Name: discrimintion and mapping of the primary and processed transcripts in maize mitochondrion using a circular rt-pcr-based strategy
Uploaded: 2019-07-29T15:00:00
Description: Watch this Scientific Journal Video about Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy at JoVE.com

这项工作得到了国家自然科学基金(授权号31600250,Y.Z.)、广州市科技项目(批号201804020015,H.N.)和中国农业研究系统(批号No.S.)的支持。CARS-04-PS09,H.N.)。

1. 底漆设计
<ol><li>根据引物设计的一般规则,使用PCR引物设计软件(材料表)设计逆转录(RT)基因专用引物。 注:RT 引录对目标转录本非常具体,它们通常固定在编码序列的 5' 部分(成熟的 mRNA 和前体 RNA),或预计的 5' 端 (18S 和 26S rRNA)的下游 ±500–600 nt。</li>
	<li>设计发散引基对,通过cRT-PCR放大循环转录。 注:成对发散引引在循环转录本的5'-3'结侧侧,它们的?...

<ol>
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M., Bonen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+wheat+mitochondrial+mRNA+termini+and+comparison+with+breakpoints+in+DNA+homology+among+plants.">Mapping of wheat mitochondrial mRNA termini and comparison with breakpoints in DNA homology among plants.</a> Plant Molecular Biology. 80 (4-5), 539-552 (2012).</li><li>Calixte, S., Bonen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmentally-specific+transcripts+from+the+ccmFN-rps1+locus+in+wheat+mitochondria.">Developmentally-specific transcripts from the ccmFN-rps1 locus in wheat mitochondria.</a> Molecular Genetics and Genomics. 280 (5), 419-426 (2008).</li><li>Kuhn, K., Weihe, A., Borner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+promoters+are+a+common+feature+of+mitochondrial+genes+in+Arabidopsis.">Multiple promoters are a common feature of mitochondrial genes in Arabidopsis.</a> Nucleic Acids Research. 33 (1), 337-346 (2005).</li><li>Jonietz, C., Forner, J., Holzle, A., Thuss, S., Binder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+PROCESSING+FACTOR2+is+required+for+5'+end+processing+of+nad9+and+cox3+mRNAs+in+mitochondria+of+Arabidopsis+thaliana.">RNA PROCESSING FACTOR2 is required for 5' end processing of nad9 and cox3 mRNAs in mitochondria of Arabidopsis thaliana.</a> ThePlant Cell. 22 (2), 443-453 (2010).</li><li>Stoll, B., Stoll, K., Steinhilber, J., Jonietz, C., Binder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transcript+length+polymorphisms+are+a+widespread+phenomenon+in+Arabidopsis+thaliana.">Mitochondrial transcript length polymorphisms are a widespread phenomenon in Arabidopsis thaliana.</a> Plant Molecular Biology. 81 (3), 221-233 (2013).</li><li>Mulligan, R. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+11+nucleotide+sequence+contains+an+essential+promoter+element+of+the+maize+mitochondrial+atp1+gene.">A conserved 11 nucleotide sequence contains an essential promoter element of the maize mitochondrial atp1 gene.</a> The EMBO Journal. 11 (3), 1065-1073 (1992).</li><li>Forner, J., Weber, B., Thuss, S., Wildum, S., Binder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+mitochondrial+mRNA+termini+in+Arabidopsis+thaliana:+t-elements+contribute+to+5'+and+3'+end+formation.">Mapping of mitochondrial mRNA termini in Arabidopsis thaliana: t-elements contribute to 5' and 3' end formation.</a> Nucleic Acids Research. 35 (11), 3676-3692 (2007).</li><li>Binder, S., Stoll, K., Stoll, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+5'+ends+of+plant+mitochondrial+RNAs.">Maturation of 5' ends of plant mitochondrial RNAs.</a> Physiologia Plantarum. 157 (3), 280-288 (2016).</li><li>Hang, R. L., Liu, C. Y., Ahmad, A., Zhang, Y., Lu, F. L., Cao, X. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+protein+arginine+methyltransferase+3+is+required+for+ribosome+biogenesis+by+affecting+precursor+ribosomal+RNA+processing.">Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16190-16195 (2014).</li><li>Wang, H. Q., 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=Maize+Urb2+protein+is+required+for+kernel+development+and+vegetative+growth+by+affecting+pre-ribosomal+RNA+processing.">Maize Urb2 protein is required for kernel development and vegetative growth by affecting pre-ribosomal RNA processing.</a> New Phytologist. 218 (3), 1233-1246 (2018).</li><li>Maloney, A. P., 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=Identification+in+maize+mitochondrial+26S+rRNA+of+a+short+5'-end+sequence+possibly+involved+in+transcription+initiation+and+processing.">Identification in maize mitochondrial 26S rRNA of a short 5'-end sequence possibly involved in transcription initiation and processing.</a> Current Genetics. 15 (3), 207-212 (1989).</li><li>Green, R. M., Sambrook, J. <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. , (2012).</li><li>Canino, 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=Arabidopsis+encodes+four+tRNase+Z+enzymes.">Arabidopsis encodes four tRNase Z enzymes.</a> Plant Physiology. 150 (3), 1494-1502 (2009).</li><li>Gobert, 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=A+single+Arabidopsis+organellar+protein+has+RNase+P+activity.">A single Arabidopsis organellar protein has RNase P activity.</a> Nature Structural, Molecular Biology. 17, 740-744 (2010).</li><li>Gutmann, B., Gobert, A., Giege, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRORP+proteins+support+RNase+P+activity+in+both+organelles+and+the+nucleus+in+Arabidopsis.">PRORP proteins support RNase P activity in both organelles and the nucleus in Arabidopsis.</a> Genes &amp; Development. 26 (10), 1022-1027 (2012).</li><li>Stern, D. 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在先前的研究中,利用阿拉伯多普西细胞悬浮培养的组蛋白和线粒体RNA,用cRT-PCR绘制线粒体转录终点,结果相似,获得12.然而,在许多其他研究中,只有富集线粒体RNA被用于绘制线粒体转录终点尼的图谱。研究发现,线粒体RNA的富集是玉米中线粒体转录终点的cRT-PCR映射的重要一步。在?...

