我们感谢Edanz Group（www.edanzediting.com）的Sarah Williams博士编辑了这份手稿的草稿。

动物实验经吉智医科大学动物伦理委员会批准，并符合《吉智医科大学实验动物指南》中的实验动物使用和护理指南。
1. 从HIGA小鼠中获取肾脏样本
注意:HIGA小鼠在8,9,10,11岁25周后显示出稳定的IgA肾病表型。应选择Balb/c小鼠作为对照组8、9、10、11。获得25周龄雌性HIGA小鼠（n = 10）和25周龄雌性Balb / c小鼠（n = 10）。有必要提前确定实验所需的小鼠数量。对于10只HIGA小鼠和10只Balb / c小鼠的样本量，此步骤需要约7-8小时。
<ol><li>准备这些物品:HIGA小鼠（25周龄，雌性），Balb / c小鼠（25周龄，雌性），吸入麻醉器，异氟醚，软木板，针，磷酸盐缓冲盐水（PBS），注射器，针头，手术剪刀和镊子。</li>
	<li>使用4%-5%的异氟醚和吸入麻醉装置麻醉HIGA小鼠，并使用销钉将其安装在软木板上的背侧位置。将异氟醚的浓度调节至2%-3%作为诱导麻醉后的维持剂量。 注意:麻醉深度保持在完全消除与侵入性手术相关的疼痛的水平。特别是，在切除胸部等组织时，异氟醚浓度应调节至4-5%。脱毛和眼膏不是必需的。如果可以快速执行该过程，则无需将鼠标安装在加热垫上。</li>
	<li>用手术剪刀和镊子在小鼠腹壁上做一个3-4厘米的中线切口，并仔细识别肾脏的两侧。</li>
	<li>用手术剪刀和镊子切开肋骨和横膈膜，露出心脏。切开右心房后，将PBS注射到左心室，直到肾脏颜色变为淡黄色（此过程意味着小鼠的整个身体都注入了PBS。</li>
	<li>切开肾动脉、肾静脉和输尿管，切除肾脏。将肾脏分成30毫克片，用于下一步。 注意:IgA肾病的病理主要涉及肾皮质（肾脏外部）中的肾小球。虽然很难在肉眼下完全区分皮层和髓质，但希望主要收集肾脏的外部。这些样品可在–80°C下储存数月。</li>
</ol>2. 纯化肾脏样本中的总RNA
注意:在此步骤中，市售miRNA分离试剂盒用于提取总RNA。此外，还使用生物聚合物粉碎离心柱。有关更多详细信息，请参阅 材料表 。miRNA 分离试剂盒包含基于硅胶膜的离心柱、基于苯酚/胍的裂解试剂、胍/乙醇洗涤缓冲液（洗涤缓冲液 1）、乙醇洗涤缓冲液（洗涤缓冲液 2）和无核酸酶的水。对于10只HIGA小鼠和10只对照小鼠的样本量，此步骤需要约3小时。
<ol><li>准备以下物品:1.5 或 2.0 mL 收集管、100% 乙醇、氯仿、硅均质器、微量移液器、移液器吸头、离心机、生物聚合物粉碎离心柱、硅胶膜离心柱、苯酚/胍基裂解试剂、胍/乙醇洗涤缓冲液（洗涤缓冲液 1）、乙醇洗涤缓冲液（洗涤缓冲液 2）和无核酸酶水。</li>
	<li>在室温下，使用硅匀浆器和 700 μL 苯酚/胍基裂解试剂匀浆 30 mg 肾脏样品。 注意:样品中的miRNA在暴露于基于苯酚/胍的裂解试剂之前是脆弱的，因此应立即执行这些步骤。</li>
	<li>将裂解物转移到生物聚合物粉碎离心柱中，并在室温下以15,000× g 离心2分钟。</li>
	<li>向滤液中加入 140 μL 氯仿并剧烈混合 15 秒。放置2-3分钟，然后在4°C下以12,000 ×g 离心15分钟。</li>
	<li>轻轻地将透明上清液不接触中间层转移到新的收集管中，并加入测定体积（见下文）的100%乙醇。涡旋5秒。 注意:需要100%乙醇，为获得的上清液体积的1.5倍。</li>
	<li>将样品（上限 700 μL）转移到基于硅胶膜的离心柱中，并在室温下以 8,000 x g 离心样品 15 秒。离心后弃去滤液。</li>
	<li>向基于硅胶膜的离心柱中加入 700 μL 洗涤缓冲液 1，并在室温下以 8,000 x g 离心柱 15 秒。离心后弃去滤液。</li>
	<li>将 500 μL 洗涤缓冲液 2 加入基于硅胶膜的离心柱中，并在室温下以 8,000 x g 离心 15 秒。离心后弃去滤液。</li>
	<li>将 500 μL 洗涤缓冲液 2 加入基于硅胶膜的离心柱中，并在室温下以 8,000 x g 离心 15 秒。离心后弃去滤液。</li>
	<li>再次离心基于硅胶膜的离心柱，而不添加任何东西以除去任何额外的乙醇，在室温下以15,000× g 离心1分钟。离心后扔掉滤液。</li>
	<li>将连接到离心柱的管更换为新的收集管。</li>
	<li>向基于硅胶膜的离心柱中加入 30 μL 无核酸酶水，并在室温下以 8,000 x g 离心 1 分钟。 注意:所得的 30 μL 溶液含有高浓度的总 RNA。该样品可在–80°C下储存数月。</li>
</ol>3. 从总RNA合成cDNA
注意:在此步骤中，使用市售的逆转录试剂盒。有关更多详细信息，请参阅 材料表 。该试剂盒包含核酸混合物、逆转录酶混合物和缓冲液。此过程必须在冰上进行，以防止反应进展。对于10只HIGA小鼠和10只对照小鼠的样本量，此步骤需要约3小时。
<ol><li>准备以下物品:1.5 mL 收集管、八孔试管、微量移液器、移液器吸头、分光光度计、无核酸酶水、冰、核酸混合物、逆转录酶混合物、缓冲液和热循环仪。</li>
