Name: an in vitro protocol for evaluating microrna levels, functions, and associated target genes in tumor cells
Uploaded: 2019-05-21T14:00:00
Description: Watch this Scientific Journal Video about An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells at JoVE.com

这项研究得到了韩国国家科学研究基金会(NRF)基础科学研究计划的支持,该基金会由韩国教育部资助(2017R1D1A3B03035662);和Hallym大学研究基金,2017年(HRF-201703-003)。

1. 成熟微RNA (miRNA) 表达分析
<ol><li>
成熟 miRNA 补充 DNA (cDNA) 合成
	<ol>
<li>加入总RNA的254纳克和4.5μL的脱氧核糖核酸I(DNase I)混合物,然后将超纯水加入PCR带管,以补制成高达18μL(图2A)。根据反应总数,使用足够量的DNase I混合物,制备从多个细胞系纯化的每个总RNA样品的反应。 注:DNase I混合物由DNase I(1.8 μL)、核糖酸酶抑制剂(0.3 μL)和25 mM MgCl 2(2.4 ?...

<ol>
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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=Epigenetic+silencing+of+MicroRNA+miR-107+regulates+cyclin-dependent+kinase+6+expression+in+pancreatic+cancer.">Epigenetic silencing of MicroRNA miR-107 regulates cyclin-dependent kinase 6 expression in pancreatic cancer.</a> Pancreatology. 9 (3), 293-301 (2009).</li><li>van Tonder, A., Joubert, A. M., Cromarty, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+the+3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium+bromide+(MTT)+assay+when+compared+to+three+commonly+used+cell+enumeration+assays.">Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays.</a> BMC Research Notes. 8, 47 (2015).</li><li>Wang, P., Henning, S. M., Heber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+MTT+and+MTS-based+assays+for+measurement+of+antiproliferative+activity+of+green+tea+polyphenols.">Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols.</a> PloS One. 5 (4), e10202 (2010).</li><li>Wu, L., Belasco, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Let+me+count+the+ways:+mechanisms+of+gene+regulation+by+miRNAs+and+siRNAs.">Let me count the ways: mechanisms of gene regulation by miRNAs and siRNAs.</a> Molecular Cell. 29 (1), 1-7 (2008).</li><li>Jin, Y., Chen, Z., Liu, X., Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+microRNA+targeting+sites+by+luciferase+reporter+gene+assay.">Evaluating the microRNA targeting sites by luciferase reporter gene assay.</a> Methods in Molecular Biology. , 117-127 (2013).</li><li>Ma, Z., 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=Gamma-synuclein+binds+to+AKT+and+promotes+cancer+cell+survival+and+proliferation.">Gamma-synuclein binds to AKT and promotes cancer cell survival and proliferation.</a> Tumour Biology. 37 (11), 14999-15005 (2016).</li><li>Pan, Z. Z., Bruening, W., Giasson, B. I., Lee, V. M., Godwin, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-synuclein+promotes+cancer+cell+survival+and+inhibits+stress-+and+chemotherapy+drug-induced+apoptosis+by+modulating+MAPK+pathways.">Gamma-synuclein promotes cancer cell survival and inhibits stress- and chemotherapy drug-induced apoptosis by modulating MAPK pathways.</a> Journal of Biological Chemistry. 277 (38), 35050-35060 (2002).</li><li>Martinez-Sanchez, A., Murphy, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+Target+Identification-Experimental+Approaches.">MicroRNA Target Identification-Experimental Approaches.</a> Biology (Basel). 2 (1), 189-205 (2013).</li><li>Lee, E. 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确定具有目标miRNA功能的真正miRNA靶点的策略对于理解miRNA的多重作用是必不可少的。miRNA靶基因的识别可以作为解释由miRNA在细胞中调节的细胞信号事件的指南。揭示miRNA的功能重要的靶基因可以为开发基于miRNA的癌症治疗提供基本知识。
微阵列、小RNA库测序、深度测序、原位PCR逆转录酶和北方印迹等多种方法,利用从细胞系和组织中分离出的总RNA,可用于探索m...