在植物线粒体中,许多成熟和前体RNA作为多种等形体积累,稳定状态转录法可以根据其5'末端1、2、3的差异被分成两组。 4.主转录有5'三磷酸盐末端,从转录启动派生。相比之下,经过处理的转录本具有5'单磷酸盐,通过转录后处理产生。两种类型的转录本的鉴别和映射对于解开转录和转录结束成熟的分子机制非常重要。
为了区分植物线粒体的主要转录和加工记录,已经开发了四个主要策略。第一种策略是用烟草酸焦磷酸酶(TAP)预处理线粒体RNA,将5'三磷酸盐转化为单磷酸盐,并使初级转录本通过RNA连带循环。然后通过快速扩增cDNA端(RACE)或圆形RT-PCR(cRT-PCR)2、3、4,比较TAP处理和未处理RNA样本的转录量。在第二种策略中,处理的转录本首先从线粒体RNA中耗尽,使用终结器5'-磷酸盐依赖外糖酶(TEX),然后通过引世扩展分析5,6映射所剩的主要转录本.第三种策略是使用甘蓝转移酶预盖初级转录,然后三磷酸盐5'termini的位置由引基延伸与核糖酸酶或S1核酸酶保护分析7,8确定 ,9.与基于5'三磷酸的存在/不存在的不同,第四种策略结合了体外转录、位点定向诱变和引物延伸分析,以表征启动子并确定转录启动站点8,10,11。通过使用这些策略,许多主要和经过处理的转录本在植物线粒体中已经确定。
然而,一些研究报告说,原发转录的5'三磷酸盐是不稳定的,他们很容易转换为单磷酸盐不明的原因2,4,12,13。这个问题阻碍了对两种类型的成绩单的明确区分,因为使用技术取决于存在/缺少 5' 三磷酸,以及以前在工厂中系统地区分初级记录和加工抄本的努力线粒体失败2,12。
在此协议中,我们将cRT-PCR、RNA 5'聚磷酸酶处理、RT-qPCR和北方斑点相结合,系统地区分玉米(ZeaMays)线粒体中稳态积累的初级和加工转录(图1)。cRT-PCR允许同时映射RNA分子的5'和3'的四肢,它通常适应在植物2,12,14,15的图谱抄本术语。RNA 5' 聚磷酸酶可以从三磷酸盐5'特尼中去除两种磷酸盐,这使得主转录本可用于RNA连结酶的自我结扎。先前的研究表明,玉米中成熟的26S rRNA已经加工了5'终点,对RNA5'聚磷酸酶1,16)不敏感。为了将不稳定的三磷酸盐对原发5'特尼的影响降至最低,5'聚磷酸酶处理和未处理RNA通过成熟的26S rRNA进行归一化,然后通过比较来区分原发和加工的转录本。从两个RNA样本中获得的cRT-PCR产物。cRT-PCR映射和鉴别结果分别由北方印子和RT-qPCR验证。最后,使用替代引液来放大在北方污点中检测到的转录本,而不是通过cRT-PCR检测到的。通过这种基于cRT-PCR的策略,对玉米线粒体中大多数稳态转录被区分和映射为1。