	<li>使用分光光度计测量总RNA的浓度。然后，计算所需体积的无核酸酶水，使 12 μL 含有 1 μg 总 RNA。</li>
	<li>制备含有以下内容的预混液:2.0 μL 核酸混合物、2.0 μL 逆转录酶混合物和 4.0 μL 缓冲液，每个样品总共 8.0 μL。</li>
	<li>向八孔试管的每个孔中加入 8 μL 预混液。</li>
	<li>稀释以3.2计算的无核酸酶水体积中的总RNA。然后，向每个八孔试管孔中加入 12 μL 总 RNA 溶液（含有 1 μg 总 RNA）。</li>
	<li>使用热循环仪将样品在37°C的8孔试管中孵育60分钟。然后，使用热循环仪在95°C孵育5分钟。 注意:孵育后的溶液含有高浓度的cDNA。</li>
	<li>将该溶液转移到 1.5 mL 管中，加入 200 μL 无核酸酶水。 注意:所得的 200 μL 溶液可用作 qRT-PCR 模板 cDNA。该样品可在–80°C下储存约1年。</li>
</ol>4. miRNA 的 qRT-PCR
注意:在此步骤中，使用市售PCR试剂盒。有关更多详细信息，请参阅 材料表 。该试剂盒包含PCR混合物、通用引物和无核酸酶水。样品应一式两份制备，并在每种情况下考虑结果的准确性。miRNA的表达水平通过ΔΔCT方法定量。对于10只HIGA小鼠和10只对照小鼠的样本量，此步骤需要约4小时。
<ol><li>准备以下物品:1.5 mL 收集管、96 孔反应板、用于 96 孔反应板的粘合膜、微量移液器、移液器吸头、PCR 混合物、通用引物、无核酸酶水、miRNA 特异性引物和实时荧光定量 PCR 仪器。</li>
	<li>制备含有以下内容的预混液:每孔 12.5 μL PCR 混合物、2.5 μL 通用引物、1.25 μL 5 μM miRNA 特异性引物和 6.25 μL 无核酸酶水。 注意:在该测定中，使用了对miRNA[RNA，U6小核2（RNU6-2），miR-155-5p，miR-146a-5p和miR-21-5p]特异性引物。RNU6-2用作内源性对照14。</li>
	<li>向 96 孔反应板的每个孔中加入 22.5 μL 预混液。</li>
	<li>将步骤 3.7 中制备的 2.5 μL cDNA 加入 96 孔板的各个孔中。</li>
	<li>用粘附膜覆盖96孔反应板，然后以1,000× g 离心30秒。</li>
	<li>运行实时荧光定量 PCR 仪器。对PCR仪器进行编程如下:在95°C下初始活化15分钟，然后在94°C下变性40个循环15秒，在55°C下退火30秒，并在70°C下延伸30秒。</li>
	<li>使用 ΔΔCT 方法定量基因表达。相对表达水平确定为 2–ΔΔCT。 注意:应考虑从结果中排除异常的PCR扩增曲线。</li>
</ol>

<ol>
	<li>Rodrigues, J. C., Haas, M., Reich, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgA+Nephropathy.">IgA Nephropathy.</a> Clinical Journal of the American Society of Nephrology. 12 (4), 677-686 (2017).</li><li>Szeto, C. C., Li, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+IgA+nephropathy.">MicroRNAs in IgA nephropathy.</a> Nature Reviews Nephrology. 10 (5), 249-256 (2014).</li><li>Wyatt, R. J., Julian, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgA+nephropathy.">IgA nephropathy.</a> The New England Journal of Medicine. 368 (25), 2402-2414 (2013).</li><li>Vishnoi, A., Rani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRNA+Biogenesis+and+Regulation+of+Diseases:+An+Overview.">MiRNA Biogenesis and Regulation of Diseases: An Overview.</a> Methods in Molecular Biology. 1509, 1-10 (2017).</li><li>Rupaimoole, R., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+therapeutics:+towards+a+new+era+for+the+management+of+cancer+and+other+diseases.">MicroRNA therapeutics: towards a new era for the management of cancer and other diseases.