微RNA(miRNA)是一种小型调节RNA,主要通过对真实靶基因1中的3'未翻译区域(3'UTR)做出反应来调节信使RNA(mRNA)的转换和降解过程。miRNA的表达可以通过转录和转录后机制调节。这种调节机制的不平衡导致包括癌症在内的众多疾病中不受控制和独特的miRNA表达水平。单个 miRNA 可以与不同的 mRNA 进行多种交互。相应地,单个 mRNA 可以由各种 miRNA 控制。因此,细胞内信号网络受到明显表达的miRNA的复杂影响,通过这种影响,生理疾病和疾病可以启动和恶化2,3,45,6.虽然在各种癌症中观察到了miRNA的改变表达,但调节癌细胞与miRNA一起进行的分子机制在很大程度上仍不为人所知。
累积的证据表明,miRNA的致癌或肿瘤抑制作用取决于癌症的类型。例如,通过瞄准叉头盒o3(FOXO3),miR-155促进结肠直肠癌7、8的细胞增殖、转移和化学抗药性。相比之下,胶质瘤细胞入侵的限制性是由miR-107通过神经原位位同源蛋白2(NOTCH2)表达9的调节引起的。评估miRNA-目标相互作用与miRNA功能是一个不可或缺的部分,更好地了解miRNA如何调节各种生物过程在健康和疾病状态10。此外,miRNA真正靶点的发现,可以进一步为基于miRNA的抗癌药物治疗提供微调的策略。然而,miRNA领域的主要挑战是确定miRNA的直接目标。在这里,详细的方法作为可重复的实验方法,用于miRNA靶基因的测定。miRNA目标鉴定的成功实验设计涉及各种步骤和注意事项(图1)。比较肿瘤细胞和正常细胞中的成熟miRNA水平是选择感兴趣的miRNA的常见程序之一(图1A)。对选定的miRNA进行功能研究,以检测miRNA对细胞增殖的影响,对于缩小感兴趣的miRNA最佳潜在候选靶点列表非常重要(图1B)。基于miRNA的实验验证功能,需要用miRNA靶向预测程序对公司文献和数据库进行系统审查,以搜索与基因功能最相关的信息(图1C)。通过实施荧光素酶测定以及基因3'UTR的克隆、实时PCR和西方印迹(图1D),可以识别感兴趣的miRNA的真实目标基因。当前协议的目标是提供关键实验、基于探针的实时聚合酶链反应 (PCR)、磺胺 B (SRB) 分析后 miRNA 模拟转染、剂量-反应曲线生成以及荧光酶测定以及基因3'UTR的克隆。目前的协议对于更好地了解单个 miRNA 的功能以及 miRNA 在癌症治疗中的含义非常有用。