<table>
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Aladdin, China</td>
			<td>A112880</td>
			<td>To prepare 1x TAE buffer</td>
		</tr>
		<tr>
			<td>Applied Biosystems 2720 Thermal Cycler</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>4359659</td>
			<td>Thermal cycler for PCR amplification</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900134</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>Biowest Agarose</td>
			<td>Biowest, Spain</td>
			<td>9012-36-6</td>
			<td>To resolve PCR products and RNAs</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-aldrich, USA</td>
			<td>A1933</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-aldrich, USA</td>
			<td>B8026</td>
			<td>For preparation of loading buffer for agarose gel electrophoresis and Northern blot</td>
		</tr>
		<tr>
			<td>DEPC</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900882</td>
			<td>Deactivation of RNase</td>
		</tr>
		<tr>
			<td>DIG Northern starter kit</td>
			<td>Roche, USA</td>
			<td>12039672910</td>
			<td>For DIG-RNA labeling and Northern blot. This kit contains the reagents for transcription-labeling of RNA with DIG and T7 RNA polymerase, hybridization and chemiluminescent detection.</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900106</td>
			<td>For preparation of extraction buffer and 1x TAE buffer</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-aldrich, USA</td>
			<td>E3889</td>
			<td>For preparation of wash buffer</td>
		</tr>
		<tr>
			<td>Gel documentation system</td>
			<td>Bio-Rad, USA</td>
			<td>Gel Doc XR+</td>
			<td>To image the agarose gel</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-aldrich, USA</td>
			<td>G5516</td>
			<td>For preparation of loading buffer for agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>GoldView II (5000x)</td>
			<td>Solarbio,. China</td>
			<td>G8142</td>
			<td>DNA staining</td>
		</tr>
		<tr>
			<td>Hybond-N+, Nylon membrane</td>
			<td>Amersham Biosciences, USA</td>
			<td>RPN119</td>
			<td>For Northern blot</td>
		</tr>
		<tr>
			<td>Image Lab</td>
			<td>Bio-Rad, USA</td>
			<td>Image Lab 3.0</td>
			<td>Image gel, and compare the abundance of PCR products.</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900041</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Aladdin, China</td>
			<td>P112284</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900399</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>Millex</td>
			<td>Millipore, USA</td>
			<td>SLHP033RB</td>
			<td>To sterile extraction and wash buffers by filtration</td>
		</tr>
		<tr>
			<td>Miracloth</td>
			<td>Calbiochem, USA</td>
			<td>475855-1R</td>
			<td>To filter the ground kernel tissues</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900306</td>
			<td>For preparation of running buffer for Northern blot</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>2000C</td>
			<td>For RNA concentration and purity assay</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900797</td>
			<td>For preparation of wash buffer</td>
		</tr>
		<tr>
			<td>pEASY-Blunt simple cloning vector</td>
			<td>TransGen Biotech, China</td>
			<td>CB111</td>
			<td>Cloning of the gel-recovered band. It contains a T7 promoter several bps upstream of the insertion site.</td>
		</tr>
		<tr>
			<td>Phanta max super-fidelity DNA polymerase</td>
			<td>Vazyme, China</td>
			<td>P505</td>
			<td>DNA polymerase for PCR amplification</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone 40</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900008</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>Primer Premier 6.24</td>
			<td>PREMIER Biosoft, USA</td>
			<td>Primer Premier 6.24</td>
			<td>To design primers for reverse transcription and PCR amplification</td>
		</tr>
		<tr>
			<td>PrimeScript II reverse transcriptase</td>
			<td>Takara, Japan</td>
			<td>2690</td>
			<td>To synthesize the first strand cDNA</td>
		</tr>
		<tr>
			<td>PureLink RNA Mini kit</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>12183025</td>
			<td>For RNA purificaion</td>
		</tr>
		<tr>
			<td>RNA 5&#39; polyphosphatase</td>
			<td>Epicentre, USA</td>
			<td>RP8092H</td>
			<td>To convert 5&#39; triphosphate to monophosphate</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>New England Biolabs, UK</td>
			<td>M0314</td>
			<td>A component of RNA self-ligation and 5&#39; polyphosphatase treatment reactions, and it is used to inhibite the activity of RNase.</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900212</td>
			<td>For preparation of running buffer for Northern blot</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900058</td>
			<td>To prepare 20x SSC</td>
		</tr>
		<tr>
			<td>SsoFas evaGreen supermixes</td>
			<td>Bio-Rad, USA</td>
			<td>1725202</td>
			<td>For RT-qPCR</td>
		</tr>
		<tr>
			<td>T4 RNA Ligase 1</td>
			<td>New England Biolabs, UK</td>
			<td>M0437</td>
			<td>For RNA circularization</td>
		</tr>
		<tr>
			<td>Tetrasodium pyrophosphate</td>
			<td>Sigma-aldrich, USA</td>
			<td>221368</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>TIANgel midi purification kit</td>
			<td>Tiangen Biotech, China</td>
			<td>DP209</td>
			<td>To purify DNA fragments from agarose gel</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Aladdin, China</td>
			<td>T110601</td>
			<td>To prepare 1x TAE buffer</td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Invitrogen, USA</td>
			<td>15596026</td>
			<td>To extract mitochondiral RNA.</td>
		</tr>
		<tr>
			<td>Universal DNA purification kit</td>
			<td>Tiangen Biotech, China</td>
			<td>DP214</td>
			<td>To recover linearized plastmids from the restriction enzyme digestion reaction</td>
		</tr>
		<tr>
			<td>Xylene cyanol FF</td>
			<td>Sigma-aldrich, USA</td>
			<td>X4126</td>
			<td>For preparation of loading buffer for agarose gel electrophoresis</td>
		</tr>
	</tbody>
</table>

