Name: detection of human immunodeficiency virus type 1 (hiv-1) antisense protein (asp) rna transcripts in patients by strand-specific rt-pcr
Uploaded: 2019-11-27T14:00:00
Description: Watch this Scientific Journal Video about Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR at JoVE.com

我们感谢帕特里齐亚·阿梅利奥、亚历山德拉·诺托、克雷格·芬威克和马蒂厄·佩雷奥随时可以讨论我们的工作，并感谢艾滋病免疫机制实验室的所有人提供的宝贵技术援助。我们还要感谢VSB联合公司的约翰和亚伦·韦德尔，他们贡献了优秀的艺术品。最后，许多特别感谢所有的病人，没有他们，这项工作是不可能的。这项工作没有得到任何供资机构的具体赠款。

这项研究得到了瓦多瓦大学中心医院机构审查委员会的批准。
1. 外周血单核细胞（PBMC）感染HIV-1HXB2菌株
<ol><li>
第 1 天：PBMC STIMULATION
	<ol>
<li>将 PBMC 从健康的捐赠者布漆中分离出。</li>
		<li>在完整的罗斯威尔公园纪念研究所（RPMI）1640培养基中，以1x106/mL的浓度计数和重新悬浮多溴联苯（PBMC），含有10%的胎儿牛血清（FBS）和1%的青霉素/链霉素（R-...

<ol>
	<li>Miller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+may+encode+a+novel+protein+on+the+genomic+DNA+plus+strand.">Human immunodeficiency virus may encode a novel protein on the genomic DNA plus strand.</a> Science. 239 (4846), 1420-1422 (1988).</li><li>Vanheebrossollet, 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=A+Natural+Antisense+Rna+Derived+from+the+Hiv-1+Env+Gene+Encodes+a+Protein+Which+Is+Recognized+by+Circulating+Antibodies+of+Hiv++Individuals.">A Natural Antisense Rna Derived from the Hiv-1 Env Gene Encodes a Protein Which Is Recognized by Circulating Antibodies of Hiv+ Individuals.</a> Virology. 206 (1), 196-202 (1995).</li><li>Briquet, S., Vaquero, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+studies+of+an+antisense+protein+in+HIV-1-infected+cells+and+viral+particles.">Immunolocalization studies of an antisense protein in HIV-1-infected cells and viral particles.</a> Virology. 292 (2), 177-184 (2002).</li><li>Clerc, I., 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=Polarized+expression+of+the+membrane+ASP+protein+derived+from+HIV-1+antisense+transcription+in+T+cells.">Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells.</a> Retrovirology. 8, 74 (2011).</li><li>Landry, 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=Detection,+characterization+and+regulation+of+antisense+transcripts+in+HIV-1.">Detection, characterization and regulation of antisense transcripts in HIV-1.</a> Retrovirology. 4, 71 (2007).</li><li>Kobayashi-Ishihara, 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=HIV-1-encoded+antisense+RNA+suppresses+viral+replication+for+a+prolonged+period.">HIV-1-encoded antisense RNA suppresses viral replication for a prolonged period.</a> Retrovirology. 9, 38 (2012).</li><li>Barbagallo, M. S., Birch, K. E., Deacon, N. J., Mosse, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+control+of+human+immunodeficiency+virus+type+1+asp+expression+by+alternative+splicing+in+the+upstream+untranslated+region.">Potential control of human immunodeficiency virus type 1 asp expression by alternative splicing in the upstream untranslated region.</a> DNA Cell Biol. 31 (7), 1303-1313 (2012).</li><li>Laverdure, 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=HIV-1+Antisense+Transcription+Is+Preferentially+Activated+in+Primary+Monocyte-Derived+Cells.">HIV-1 Antisense Transcription Is Preferentially Activated in Primary Monocyte-Derived Cells.</a> Journal of Virology. 86 (24), 13785-13789 (2012).</li><li>Zapata, J. 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=The+Human+Immunodeficiency+Virus+1+ASP+RNA+promotes+viral+latency+by+recruiting+the+Polycomb+Repressor+Complex+2+and+promoting+nucleosome+assembly.">The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by recruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly.</a> Virology. 506, 34-44 (2017).</li><li>Haist, K., Ziegler, C., Botten, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strand-Specific+Quantitative+Reverse+Transcription-Polymerase+Chain+Reaction+Assay+for+Measurement+of+Arenavirus+Genomic+and+Antigenomic+RNAs.">Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs.</a> PLoS One. 10 (5), 0120043 (2015).</li><li>Lerat, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+detection+of+hepatitis+C+virus+minus+strand+RNA+in+hematopoietic+cells.">Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.</a> The Journal of Clinical Investigation. 97 (3), 845-851 (1996).</li><li>Tuiskunen, 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=Self-priming+of+reverse+transcriptase+impairs+strand-specific+detection+of+dengue+virus+RNA.">Self-priming of reverse transcriptase impairs strand-specific detection of dengue virus RNA.</a> J Gen Virol. 91, 1019-1027 (2010).</li><li>Mancarella, 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=Detection+of+antisense+protein+(ASP)+RNA+transcripts+in+individuals+infected+with+human+immunodeficiency+virus+type+1+(HIV-1).">Detection of antisense protein (ASP) RNA transcripts in individuals infected with human immunodeficiency virus type 1 (HIV-1).</a> Journal of General Virology. , (2019).</li><li>Peyrefitte, C. N., Pastorino, B., Bessaud, M., Tolou, H. J., Couissinier-Paris, P. <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+in+vitro+falsely-primed+cDNAs+that+prevent+specific+detection+of+virus+negative+strand+RNAs+in+dengue-infected+cells:+improvement+by+tagged+RT-PCR.">Evidence for in vitro falsely-primed cDNAs that prevent specific detection of virus negative strand RNAs in dengue-infected cells: improvement by tagged RT-PCR.</a> J Virol Methods. 113 (1), 19-28 (2003).</li><li>Boncristiani, H. F., Di Prisco, G., Pettis, J. S., Hamilton, M., Chen, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+approaches+to+the+analysis+of+deformed+wing+virus+replication+and+pathogenesis+in+the+honey+bee,+Apis+mellifera.">Molecular approaches to the analysis of deformed wing virus replication and pathogenesis in the honey bee, Apis mellifera.</a> Virol J. 6, 221 (2009).</li><li>Boncristiani, H. F., Rossi, R. D., Criado, M. F., Furtado, F. M., Arruda, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+purification+of+biotinylated+cDNA+removes+false+priming+and+ensures+strand-specificity+of+RT-PCR+for+enteroviral+RNAs.">Magnetic purification of biotinylated cDNA removes false priming and ensures strand-specificity of RT-PCR for enteroviral RNAs.</a> J Virol Methods. 161 (1), 147-153 (2009).</li><li>Craggs, J. K., Ball, J. K., Thomson, B. J., Irving, W. L., Grabowska, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+strand-specific+RT-PCR+based+assay+to+detect+the+replicative+form+of+hepatitis+C+virus+RNA.">Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA.</a> J Virol Methods. 94 (1-2), 111-120 (2001).</li><li>Barbeau, B., Mesnard, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+sense+out+of+antisense+transcription+in+human+T-cell+lymphotropic+viruses+(HTLVs).">Making sense out of antisense transcription in human T-cell lymphotropic viruses (HTLVs).</a> Viruses. 3 (5), 456-468 (2011).</li></ol>