<table>
	<tbody>
		<tr>
			<td>15 mL conical tube</td>
			<td>SPL Life Sciences</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>SPL Life Sciences</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>6X DNA loading dye</td>
			<td>Real Biotech Corporation</td>
			<td>RD006</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>8-cap strip</td>
			<td>Applied Biosystems</td>
			<td>N8010535</td>
			<td>For cDNA synthesis</td>
		</tr>
		<tr>
			<td>8-tube strip</td>
			<td>Applied Biosystems</td>
			<td>N8010580</td>
			<td>For cDNA synthesis</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma</td>
			<td>A6283-1L</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Agarose A</td>
			<td>Bio Basic</td>
			<td>D0012</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase</td>
			<td>New England Biolabs</td>
			<td>M0290S</td>
			<td>10,000 U/mL</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Bio basic Canada Inc</td>
			<td>AB0028</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>AriaMx 96 tube strips</td>
			<td>Agilent Technologies</td>
			<td>401493</td>
			<td>For real time PCR</td>
		</tr>
		<tr>
			<td>AriaMx real-time PCR system</td>
			<td>Agilent Technologies</td>
			<td>G8830A</td>
			<td>qPCR amplification, detection, and data analysis</td>
		</tr>
		<tr>
			<td>AsiSI</td>
			<td>New England Biolabs</td>
			<td>R0630</td>
			<td>10,000 units/mL</td>
		</tr>
		<tr>
			<td>CAPAN-1 cells</td>
			<td>ATCC</td>
			<td>HTB-79</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture hood</td>
			<td>Labtech</td>
			<td></td>
			<td>Model: LCB-1203B-A2</td>
		</tr>
		<tr>
			<td>Counting chambers with V-slash</td>
			<td>Paul Marienfeld</td>
			<td>650010</td>
			<td>Cells counter</td>
		</tr>
		<tr>
			<td>CutSmart buffer</td>
			<td>New England Biolabs</td>
			<td>B7204S</td>
			<td>10X concentration</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>DNA gel extraction kit</td>
			<td>Bionics</td>
			<td>DN30200</td>
			<td>200 prep</td>
		</tr>
		<tr>
			<td>DNA ladder</td>
			<td>NIPPON Genetics EUROPE</td>
			<td>MWD1</td>
			<td>1 Kb ladder</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Invitrogen</td>
			<td>18068015</td>
			<td>100 units</td>
		</tr>
		<tr>
			<td>Dual-luciferase reporter assay system</td>
			<td>Promega</td>
			<td>E1910</td>
			<td>100 assays</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>HIT competent cells</td>
			<td>Real Biotech Corporation(RBC)</td>
			<td>RH617</td>
			<td>Competent cells</td>
		</tr>
		<tr>
			<td>HPNE cells</td>
			<td>ATCC</td>
			<td>CRL-4023</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar broth</td>
			<td>Bio Basic</td>
			<td>SD7003</td>
			<td>250 g</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td>0.75 mL</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778-075</td>
			<td>0.75 mL</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td></td>
			<td>Model: E5311</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>TECAN</td>
			<td></td>
			<td>Infinite F50</td>
		</tr>
		<tr>
			<td>miRNA control mimic</td>
			<td>Ambion</td>
			<td>4464058</td>
			<td>5 nmole</td>
		</tr>
		<tr>
			<td>miRNA-107 mimic</td>
			<td>Ambion</td>
			<td>4464066</td>
			<td>5 nmole</td>
		</tr>
		<tr>
			<td>miRNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>50 prep</td>
		</tr>
		<tr>
			<td>Mupid-2plus (electrophoresis system)</td>
			<td>TaKaRa</td>
			<td></td>
			<td>Model: AD110</td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>New England Biolabs</td>
			<td>R3189</td>
			<td>20,000 units/mL</td>
		</tr>
		<tr>
			<td>Oligo explorer program</td>
			<td>GeneLink</td>
			<td></td>
			<td>For primer design</td>
		</tr>
		<tr>
			<td>Optical tube strip caps (8X Strip)</td>
			<td>Agilent Technologies</td>
			<td>401425</td>
			<td>For real time PCR</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td>500 Ml</td>
		</tr>
		<tr>
			<td>PANC-1 cells</td>
			<td>ATCC</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Gibco</td>
			<td>14040117</td>
			<td>1000 mL</td>
		</tr>
		<tr>
			<td>Plasmid DNA miniprep S&#38; V kit</td>
			<td>Bionics</td>
			<td>DN10200</td>
			<td>200 prep</td>
		</tr>
		<tr>
			<td>PrimeSTAR GXL DNA polymerase</td>
			<td>TaKaRa</td>
			<td>R050A</td>
			<td>250 units</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>TECAN</td>
			<td></td>
			<td>Shaking platform</td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Labtech</td>
			<td></td>
			<td>Model: LSI-3016A</td>
		</tr>
		<tr>
			<td>Sigmaplot 14 software</td>
			<td>Systat Software Inc</td>
			<td></td>
			<td>For dose-response curve generation</td>
		</tr>
		<tr>
			<td>Sulforhodamine B powder</td>
			<td>Sigma</td>
			<td>S1402-5G</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>SYBR green master mix</td>
			<td>Smobio</td>
			<td>TQ12001805401-3</td>
			<td>Binding fluorescent dye for dsDNA</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>TaKaRa</td>
			<td>2011A</td>
			<td>25,000 U</td>
		</tr>
		<tr>
			<td>TaqMan master mix</td>
			<td>Applied Biosystems</td>
			<td>4324018</td>
			<td>200 reactions, no AmpErase UNG</td>
		</tr>
		<tr>
			<td>TaqMan microRNA assay (hsa-miR-107)</td>
			<td>Applied Biosystems</td>
			<td>4427975</td>
			<td>Assay ID: 000443 (50RT, 150 PCR rxns)</td>
		</tr>
		<tr>
			<td>TaqMan microRNA assay (hsa-miR-301)</td>
			<td>Applied Biosystems</td>
			<td>4427975</td>
			<td>Assay ID: 000528 (50RT, 150 PCR rxns)</td>
		</tr>
		<tr>
			<td>TaqMan miR RT kit</td>
			<td>Applied Biosystems</td>
			<td>4366597</td>
			<td>1000 reactions</td>
		</tr>
		<tr>
			<td>Thermo CO2 incubator (BB15)</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>37 &#176;C and 5% CO2 incubation</td>
		</tr>
		<tr>
			<td>Trichloroacetic acid</td>
			<td>Sigma</td>
			<td>91228-100G</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T4661-100G</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Veriti 96 well thermal cycler</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>For amplification of DNA (or cDNA)</td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>New England Biolabs</td>
			<td>R0146</td>
			<td>20,000 units/mL</td>
		</tr>
	</tbody>
</table>

an in vitro protocol for evaluating microrna levels, functions, and associated target genes in tumor cells

