Name: lung microrna profiling across the estrous cycle in ozone-exposed mice
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice at JoVE.com

这项研究得到了 nih k01hl133520 (ps) 和 k12hd055882 (ps) 的资助。作者感谢乔安娜·弗洛罗斯博士在臭氧暴露实验方面的帮助。

这里描述的所有方法都得到了宾州立大学动物护理和使用机构委员会 (iacuc) 的批准。
1. 对激烈循环阶段的评估
<ol><li>使用 machholz 等人描述的单手小鼠约束技术, 适当约束雌性 c57bl6 小鼠 (8-9周大).</li>
	<li>用10μl 的超纯水填充无菌塑料移液器。</li>
	<li>将塑料移液器的尖端引入阴道。</li>
	<li>轻轻冲洗液体4-5 以收集样品。</li>
	<li>将含有阴道液的最后冲洗放在...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+as+biomarkers+for+inflammatory+diseases.">Circulating microRNAs as biomarkers for inflammatory diseases.</a> Microrna. 2 (1), 63-71 (2013).</li><li>Sessa, R., Hata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+microRNAs+in+lung+development+and+pulmonary+diseases.">Role of microRNAs in lung development and pulmonary diseases.</a> Pulmonary Circulation. 3 (2), 315-328 (2013).</li><li>Fuentes, N., Roy, A., Mishra, V., Cabello, N., Silveyra, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-specific+microRNA+expression+networks+in+an+acute+mouse+model+of+ozone-induced+lung+inflammation.">Sex-specific microRNA expression networks in an acute mouse model of ozone-induced lung inflammation.</a> Biology of Sex Differences. 9 (1), 18 (2018).</li><li>Aris, R. 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=Ozone-induced+airway+inflammation+in+human+subjects+as+determined+by+airway+lavage+and+biopsy.">Ozone-induced airway inflammation in human subjects as determined by airway lavage and biopsy.</a> American Review of Respiratory Disease. 148 (5), 1363-1372 (1993).</li><li>Ritchie, M. E., 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=limma+powers+differential+expression+analyses+for+RNA-sequencing+and+microarray+studies.">limma powers differential expression analyses for RNA-sequencing and microarray studies.</a> Nucleic Acids Research. 43 (7), e47 (2015).</li><li>Kr&#228;mer, A., Green, J., Pollard, J., Tugendreich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causal+analysis+approaches+in+Ingenuity+Pathway+Analysis.">Causal analysis approaches in Ingenuity Pathway Analysis.</a> Bioinformatics. 30 (4), 523-530 (2014).</li><li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+restraint+and+common+compound+administration+routes+in+mice+and+rats.">Manual restraint and common compound administration routes in mice and rats.</a> Journal of Visualized Experiments. (67), (2012).</li><li>Umstead, T. M., Phelps, D. S., Wang, G., Floros, J., Tarkington, B. K. <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+exposure+of+proteins+to+ozone.">In vitro exposure of proteins to ozone.</a> Toxicology Mechanisms and Methods. 12 (1), 1-16 (2002).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Phipson, B., Lee, S., Majewski, I. J., Alexander, W. S., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+hyperparameter+estimation+protects+against+hypervariable+genes+and+improves+power+to+detect+differential+expression.">Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression.</a> Annals of Applied Statistics. 10 (2), 946-963 (2016).</li><li>Smyth, G. K., 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=Linear+Models+for+Microarray+and+RNA-Seq+Data+User's+Guide.">Linear Models for Microarray and RNA-Seq Data User's Guide.</a> Bioconductor. , (2002).</li><li>Benjamini, Y., Hochberg, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+False+Discovery+Rate:+A+Practical+and+Powerful+Approach+to+Multiple+Testing.">Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing.</a> Journal of the Royal Statistical Society. Series B (Methodological). 57 (1), 289-300 (1995).</li><li>Byers, S. L., Wiles, M. V., Dunn, S. L., Taft, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+estrous+cycle+identification+tool+and+images.">Mouse estrous cycle identification tool and images.</a> Public Library of Science ONE. 7 (4), e35538 (2012).</li><li>Alves, M. 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=Comparison+of+RNA+Extraction+Methods+for+Molecular+Analysis+of+Oral+Cytology.">Comparison of RNA Extraction Methods for Molecular Analysis of Oral Cytology.</a> Acta Stomatologica Croatica. 50 (2), 108-115 (2016).</li><li>Wilfinger, W. W., Mackey, K., Chomczynski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+and+ionic+strength+on+the+spectrophotometric+assessment+of+nucleic+acid+purity.">Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity.</a> Biotechniques. 22 (3), 478-481 (1997).</li><li>Bustin, S. 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=The+MIQE+guidelines:+minimum+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.</a> Clinical Chemistry. 55 (4), 611-622 (2009).</li><li>Walker, S. E., Lorsch, 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+purification-+precipitation+methods.">RNA purification- precipitation methods.</a> Methods in Enzymology. 530, 337-343 (2013).</li><li>Git, 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=Systematic+comparison+of+microarray+profiling,+real-time+PCR,+and+next-generation+sequencing+technologies+for+measuring+differential+microRNA+expression.">Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression.</a> RNA. 16 (5), 991-1006 (2010).</li><li>Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limma:+linear+models+for+microarray+data.">Limma: linear models for microarray data.</a> Bioinformatics and Computational Biology Solutions Using R and Bioconductor. , 397-420 (2005).</li><li>Griffiths-Jones, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+the+microRNA+sequence+database.">miRBase: the microRNA sequence database.</a> Methods Mol Biol. 342, 129-138 (2006).</li><li>Sethupathy, P., Corda, B., Hatzigeorgiou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TarBase:+A+comprehensive+database+of+experimentally+supported+animal+microRNA+targets.">TarBase: A comprehensive database of experimentally supported animal microRNA targets.</a> RNA. 12 (2), 192-197 (2006).</li><li>Agarwal, V., Bell, G. W., Nam, J., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+effective+microRNA+target+sites+in+mammalian+mRNAs.">Predicting effective microRNA target sites in mammalian mRNAs.</a> eLife. 4, e05005 (2015).</li><li>Xiao, F., Zuo, Z., Cai, G., Kang, S., Gao, X., Li, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRecords:+an+integrated+resource+for+microRNA-target+interactions.">miRecords: an integrated resource for microRNA-target interactions.</a> Nucleic Acids Res. 37, D105-D110 (2009).</li><li>Mullany, L. E., Wolff, R. K., Slattery, M. 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微 rna 分析是疾病诊断和机械研究的有利技术。在这篇手稿中, 我们定义了一个评估 mirna 表达的协议, 预测它将调节在不同发情周期阶段暴露在臭氧下的雌性小鼠肺部的炎症基因。介绍了对强度周期的测定方法, 如视觉检测方法, 共16种.但是, 这些数据依赖于一次性测量, 因此是不可靠的。为了准确地识别女性定期周期中的所有有缺陷的周期阶段, 建议采用此处描述的方法。此外, 这...