在本报告中，我们描述了一种链特异性RT测定，用于检测从HIV-1感染者中分离的CD4+T细胞中的ASPRNA。RT 期间发生非特异性吸注会妨碍具有正确极性的 RNA 转录本的检测，从而导致对结果的误解。以前的研究小组已经制定了几种策略，旨在防止在RT反应期间与引物无关的cDNA合成。在3'末端标记反向引种与与HIV无关的序列已被证明能有效地实现股特异性扩增6，8，9。<sup class="xref...

反义蛋白（ASP）基因是一个开放阅读框架（ORF），位于人类免疫缺陷病毒1型（HIV-1）包络（env）基因的负链上，跨越结gp120/gp411。过去30年来，有多份报告显示，HIV ASP基因确实被转录和翻译了2，3，4，5，6，7，8，9。虽然ASP反义转录本在体外已经完全特征化，但直到最近，关于ASPRNA在患者中实际产生的信息仍然缺失。
ASP 的顺序是反向的，并且与 env 互补。这是尝试检测 ASP 脚本时的主要障碍。标准逆转录聚合酶链反应（RT-PCR）方法使用基因特异性反义引物合成正确极性的互补DNA（cDNA）。然而，这种方法不允许确定初始RNA模板的方向（感觉或反义），因为RNA发夹或环可以在没有引源10的情况下在两个方向上对RT进行素脚，这种现象称为RT自吸。大多数ASP调查员回避了RT自吸问题，使用带有与HIV-11、12无关的序列标记的引注。然而，这一战略并没有消除这一现象的发生，并可能导致非特异性cDNA可能带入PCR11。
我们最近开发了一种用于反义RNA研究的新型股特异性RT-PCR测定，并用它来检测6名艾滋病毒感染者的ASPRNA，如表1所示。下面描述的程序以前曾由安东尼奥·曼卡雷拉等人发表。在我们的协议中，我们避免通过双种方法生产非特定 cDNA。首先，我们在高温（94°C）下变性RNA来消除RNA二次结构;其次，我们使用生物素化ASP特异性底漆逆转转录ASPRNA，并亲和-纯化产生的cDNA。通过这种方法，我们能够仅扩增我们的目标cDNA，因为其他非特异性RT产品要么被阻止生成（RNA的高温变性），要么在PCR（亲和纯化）之前消除。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD LSR II</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BigDye Terminator v1.1 Cycle Sequencing Kit</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4337450</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Set (100 mM)</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>10297018</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-280 Streptavidin</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>11205D</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human CD4+ T Cell Isolation Kit</td>
			<td>Stemcell Technologies</td>
			<td>19052</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>S1010-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Solution Kit</td>
			<td>Becton Dickinson</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>HIV Gag p24 flow cytometry antibody - Kc57-FITC</td>
			<td>Beckman Coulter</td>
			<td>6604665</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-2</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-743</td>
			<td></td>
		</tr>
		<tr>
			<td>Lectin from Phaseolus vulgaris (PHA)</td>
			<td>Sigma-Aldrich</td>
			<td>61764-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Yellow Dead Cell Stain Kit, for 405 nm excitation</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>L34967</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human CD28</td>
			<td>Becton Dickinson</td>
			<td>55725</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human CD3</td>
			<td>Becton Dickinson</td>
			<td>55329</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and Probes</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>BioConcept</td>
			<td>4-01F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Taq DNA Polymerase High Fidelity</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>11304011</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene Infection / Transfection Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 Medium</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR System</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4376600</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4369016</td>
			<td></td>
		</tr>
		<tr>
			<td>TOPO TA Cloning Kit for Subcloning, with One Shot TOP10 chemically competent E. coli cells</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>K450001</td>
			<td></td>
		</tr>
		<tr>
			<td>TURBO DNase (2 U/&#181;L)</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>AM2238</td>
			<td></td>
		</tr>
		<tr>
			<td>Veriti Thermal Cycler</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4375786</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of human immunodeficiency virus type 1 (hiv-1) antisense protein (asp) rna transcripts in patients by strand-specific rt-pcr

在逆转录病毒中，在人体免疫缺陷病毒1型（HIV-1）和人类T-淋巴病毒1（HTLV-1）中都描述了反义转录。在HIV-1中，反义蛋白ASP基因位于env的负链上，在读帧-2中，跨越连接gp120/gp41。在感向上，ASP 开放读取帧的 3' 端与 gp120 超可变区域 V4 和 V5 重叠。ASP RNA的研究因一种称为RT自吸的现象而受阻，即RNA二级结构能够在没有特异性引物的情况下对RT进行质感，从而产生非特异性cDNA。在RT反应中结合高RNA变性与生物素化逆转引物，结合将cDNA亲和纯化到链球菌素涂层磁珠上，使我们能够选择性地扩增从HIV-1感染者中提取的CD4+T细胞中的ASP RNA。我们的方法成本相对较低，操作简单，可靠性高，易于重现。在这方面，它不仅在HIV-1中，而且在其他生物系统中，是研究反义转录的有力工具。

高温RNA变性与生物素化cDNA的亲亲纯化相结合，可防止在体外感染的PBMC和从患者分离的CD4+T细胞中扩增非特异性ASP产物。RT自吸已证明发生在反义RNA10，14，15，16，17的逆转录过程中。为了防止这种现象，我们开发了一种新方法，使用最初由Heist等人10描...

Watch this Scientific Journal Video about Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR at JoVE.com

Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR

在没有序列特异性引录的情况下，RNA发夹和环可作为逆转录（RT）的引种，干扰重叠反义转录本的研究。我们已经开发出一种能够识别股特异性RNA的技术，并用它来研究HIV-1反义蛋白ASP。

通过斯特兰德特异性RT-PCR检测患者人类免疫缺陷病毒类型1（HIV-1）反义蛋白（ASP）RNA转录本

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). ...