微RNA (miRNA) 是小型调节RNA,可识别调节包括癌症在内的多种疾病中的许多细胞内信号通路。这些小型调节RNA主要与其目标信使RNA(mRNA)的3'未翻译区域(3'UTR)相互作用,最终导致mRNA解码过程的抑制和目标mRNA降解的增强。基于表达水平和细胞内功能,miRNA能够作为致癌和肿瘤抑制性mRNA的调节因子。在数百甚至数千个计算预测目标中识别 miRNA 的真正目标基因是识别感兴趣的 miRNA 的作用和基本分子机制的关键步骤。各种miRNA目标预测程序可用于搜索可能的miRNA-mRNA相互作用。然而,最具挑战性的问题是如何验证感兴趣的miRNA的直接靶基因。该协议描述了如何识别与miRNA功能相关的miRNA靶点的关键方法的可重复策略。该协议提供了一个实用的指南,用于使用基于探针的实时聚合酶链反应(PCR)、磺胺B(SRB)测定,在miRNA模拟转染后发现miRNA水平、功能和相关目标mRNA。,剂量反应曲线生成,和荧光素酶测定以及基因的3'UTR克隆,这是正确理解单个miRNA的作用所必需的。

成功和准确地确认miRNA水平对于解释基于miRNA在疾病发展和进展中预期作用的miRNA分类的数据非常重要。使用基于探针的定量PCR在三个胰腺细胞系中测量了miRNA-107和miRNA-301的水平。在相同反应中合成特定miRNA和参考基因的cDNA可以提高数据的可重复性。PANC-1和CAPAN-1是人类胰腺导管腺癌细胞系,而HPNE是一种不朽的胰腺导管细胞系,通过携带人类端粒酶逆转录酶(hTERT)基因的逆转录病毒表达载体进行转导。?...

Watch this Scientific Journal Video about An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells at JoVE.com

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

该协议使用基于探针的实时聚合酶链反应(PCR)、磺胺胺B(SRB)测定、3'未翻译区域(3'UTR)克隆和荧光素酶测定来验证感兴趣的miRNA的目标基因,并了解miRNA的功能。