</a> Nature Reviews Drug Discovery. 16 (3), 203-222 (2017).</li><li>Serino, G., Sallustio, F., Cox, S. N., Pesce, F., Schena, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+miR-148b+expression+promotes+aberrant+glycosylation+of+IgA1+in+IgA+nephropathy.">Abnormal miR-148b expression promotes aberrant glycosylation of IgA1 in IgA nephropathy.</a> Journal of the American Society of Nephrology. 23 (5), 814-824 (2012).</li><li>Serino, 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=In+a+retrospective+international+study,+circulating+miR-148b+and+let-7b+were+found+to+be+serum+markers+for+detecting+primary+IgA+nephropathy.">In a retrospective international study, circulating miR-148b and let-7b were found to be serum markers for detecting primary IgA nephropathy.</a> Kidney International. 89 (3), 683-692 (2016).</li><li>Muso, 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=Enhanced+production+of+glomerular+extracellular+matrix+in+a+new+mouse+strain+of+high+serum+IgA+ddY+mice.">Enhanced production of glomerular extracellular matrix in a new mouse strain of high serum IgA ddY mice.</a> Kidney International. 50 (6), 1946-1957 (1996).</li><li>Kurano, M., Yatomi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+gas+chromatography+mass+spectrometry+to+elucidate+metabolites+predicting+the+phenotypes+of+IgA+nephropathy+in+hyper+IgA+mice.">Use of gas chromatography mass spectrometry to elucidate metabolites predicting the phenotypes of IgA nephropathy in hyper IgA mice.</a> Plos One. 14 (7), 0219403 (2019).</li><li>Hyun, Y. Y., 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=Adipose-derived+stem+cells+improve+renal+function+in+a+mouse+model+of+IgA+nephropathy.">Adipose-derived stem cells improve renal function in a mouse model of IgA nephropathy.</a> Cell Transplantation. 21 (11), 2425-2439 (2012).</li><li>Katsuma, 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=Genomic+analysis+of+a+mouse+model+of+immunoglobulin+A+nephropathy+reveals+an+enhanced+PDGF-EDG5+cascade.">Genomic analysis of a mouse model of immunoglobulin A nephropathy reveals an enhanced PDGF-EDG5 cascade.