微 rna (mirna) 是短的 (19 到25个核苷酸), 自然存在的, 非编码 rna 分子。mirna 的序列在物种间是进化守变的, 这表明 mirna 在调节生理功能方面的重要性1。微 rna 表达谱已被证明有助于识别在调节各种过程中非常重要的 mirna, 包括免疫反应、细胞分化、发育过程和凋亡2。最近, mirna 因其在疾病诊断和治疗中的潜在用途而被认为是公认的。对于研究基因调控机制的研究人员来说, 测量 mirna 表达可以启发系统级调控过程模型, 特别是当 mirna 信息与 mrna 特征分析和其他基因组规模数据合并时3。另一方面, mirna 在一系列标本类型中也被证明比 mirna 更稳定, 并且也比蛋白质4具有更大的灵敏度。这引起了人们对 mirna 作为包括肺部疾病在内的各种分子诊断应用的生物标志物的开发的极大兴趣。
在肺中, mirna 在发育过程中起着重要作用, 维持稳态。此外, 它们的异常表达与各种肺病的发展和进展有关5。空气污染引起的炎症性肺病在女性中表现出更大的严重程度和更差的预后, 这表明激素和雌激素循环可以调节肺先天免疫和 mirna 的表达, 以应对环境挑战6. 在本议定书中, 我们利用臭氧接触----空气污染的一个主要组成部分----在没有适应性免疫力的情况下, 在雌性小鼠中诱发某种形式的肺部炎症。通过使用臭氧, 我们正在诱导气道高反应性的发展, 这与气道上皮细胞损伤和增加中性粒细胞和炎症介质在近端气道 7.目前, 还没有描述良好的协议来描述和分析整个有光泽的周期的 mirna 暴露在臭氧暴露的小鼠。
下面, 我们描述了一个简单的方法来识别有偏离周期阶段和 mirna 表达的女性小鼠的肺组织暴露臭氧。我们还讨论了 mirna 发现和目标识别的有效生物信息学方法, 重点是计算生物学。我们使用limma分析微阵列数据, limma 是一个 r/bicom导体软件, 它为分析基因表达实验8中的数据提供了一个集成的解决方案.在使用少量 arrays2 样本比较表达时, 对limma pcr 阵列数据的分析在功率优于基于 t 检验的程序方面具有优势。为了理解 mirna 表达结果的生物学背景, 我们使用了功能分析软件。为了了解调节转录变化的机制并预测可能的结果, 该软件结合了 mirna 表达集和文献9中的知识。与只在与 mirna 集合重叠的情况下寻求统计丰富的软件相比, 这是一个优势。

