通常用于研究RNA。变性凝胶通常用于判断RNA大小或RNA处理,但通常,在北方斑点的情况下,需要几个缓慢和连续的步骤才能生成图像。虽然其他RNA致氟的贴合剂,如RNA菠菜和西兰花,已经成功地用于凝胶成像9,但迄今为止没有氟化贴合剂系统具有芒果系统的高亮度和亲和力,因此具有相当的兴趣调查芒果的凝胶成像能力在这项研究中,我们想知道RNA芒果系统是否可以简单地扩展到凝胶成像,因为TO1-Biotin的激发和发射波长(分别为510nm和535nm)适合在大多数荧光器常见的eGFP通道中成像凝胶扫描仪器。
此处介绍的凝胶后染色方案提供了一种快速方法,用于专门检测原生和变性聚丙烯酰胺凝胶电泳 (PAGE) 凝胶中的芒果标记 RNA 分子。这种染色方法涉及在含有钾和TO1-生物的缓冲液中浸泡凝胶。RNA芒果贴切剂以G-四重体为基础,需要钾来稳定这种结构。使用从最小的芒果编码DNA模板转录的RNA(参见协议部分),我们可以使用简单的染色方案,在原生凝胶中检测至多62.5 fmol的RNA和125 fmol的RNA。与常见的非特异性核酸污渍(见材料表,本文中引用SG),我们可以清楚地识别芒果标记的RNA,即使样品中存在高浓度的未标记总RNA。

<table border="0" cellpadding="0" cellspacing="0" style="width:924px;" width="924">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">0.8mm Thick Comb 14 Wells for 30 mL PAGE gels</td>
			<td style="width:192px;">LabRepCo</td>
			<td style="width:147px;">11956042</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">101-1000 &#181;L&#160; tips</td>
			<td>Fisher</td>
			<td>02-707-511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20-200 &#181;L low retention&#160; tips</td>
			<td>Fisher Scientific</td>
			<td>02-717-143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrylamide:N,N&#39;-methylenebisacrylamide (40% 19:1)</td>
			<td>Bioreagents</td>
			<td>BP1406-1</td>
			<td>Acute toxicity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrylamide:N,N&#39;-methylenebisacrylamide (40% 29:1)</td>
			<td>Fisher</td>
			<td>BP1408-1</td>
			<td>Acute toxicity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Anachemia</td>
			<td>02116-380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminium backed TLC plate</td>
			<td>Sigma-Aldrich</td>
			<td>1164840001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham Imager 600</td>
			<td>GE Healthcare Lifesciences</td>
			<td>29083461</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Persulfate</td>
			<td>Biorad</td>
			<td>161-0700</td>
			<td>Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BL21 cells</td>
			<td>NEB</td>
			<td>C2527H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric Acid</td>
			<td>ACP</td>
			<td>B-2940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue sodium salt</td>
			<td>Sigma</td>
			<td>B8026-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloloform</td>
			<td>ACP</td>
			<td>C3300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol</td>
			<td>Sigma Aldrich Alcohols</td>
			<td>D0632-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>ThermoFisher</td>
			<td>EN0525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA Disodium Salt</td>
			<td>ACP</td>
			<td>E-4320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Commerial&#160;</td>
			<td>P016EAAN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flat Gel Loading tips</td>
			<td>Costar</td>
			<td>CS004854</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formamide 99%</td>
			<td>Alfa Aesar</td>
			<td>A11076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel apparatus set with spacers and combs</td>
			<td>LabRepCo</td>
			<td>41077017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Dish with Plastic lid</td>
			<td>Pyrex</td>
			<td>1122963</td>
			<td style="width:275px;">Should be large enough to fit your gel piece</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Anachemia</td>
			<td>43567-540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Anachemia</td>
			<td>464140468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageQuanTL</td>
			<td>GE Healthcare Lifesciences</td>
			<td>29000605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG</td>
			<td>Invitrogen</td>
			<td>15529-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>ACP</td>
			<td>P-2940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Caledron</td>
			<td>4903-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M3409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>ACP</td>
			<td>S-2830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>BDH</td>
			<td>BDH9292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orbital Rotator</td>
			<td>Lab-Line</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol</td>
			<td>Invitrogen</td>
			<td>15513-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round Gel Loading tips</td>
			<td>Costar</td>
			<td>CS004853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate dibasic</td>
			<td>Caledron</td>
			<td>8120-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate monobasic</td>
			<td>Caledron</td>
			<td>8180-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBRGold</td>
			<td>ThermoFisher</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 RNA Polymerase</td>
			<td>ABM</td>
			<td>E041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T7024-50 ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TO1-3PEG-Biotin Fluorophore</td>
			<td>ABM</td>
			<td>G955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher</td>
			<td>BP152-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>Fisher</td>
			<td>BP1421-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Sigma</td>
			<td>P9496-100</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Urea</td>
			<td>Fisher</td>
			<td>U15-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene Cyanol</td>
			<td>Sigma</td>
			<td>X4126-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Bioshop</td>
			<td>YEX401.500</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescent visualization of mango-tagged rna in polyacrylamide gels via a poststaining method