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

获得神经微RNA目标与微RNA功能的相互作用对于了解微RNA如何调节健康状态和患病状态的各种生物过程至关重要。该协议描述了通过策略识别微RNA靶点和功能性微RNA的复制，因为微RNA直接目标的验证具有挑战性。我们的协议可以提供关于单个微RNA的功能和微RNA在癌症治疗中的含义的见解。计算半最大抑制浓度是一种重要的方法，不仅对于微RNA研究，而且对于其他抗癌药物的疗效评价。我们的协议将有助于新的研究人员，因为我们描述了了解细胞中微RNA水平的基本方法。要开始协议，请用单元格计数器计数单元格。将细胞板放在96井板中。第二天，准备一组转染混合物，在微RNA对照模拟的几种最终浓度中转染细胞，并模拟微RNA-107。从库存的25微摩尔微RNA控制模仿，或微RNA-107模仿，稀释和添加控制模仿或微RNA-107模仿在减少的血清介质以及微培养管中的转染试剂。使用微管轻轻混合含寡分的混合物。在细胞培养罩中孵育10分钟后，再次轻轻混合含寡糖的混合物，然后将50微升混合物加入到每个井中。将转染细胞留在细胞培养箱中。小心地吸进板的每个井中的细胞培养基，并及时将100微升10%的三氯乙酸（TCA）填充到每个井中。将含有 10%TCA 的板在 4 摄氏度下保持一小时。接下来，将盘子淹没在一个带自来水的浴缸里，洗几次。通过敲击板，直到没有水，从井内去除多余的水，然后擦干板。将 50 微升 0.4% SRB 溶液移入每口井，包括空井。轻轻摇动板，直到 0.4% SRB 溶液均匀地覆盖井底。然后，孵育板40至60分钟。孵育后，用1%醋酸清洗板，直到未绑定染料被完全冲走。移液100微升10毫摩尔三口基溶液进入相应的井，包括空白井。将盘子放在摇床上 10 分钟。测量 492 纳米的吸光度。使用 SRB 测定的吸收度值计算每个组的平均吸收率百分比和标准偏差。通过垂直对齐数据，将原始数据（包括处理浓度、平均吸收度和标准偏差）导入软件。单击"创建图形"选项卡并选择"简单散点错误栏"。选择工作表列作为符号值，然后单击"下一步"。在"数据格式"面板中，选择 X-Y 对并单击下一步。在"选择数据"面板中选择相应的数据列。单击"完成"以创建绘图。双击 X 轴可修改轴缩放中的缩放类型。将比例类型从线性更改为日志。将开始和结束范围编号分别修改为 0.01 和 200。右键单击任何散点图，选择"曲线拟合"，然后转到已定义的子类别用户。选择剂量响应曲线。单击下一个按钮，然后单击"完成"。转到报表选项卡，然后检查 n、k 和 R 值。运行 PCR，详见随附的文本协议中。然后通过组合反应混合物，包括限制酶Exo1和 Not1在管中加入双消化。在37摄氏度的水浴中孵育混合物3至4小时。在 1%的加糖凝胶上运行双消化产品。然后在紫外线下切割带子。从切除频段中净化双消化的PCR产品和荧光素向量。通过制备20微升的结扎反应混合物（包括DNA连接酶），继续将PCR产品与荧光素酶载体连接。将管子短暂离心10至15秒。使用热循环器在16摄氏度下孵育结扎过夜。第二天，通过将结扎混合物加入含有称职细胞的管中进行转化。轻轻敲按管子，在冰上保持20分钟。快速、轻轻地将管转移到 42 摄氏度的热块中，30 秒到 1 分钟。热冲击后，将管子放在冰上20分钟。将称职的细胞铺在 LB 阿加板上。在37摄氏度的培养箱中一夜之间生长称职的细胞。第二天，在含有超纯水的8条管中挑选一个个体菌落并重新分配大肠杆菌，然后重复这一步骤，将大肠杆菌从4个随机选择的菌落重新暂停到8个。将 25 微升大肠杆菌悬浮液转移到另一组 8 条管中。使用大肠杆菌悬浮液执行菌落 PCR。在24井板中准备荧光素酶测定。在每一个井的500微升细胞培养培养的四个细胞中，将1到2倍的10倍。经过一夜的孵育，将50纳米的荧光素酶载体转染到细胞中，使用转染试剂进行对照模仿或特定的微RNA模拟。第二天，用磷酸盐缓冲盐水洗两次井内。在测量荧光素酶活性之前，将200微升的解解试剂放入井中，并充分进行细胞解解。保持板抖至少 15 分钟。将5至10微升的细胞解酶转移到新管中，并加入100微升试剂，然后通过移液立即混合溶液。然后使用光照仪读取萤火虫荧光素酶活性 10 至 15 秒。在同一管中加入100微升试剂，然后通过移液混合两次。最后，使用光照仪读取 10 到 15 秒的 renilla 荧光素酶活性。RTPCR显示，与HPNE细胞相比，微RNA-107、PANC-1和CAPAN-1细胞显著减少。与HPNE细胞相比，PANC-1和CAPAN-1细胞的微RNA-301水平显著上升。这项研究中的SRB测定清楚地表明，在微RNA-107模拟转染后，PANC-1细胞的增殖减少。对微RNA-107的11个潜在预测靶点进行筛选，清楚地表明，只有突核素伽马（SNCG）是肿瘤细胞生长的正调节剂，直接与微RNA-107和PANC-1细胞相互作用，表明微RNA-107可以通过调节SNCG表达对PANC-1细胞的增殖产生负性。使用四氢基实体和 CFSE 的成人细胞增殖检测适用于筛选微RNA模拟等效果，具体取决于细胞类型。基于微RNA的实验验证功能，需要对数据库和目标预测程序进行系统审查，以缩小荧光素分析前候选目标列表。对于微RNA与抗癌药物的结合效率的评价，根据组合指数评价方案计算调整后的IC值是有利的。

Research

JoVE Journal

Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

科研

遗传学

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

重组腺相关病毒9型 MicroRNA 系统分娩治疗啮齿动物神经肌肉疾病

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

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 ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...