discrimintion and mapping of the primary and processed transcripts in maize mitochondrion using a circular rt-pcr-based strategy

在植物线粒体中,一些稳态转录本具有从转录启动(主转录)衍生的5'三磷酸盐,而其他含有5'单磷酸盐产生的转录后(处理转录)。为了区分这两种类型的成绩单,已经制定了几种策略,其中大多数取决于存在/缺少5'三磷酸盐。然而,小学5年级的三磷酸盐是不稳定的,它阻碍了对两种类型的抄本的明显区分。为了系统地区分和绘制玉米线粒体中稳定积累的初级和加工转录本,我们开发了基于CRT-PCR(cRT-PCR)的循环RT-PCR(cRT-PCR)策略,将cRT-PCR、RNA 5'多磷酸酶处理、定量RT-PCR (RT-qPCR) 和北方污点。作为改进,该策略包括RNA正常化步骤,以尽量减少不稳定的5'三磷酸盐的影响。
在此协议中,富集线粒体RNA由RNA 5'聚磷酸酶预处理,将5'三磷酸酯转化为单磷酸盐。经过循环和逆转录后,从5'聚磷酸酶处理和未处理RNA衍生的两种cDNA由玉米26S成熟rRNA归一化,其加工为5'结束,对5'聚磷酸酶不敏感。规范化后,通过比较从处理过的和未经处理的RNA获得的cRT-PCR和RT-qPCR产物,对初级和加工的转录本进行区分。转录端验通过cRT-PCR产物的克隆和测序确定,然后由北方印块验证。
通过采用这一策略,确定了玉米线粒体中大多数稳态记录。由于一些线粒体基因的转录模式复杂,一些稳态转录图没有被区分和/或映射,尽管它们是在北方的污点中检测到的。我们不确定此策略是否适合在其他植物线粒体或石膏中区分和映射稳态记录。

线粒体RNA循环效率估计
在以前的研究中,总和线粒体RNA都用于cRT-PCR映射线粒体转录在阿拉伯拉多普西斯(阿拉伯拉多普西斯)的线粒体转录终点,两种类型的RNA给出了类似的映射结果12。最初,我们还使用总RNA在玉米中线粒体转录终点的cRT-PCR映射。经过多次试验,我们发现目标记录很难被?...

Watch this Scientific Journal Video about Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy at JoVE.com

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

使用基于循环 RT-PCR 的策略对玉米 Mitochondrion 中初级和已处理转录的区分和映射

通过结合循环RT-PCR、定量RT-PCR、RNA 5'聚磷酸酶处理和北方斑点,提出了基于RT-PCR的循环RT-PCR策略。该协议包括一个标准化步骤,以尽量减少不稳定5'三磷酸酯的影响,它适用于对玉米线粒体中稳定积累的初级和加工转录转录进行鉴别和映射。