<table>
	<tbody>
		<tr>
			<td>C57BL/6J mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td>8 weeks old</td>
		</tr>
		<tr>
			<td>UltraPure Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>10813012</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-25</td>
			<td>Capacity: 1.7mL</td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>2951TS</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Microscope World</td>
			<td>MW3-H5</td>
			<td>10X and 20X objective</td>
		</tr>
		<tr>
			<td>Ketathesia- Ketamine HCl Injection USP</td>
			<td>Henry Schein Animal Health</td>
			<td>55853</td>
			<td>90 mg/kg. Controlled drug.</td>
		</tr>
		<tr>
			<td>Xylazine Sterile Solution</td>
			<td>Lloyd Laboratories</td>
			<td>139-236</td>
			<td>10mg/kg. Controlled Drug.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td>Dilute to 70% ethanol with water.</td>
		</tr>
		<tr>
			<td>21G gauge needle</td>
			<td>BD Biosciences</td>
			<td>305165</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Fisher Scientific</td>
			<td>329654</td>
			<td>1mL</td>
		</tr>
		<tr>
			<td>Operating Scissors</td>
			<td>World Precision Instruments</td>
			<td>501221, 504613</td>
			<td>14cm, Sharp/Blunt, Curved and 9 cm, Straight, Fine Sharp Tip</td>
		</tr>
		<tr>
			<td>Tweezer Kit</td>
			<td>World Precision Instruments</td>
			<td>504616</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#730;C freezer</td>
			<td>Forma</td>
			<td>7240</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrum&#160;Bessman Tissue Pulverizers</td>
			<td>Fisher Scientific</td>
			<td>08-418-1</td>
			<td>Capacity: 10 to 50mg</td>
		</tr>
		<tr>
			<td>RNase-free Microfuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM12400</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct-zol RNA MiniPrep Plus</td>
			<td>Zymo Research</td>
			<td>R2071</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript II RT kit</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Inflammatory Response &#38; Autoimmunity miRNA PCR Array</td>
			<td>Qiagen</td>
			<td>MIMM-105Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin-walled, DNase-free, RNase-free PCR tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM12225</td>
			<td>for 20 &#956;l reactions</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in Control</td>
			<td>Qiagen</td>
			<td>219610</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td>https://office.microsoft.com/excel/</td>
		</tr>
		<tr>
			<td>Ingenuity Pathway Analysis</td>
			<td>Qiagen</td>
			<td></td>
			<td>https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/</td>
		</tr>
		<tr>
			<td>R Software</td>
			<td>The R Foundation</td>
			<td></td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>Thermal cycler or chilling/heating block</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time PCR cycler</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipettor</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNA wash buffer</td>
			<td>Zymo Research</td>
			<td>R1003-3-48</td>
			<td>48 mL</td>
		</tr>
		<tr>
			<td>DNA digestion buffer</td>
			<td>Zymo Research</td>
			<td>E1010-1-4</td>
			<td>4 mL</td>
		</tr>
		<tr>
			<td>RNA pre-wash buffer</td>
			<td>Zymo Research</td>
			<td>R1020-2-25</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Ultraviolet ozone analyzer</td>
			<td>Teledyne API</td>
			<td>Model T400</td>
			<td>http://www.teledyne-api.com/products/oxygen-compound-instruments/t400</td>
		</tr>
		<tr>
			<td>Mass flow controllers</td>
			<td>Sierra Instruments Inc</td>
			<td>Flobox 951/954</td>
			<td>http://www.sierrainstruments.com/products/954p.html</td>
		</tr>
	</tbody>
</table>