传统的RT-PCR不允许在检测由重叠基因编码的RNA时对感觉序列和反感觉序列进行区分。该协议使得可以检测特别反感觉RNA。使用生物仿制品底剂和高RNA变性温度，可以扩增抗感cDNA，防止非特异性RT产品的形成。我们用这种方法来证明，ASP无论是作为RNA抗感觉还是作为蛋白质前体，它都成为一种新的HIV抗原，它有可能成为新的HIV治疗靶点。此方法可应用于任何系统，其中可检测重叠基因中的抗感觉RNA。首先使用 PanASP 底转本内部的亲病毒 DNA 序列设计患者特定的底转。接下来，使用DNase处理样品，量化RNA并消除DNA污染。将 0.1 微克总 RNA 转移到 96 孔 PCR 板上的适当孔中。然后根据手稿指示设置反向转录或 RT 反应。通过添加五微升无核酸酶无水代替 ASP 反向底原，准备内源性 RT 控制。将板放入热循环器中，将RNA加热至94摄氏度5分钟。然后立即在冰水中冷却至少一分钟。如文本手稿所述，在 1.5 毫升管中准备反应混合物，然后将 17.5 微升转移到每个含有孔的 RNA 中。RNA变性必须在94摄氏度下进行。如果没有完全变性，可能会发生来自感觉RNA的非特异性产品的合成，导致非特异性cDNA的扩增。轻轻混合井内装物，在55摄氏度下孵育板60分钟。然后，将板加热至70摄氏度15分钟，使反应灭活。根据手稿说明准备一升 2X 洗涤和装订缓冲液，并过滤溶液。接下来，使用PCR级水准备50毫升1X洗涤和装订缓冲液。将适当数量的链球蛋白结合磁珠转移到1.5毫升管中，然后通过添加2X洗涤和结合缓冲液的等量进行洗涤，并旋转管15秒。将管子放入磁性分离机架中，并在室温下孵育三分钟。然后，小心地取出上流液，而不干扰珠子，并添加与珠子初始体积相同的2X洗涤和装订缓冲液的体积。旋转珠子30秒，将悬浮液的10微升转移到适当数量的1.5毫升管。将管子放在磁性分离架中，并在室温下孵育三分钟。然后，丢弃上流液，并添加50微升的2X洗涤和装订缓冲液。将50微升生物仿生cDNA加入相应的管子中，用珠子孵育30分钟，轻轻旋转。孵育后，将管子放在磁性分离架上三分钟，然后用200微升1X洗涤和装订缓冲液清洗珠子，然后在分离架上孵育三分钟。丢弃上一杯，重复洗涤两次。然后，将珠子重新在PCR级水的10微升中重新浇水。根据手稿说明准备适当的 PCR 主混合卷。混合好，并迅速旋转下来，然后转移49微升每井到96井PCR板。用纯化的cDNA小心地旋转管15秒，并将悬浮液的微升添加到相应的孔中。运行 PCR 并在 1%的加糖凝胶上分离产品。从凝胶中切割带，将放大的产品克隆成PCR 2.1质粒。然后对克隆进行测序并分析每个序列。初始RT反应使用常规非生物化抗感觉底原，成功扩增了SATP。然而，相同分子量和底数减去对联的波段也被放大。为了绕过这个问题，特定的抗感觉底像被标记为生物tyn，由此产生的抗感觉cDNA在PCR之前被净化。这使得扩大 ASP 序列成为可能，从而大大减少了底流减控制中非特异性 cDNA 的污染。该方法在RT前在94摄氏度下完全变性，随后在冰水中立即冷却，从而有效放大了SAMP波段，没有非特异性产品，从而实现了该方法的进一步优化。测量了 ASP RNA 的动力学，从三个HIV阳性受试者中分离出CD4细胞，这些受试者具有可检测的病毒血症，并且在用抗CD3/CD28刺激后没有治疗。在接受治疗治疗的患者中，ASP RNA的定量也是可能的，具有检测性极小。在三分之二的患者中，ASP RNA 在刺激后三到五天的低水平被检测出来。对两名患者进行了 ASP 和 env RNA 分析，一名未经治疗，一名治疗。对于未经治疗的患者，可以检测到两个RNA。对于治疗的患者，ASP 和 env 在经过几天的刺激后几乎无法检测到。在尝试此程序时，重要的是要记住变性使RNA线性化，以便能够以RT为底的二级结构被破坏。然而，洗涤珠子会冲走非生物化的cDNA。在用正确的极性纯化cDNA后，可以执行qPCR来分析基因表达。此外，也可以克隆cDNA并用于序列分析。这种方法允许研究基因重叠在相反的方向，并可能可用于阐明细菌和病毒相关疾病，包括某些类型的癌症的调控和发病机制。

Research

JoVE Journal

Genetics

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

使用BrUTP链特异性转录运行的（TRO）方法终止转录分析

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

科研

遗传学

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

人端粒酶逆转录蛋白 rna 依赖性 rna 聚合酶活性的半定量检测

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

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 ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

hiv-1 反向转录过程中鼻部单链病毒 dna 分子的 3 '-特米尼和序列的测定

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

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 ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

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

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