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

该协议用于绘制和歧视玉米线粒体的主要和加工成绩单。该技术包括RNA规范化步骤，它最大限度地减少了不稳定的五个原三磷酸盐的影响，这妨碍了主抄本和加工成绩单之间的明显区分。除玉米线粒体外，该方法可应用于植物线粒体和其他植物线粒体。首先收集20克开发内核到50毫升的冰管。将内核转移到预冷却的砂浆中，并添加 10 到 20 毫升的冰冷萃取缓冲液。完全研磨内核，添加更多的萃取缓冲液，并通过两层滤布过滤地面组织。将滤金物在8000倍G下离心10分钟。然后将上一液转移到新管中，然后丢弃滤液。将管子在20，000次G下离心10分钟。从上流液中倒开，将颗粒重新悬浮在六毫升的洗涤缓冲液中。将悬浮液放入5个1.5毫升无RNase管中，并在14000次G下离心5分钟。丢弃上清液，将液氮中的线粒冷冻。根据制造商的说明使用商业试剂提取线粒体RNA，并设置RNA五主要多磷酶处理。然后，使用RNA纯化试剂盒回收RNA。用于RNA提取的试剂是危险的。始终在烟罩中工作，始终穿着实验室外套和手套。使用相同量的五个素多磷酶处理和非治疗线粒体RNA准备两个循环反应。在16摄氏度下孵育两种反应12至16小时，然后用以前使用的RNA纯化试剂盒恢复自结RNA。首先制备一个底转混合物，其比率为26 SCRT，最多为7个其他逆转录底转。根据稿稿方向组装两个逆转录反应系统，并在42摄氏度下孵育50分钟。要使cDNA正常化，从五种原磷多磷酶或非处理RNA中，用相同体积的cDNA准备两个PCR反应，并在手稿中描述的热循环条件下运行反应。26S 成熟 rRNA 的规范化是区分主抄本和加工成绩单的关键步骤。比较两个 PCR 产品的丰度，如有必要，通过调整模板 cDNA 的量来优化规范化。使用适当体积的规范化 cDNA 和一对发散底转（如目标成绩单的五个原点到三个主要交汇点）准备 PCR 反应，并根据手稿指示执行 PCR。然后，使用凝胶DNA恢复套件来分离由嵌套PCR放大的突出带。使用标准技术将凝胶纯化 PCR 产品克隆到载体中，并执行聚落 PCR 以选择包含目标刀片的正克隆。使用基本的局部对齐搜索工具或 BLAST 对 PCR 产品进行测序，使测序数据与玉米线粒体基因组对齐。选择有机体玉米和搜索数据库核苷酸收集。查找循环成绩单的五个黄金到三个主要交汇点，并确定五个底数和三个主要成绩单的术语的位置。放大用于准备RNA探针的DNA片段，并克隆到先前使用的载体，该载体包含T7启动子，插入位点上游有17个碱基对。使用 dig-11-UTP 标记 RNA 探头，并使用商用试剂盒进行RNA杂交。通过比较从经过的5个原磷酶和非处理RNA中获得的圆形RT-PCR产品，区分原端和加工的五个素端。然后，根据映射结果设计定量RT-PCR底因，用RT-PCR验证歧视结果。COX-2基因作为例子，用基于圆形RT-PCR的策略来演示玉米线粒体转录本的映射和歧视。规范化的cDNA被用作PCR的模板，从而从经过处理的五个原磷酶样本中放大了两个突出波段。这两个波段被命名为COX-2一和两个从凝胶中恢复，并克隆成载体。殖民地PCR结果表明，正克隆包含可变大小的插入物，这意味着术语具有异构的五个原数或三个原表。测序结果表明，COX-2 1和2在停止胶东下游的39个核苷酸具有相同的三个主端，而其五个主端分别位于992至1，030和1，276至1，283核苷酸的启动科顿上游。RNA凝胶印迹杂交用于验证圆形RT-PCR映射结果，并检测到两个大小与COX-2一和2相似的主要波段。另一对发散底剂被设计成放大用RNA凝胶印迹检测的COX-2三和四波段，但不包括第一次圆形RT-PCR。定量RT-PCR结果证实COX-2一、二、三和四对五种主要多磷酶敏感，并且它们有五个主要的五主要术语。可以执行 Primer 扩展分析来验证五个主要映射结果，尽管它未包含在此协议中。

Research

JoVE Journal

Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

科研

遗传学

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL定位和CRISPR / Cas9编辑识别抗药性基因<em&gt;弓形虫</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

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 ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

ATAC 基因组在原发性人类 T 淋巴细胞中的染色体全易接近染色质

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

3 ' 快速扩增 CDNA 末端, 以图记录癌症

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

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 Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

使用乌斯蒂拉戈梅迪斯作为特洛伊木马在现场交付玉米蛋白

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

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 ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...