lung microrna profiling across the estrous cycle in ozone-exposed mice

微 rna (mirna) 分析已成为研究人员对生物学和医学研究领域的兴趣。目前的研究表明, 在肺部疾病的诊断和护理中使用 mirna 是一个有希望的前景。在这里, 我们定义了一个 mirna 分析的协议, 以测量一组 mirna 的相对丰度, 预测从一个臭氧引起的气道炎症小鼠模型调节肺组织中的炎症基因。因为已经证明, 循环性激素水平会影响女性肺先天免疫的调节, 这种方法的目的是描述在雌性小鼠体内的炎症性 mirna 特征分析协议, 同时考虑到发情周期在臭氧暴露时, 每种动物的阶段。我们还讨论了适用于 mirna 发现的生物信息学方法, 并使用limma、r/bit导体软件和功能分析软件进行目标识别, 以了解与之相关的生物背景和途径。差异 mirna 表达。

在涂片中观察到的不同细胞类型用于识别小鼠的有嗅觉周期阶段 (图 1)。这些都是通过细胞形态来识别的。在前体过程中, 细胞几乎完全是圆形、形成良好的有核上皮细胞 (图 1a)。当小鼠处于雌激素阶段时, 细胞是有组织的鳞状上皮细胞, 存在于密集的集群中 (图 1 b)。在流星层, 有组织的上皮细胞和多态核?...

Watch this Scientific Journal Video about Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice at JoVE.com