</a> The Pharmacogenomics Journal. 1 (3), 211-217 (2001).</li><li>Morse, S. M., Shaw, G., Larner, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+mRNA+and+protein+extraction+from+the+same+experimental+sample+using+a+commercially+available+column-based+RNA+preparation+kit.">Concurrent mRNA and protein extraction from the same experimental sample using a commercially available column-based RNA preparation kit.</a> BioTechniques. 40 (1), 54-58 (2006).</li><li>Mestdagh, 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=Evaluation+of+quantitative+miRNA+expression+platforms+in+the+microRNA+quality+control+(miRQC)+study.">Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.</a> Nature Methods. 11 (8), 809-815 (2014).</li><li>Sauer, E., Babion, I., Madea, B., Courts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evidence+based+strategy+for+normalization+of+quantitative+PCR+data+from+miRNA+expression+analysis+in+forensic+organ+tissue+identification.">An evidence based strategy for normalization of quantitative PCR data from miRNA expression analysis in forensic organ tissue identification.</a> Forensic Science International: Genetics. 13, 217-223 (2014).</li><li>Wang, 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=Elevated+levels+of+miR-146a+and+miR-155+in+kidney+biopsy+and+urine+from+patients+with+IgA+nephropathy.">Elevated levels of miR-146a and miR-155 in kidney biopsy and urine from patients with IgA nephropathy.</a> Disease Markers. 30 (4), 171-179 (2011).</li><li>Hennino, M. F., 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=miR-21-5p+renal+expression+is+associated+with+fibrosis+and+renal+survival+in+patients+with+IgA+nephropathy.">miR-21-5p renal expression is associated with fibrosis and renal survival in patients with IgA nephropathy.</a> Scientific Reports. 6, 27209 (2016).</li><li>Green, M. R., 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> How to Win the Battle with RNase. 2019 (2), (2019).</li></ol>

提交人声明他们没有利益冲突。

我们能够使用这种新方法测量IgA肾病小鼠模型（HIGA小鼠）肾脏中miRNA的表达水平。IgA肾病是一种无法解释的疾病，需要进一步研究以阐明其病因和治疗靶点1,3。然而，获取人类肾脏样本是高度侵入性的。这种新技术的优点在于它允许使用小动物研究IgA肾病，并且应该有助于未来对IgA肾病和miRNA的研究。在未来的研究中，该方法可以应用于治疗IgA肾病的新...