原生和变性聚丙烯酰胺凝胶通常分别用于表征核糖核蛋白(RNP)复杂流动性和测量RNA大小。由于许多凝胶成像技术使用非特异性污渍或昂贵的荧光探针,因此非常需要灵敏、鉴别和经济的凝胶成像方法。RNA芒果核心序列是小(19~22 nt)序列图案,当被任意RNA茎关闭时,可以简单而廉价地附着在感兴趣的RNA上。这些芒果标签与一种名为TO1-Biotin的硫磷-橙色荧光配体结合,具有高亲和力和特异性,结合后荧光会增加数千倍。在这里,我们表明芒果I,II,III和IV可用于在高灵敏度的凝胶中专门成像RNA。在原生凝胶中仅62.5 fmol和变性凝胶中125 fmol的RNA,可以通过在含有钾和20 nM TO1-Biotin的成像缓冲液中浸泡凝胶30分钟来检测。通过在总细菌RNA的复杂混合物中成像芒果标记的6S细菌RNA,我们证明了芒果标记系统的特异性。

短芒果标记的RNA是编写在协议部分所述。假设由于凝胶中存在尿素,在变性条件下的荧光是最难观察到的,我们首先研究了芒果贴合剂对尿素的抗药性,尿素作为核酸变性剂。我们发现芒果贴合剂对变性具有实质性抗药性,尿素浓度约为1M(图2A)。在将凝胶染色溶液添加到变性凝胶之前,凝胶中尿素的最终浓度为6M。在实践中,染色协议达到的完全最佳结果要少,但如果?...

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Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

在这里,我们提出了一种敏感、快速和鉴别的凝胶后染色方法,使用原生或变性聚丙烯酰胺凝胶电泳(PAGE)凝胶(PAGE)凝胶,将带有RNA芒果贴片I、II、III或IV标记的RNA成像。运行标准 PAGE 凝胶后,芒果标记的 RNA 可轻松与 TO1-Biotin 染色,然后使用常用的荧光读卡器进行分析。

通过后染色方法在聚丙烯酰胺凝胶中对芒果标记RNA的荧光可视化

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

此处演示的芒果标记凝胶成像方法非常坚固，预计在灵敏度和特异性方面可以简单地扩展。这将进一步简化生物重要RNA和RNA复合物的常规纯化，只需在感兴趣的RNA中加入芒果标签的简单权宜之计。RNA 被牵连到越来越多的疾病中，因为这种后约会方法是一种能够跟踪和可视化RNA的简便而快速的方法。将凝胶从一个地方转移到另一个地方时需要格外小心，因为凝胶很脆弱，容易断裂。要准备 30 毫升变性凝胶，请首先选择具有适当溴酚蓝色和二甲苯氰醇染料的聚丙烯酰胺百分比，与感兴趣的 RNA 进行比较，以确保高波段分离。然后，根据表 1 混合解决方案 A、B 和 C，并添加 APS 和 timud。立即将凝胶溶液倒入适当的凝胶铸造装置中。插入所需的圆锥体，将凝胶留在聚合酶中约 30 分钟。使用 1 个 XTBE 溶液制作凝胶罐，并小心地拆下圆锥体。小心地提起凝胶并安装到凝胶装置上，并使用粘合剂夹固定。使用注射器，在样品装载前立即吸出油井。通过将2X变性凝胶加载溶液加入感兴趣的RNA样品，准备1XRNA样品。使用热循环器将样品加热 95 摄氏度 5 分钟。然后，使用凝胶加载技巧将冷却样品装载到每个井的底部。以及凝胶在室温下确保功率有效低，不破裂凝胶装置的玻璃板。根据手稿在干净的玻璃容器中准备 1X 凝胶染色溶液，该容器足够宽，可以舒适地适合凝胶。向容器中加入足够的 1X 凝胶染色溶液，使凝胶在放置在轨道旋转器上时完全覆盖溶液。凝胶完成运行后，从仪器上取出凝胶，切断其"孔"和凝胶的一角，以帮助确定方向。然后，小心地将凝胶转移到 1X 凝胶染色溶液中。要执行成像，请小心地将染色溶液排掉，然后用水快速冲洗凝胶。最后，小心地将凝胶转移到托盘上，以放置在成像器中。将玻璃移液器卷到凝胶上，以去除捕获的气泡和多余的液体。然后继续拍摄凝胶图像。芒果一、二、三、四极对变性有实质性的抵制，高达约一个摩尔尿素浓度。20 至 40 分钟的染色时间足以达到最大凝胶荧光。较长的时间不适合本研究中使用的小RNA，因为它们开始扩散出凝胶。荧光仪分析表明，芒果一、二、三在乌雷亚的存在，其折叠速度比四人更容易。在没有尿素的情况下，折叠的速度比预期的要快得多。后染色方法的灵敏度由单个波段显示，对应于本地和变性凝胶中每个芒果变体的折叠良好的RNA，对后一种的灵敏度稍低。原生凝胶的定量与芒果一、二、四的线性度比芒果三号更线性。变性凝胶的定量更线性，表明变性凝胶中存在尿素可能提供一种更均匀的方式，一旦它们被放置在 TO1 生物素染色溶液中，就将其折叠起来。与 SG 染色相比，使用 TO1 生物素染色，在总RNA存在的情况下可以检测芒果标记的RNA。请记住，确保凝胶染色容器足够大，足以容纳凝胶，否则凝胶可能会折叠到自身。RNA样本将从一个地方转移到另一个地方。与变性凝胶类似，此方法可用于原生凝胶。在较低的功率下在冷室中运行凝胶，以保持原生条件。

Research

JoVE Journal

Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

一种测量方法RNA<em&gt;ñ</em<sup&gt; 6</sup&gt; -methyladenosine细胞和组织修改

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

科研

生物化学

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

A Colorimetric Method for Measuring Iron Content in Plants

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

聚丙烯酰胺凝胶中蛋白质的荧光银染色

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using ÃƒÂÃ¢â‚¬Â¡CRAC

使用χCRAC以高时间分辨率监测体内蛋白质-RNA相互作用动力学

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

大鼠胰腺组织快速、经济高效的RNA提取

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...