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

在暴露于臭氧的小鼠的弹性周期中的肺微 rna 分析

在这里, 我们描述了一种方法来评估肺部表达 mirna, 预测调节炎症基因使用小鼠暴露在臭氧或过滤空气在不同阶段的雌周期。

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

这种方法可以帮助回答肺免疫领域关于如何评估微RNA的表达，预测调节肺部炎症基因的关键问题。该技术为确定循环激素对肺微RNA表达的贡献提供了简单的方法。要评估雌性周期阶段，手动约束一只八到九周大的雌性小鼠，并引入充满超纯水的塑料 10 微升移液器的尖端。轻轻冲洗四到五次以收集样品，并将阴道液的最终冲洗沉积到玻璃滑梯上。然后在光学显微镜下以 20 倍的放大率观察未污染的阴道冲洗。对于臭氧和过滤空气暴露，将最多四只小鼠放入两个单独的 1.2 升玻璃容器中，其中带网状盖。将一个玻璃容器放入臭氧室，将一个放在过滤的空气暴露室中。然后将臭氧浓度调整为百万分之二，三小时后取出容器。暴露4小时后，用70%乙醇对第一只实验鼠的暴露皮肤表面进行消毒，并切开肋骨笼，露出心脏和肺部。使用钳子将 1.5 毫升无 RNase 微离心管淹没在液氮中，并在液氮中捕捉冷冻收获的肺。然后使用不锈钢组织粉碎机对整个肺进行毛化，并在两个1.5毫升的微离心管之间分离粉碎的组织。以同样的方式从所有动物身上采集和粉碎肺部后，在每个样品中加入500微升硫化硫化物，并连续用18、21和23个量表针使每个组织均匀化。上次均质化后，在每个样品中加入500微升乙醇，然后涡旋15秒。将混合物加载到单个收集管中的单个旋转柱中，用于离心并丢弃流水线。对于DNase 1治疗，在每列中加入400微升RNA洗涤缓冲液，用于另一个离心，并在无RNase管中混合5微升DNase 1和75微升DNA消化缓冲液。将混合物直接加入柱基，在室温下孵育15分钟，然后通过离心洗涤两个400微升RNA预洗溶液。为了对RNA进行测，将35微升无DNase无水直接添加到每个柱基中，进行最终离心，并在分光光度计上测量每个样品的总RNA浓度和纯度。然后将RNA储存在零下80摄氏度。对于小RNase的逆转，将200纳米的总RNA添加到10微升预洗溶液中，加入每个样品的一管逆转录反应混合物。混合后，将样品短暂离心，放在冰上，直到孵育。接下来，在37摄氏度下孵育管子60分钟，随后在95摄氏度下孵育5分钟。在第二次孵育结束时，每20微升逆转录反应用200微升无RNase水稀释CDNA，并在预加载小鼠炎症反应和自身免疫性微RNA PCR阵列的每个井中加入10微升每个反应混合样品。通过圆形和形状良好的核上皮细胞的存在，可以识别前体阶段。当小鼠处于雌激素阶段时，在涂片中观察到密集的聚集被感染、玉米化和鳞状上皮细胞。在三聚体中，可以看到玉米化上皮细胞和多态核白细胞。在死细胞中，白细胞通常更为普遍。如证明，从四个小鼠肺中提取的RNA每微升1198至2178南克，平均 A260 至 A280 比 2.01 和 2.02 之间，平均 A260 至 A230 比 2.139 和 2.223 之间。在此表中，显示了臭氧和被过滤空气暴露的小鼠之间微RNA表达的两倍变化。在微RNA靶点滤波器和14个微RNA核心分析后，获得显著的表达对数比和P值，这些数据可以通过微RNA靶点滤波器进行映射。核心分析还提供有关规范途径、疾病和功能、调节器和网络的信息。此外，功能分析软件可用于生成网络分析，显示感兴趣的微RNA与其他分子之间的关系。在尝试此过程时，在进行实验之前，每天至少连续检查三个周期的酯循环非常重要。按照这个程序，可以执行其他方法来回答有关肺炎基因表达在整个雌激素周期的其他问题。开发后，该技术为肺免疫领域的其他研究人员利用其他模型探索性激素调节机制铺平了道路。

Research

JoVE Journal

Immunology and Infection

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

科研

免疫与感染

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

右心室收缩压测量组合在小鼠的肺和免疫组织样品的收获

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Measuring Local Anaphylaxis in Mice

小鼠量度本地过敏反应

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Influenza Virus Propagation in Embryonated Chicken Eggs

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

MicroRNA-based Regulation of Picornavirus Tropism

小核糖核酸病毒取向的微小RNA基于调控

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

嗜肺军团菌外膜囊泡：隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...