免疫球蛋白A（IgA）肾病是一种原发性肾小球肾炎，其特征是IgA在肾小球系膜区域1,2中的异常沉积。它是最常见的原发性肾小球肾炎，在20%-40%的患者中导致终末期肾衰竭2。病因尚不清楚，但与持续性黏膜感染有关1,3。皮质类固醇、免疫抑制剂和肾素-血管紧张素系统抑制剂已被提出作为治疗方法1,3，但尚未完全确立3。因此，需要进一步研究明确治疗IgA肾病的病因和治疗方法。
microRNA（miRNA）是小的非编码RNA，在调节基因表达中起重要作用4,5。据报道，miRNA参与各种疾病的发病机制，有些已被确定为疾病生物标志物和治疗剂4,5。近年来，miRNA与IgA肾病发病机制之间的关联也有报道2,6,7。例如，miR-148b被证明与IgA肾病2,6,7患者的IgA结构异常有关，而miR-148b和let-7b被记录为检测IgA肾病的新型生物标志物7。尽管了解miRNA对IgA肾病的影响可能有助于进一步阐明病因和治疗2，但尚未建立使用小动物模型分析IgA肾病中miRNA的标准方法2。
我们在此开发了一种简单可靠的方法来测量IgA肾病小鼠模型（HIGA小鼠）肾脏中的miRNA表达水平。HIGA小鼠是一种特征性的ddY菌株，显示血清IgA水平特别高，肾小球8,9,10,11中IgA的异常沉积。因此，HIGA小鼠可用作IgA肾病小鼠模型8,9,10,11。我们的方法包括四个主要步骤:首先，通过手术从HIGA小鼠获得肾脏样本;其次，使用基于硅胶膜的离心柱均质化样品并纯化总RNA;第三，使用逆转录从总RNA合成互补DNA（cDNA）;第四，通过定量逆转录聚合酶链反应（qRT-PCR）检测miRNA的表达水平。这种方法的基本原理和结果的可靠性基于以前的报告12,13。我们表明，这是一种准确测量IgA肾病小鼠模型中miRNA表达水平的有用技术，并且可用于促进未来对IgA肾病中miRNA的研究。

<table>
	<tbody>
		<tr>
			<td>BALB/cCrSlc (25-week-old, female)</td>
			<td>Japan SLC, Inc.</td>
			<td>none</td>
			<td>Mouse for control</td>
		</tr>
		<tr>
			<td>HIGA/NscSlc (25-week-old, female)</td>
			<td>Japan SLC, Inc.</td>
			<td>none</td>
			<td>IgA nephropathy mouse model</td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96 well reaction plate for qRT-PCR</td>
			<td>Thermo Fisher Scientific</td>
			<td>4316813</td>
			<td>96-well reaction plate</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>adhesive film for 96-well reaction plate</td>
		</tr>
		<tr>
			<td>miScript II RT kit</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Experimental kit for synthesis of cDNA</td>
		</tr>
		<tr>
			<td>miRNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>Experimental kit fot extraction of total RNA</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (RNU6-2)</td>
			<td>Qiagen</td>
			<td>MS00033740</td>
			<td>miRNA-specific primer</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (miR-155-5p)</td>
			<td>Qiagen</td>
			<td>MS00001701</td>
			<td>miRNA-specific primer</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (miR-146a-5p)</td>
			<td>Qiagen</td>
			<td>MS00001638</td>
			<td>miRNA-specific primer</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (miR-21-5p)</td>
			<td>Qiagen</td>
			<td>MS00009079</td>
			<td>miRNA-specific primer</td>
		</tr>
		<tr>
			<td>miScript SYBR Green PCR kit</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Experimental kit for real-time PCR</td>
		</tr>
		<tr>
			<td>QIA shredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>biopolymer-shredding spin column</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex Flex Real-Time PCR system</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>real-time PCR instrument</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex Software version 1.2.1.</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>real-time PCR instrument software</td>
		</tr>
		<tr>
			<td>takara biomasher standard</td>
			<td>Takara Bio</td>
			<td>9790B</td>
			<td>silicon homogenizer</td>
		</tr>
	</tbody>
</table>

detection of microrna expression in the kidneys of immunoglobulin a nephropathic mice

免疫球蛋白A（IgA）肾病是一种原发性肾小球肾炎，其特征是IgA沉积异常，导致终末期肾衰竭。近年来，在IgA肾病的发病机制中报道了microRNA（miRNA）的参与。然而，没有使用小动物模型分析IgA肾病中miRNA的既定方法。因此，我们开发了一种可靠的方法来分析IgA小鼠模型（HIGA小鼠）肾脏中的miRNA。该协议的目标是检测与对照小鼠肾脏中的水平相比，HIGA小鼠肾脏中miRNA的表达水平改变。简而言之，该方法包括四个步骤:1）从HIGA小鼠获得肾脏样本;2）从肾脏样本中纯化总RNA;3）从总RNA合成互补DNA;4）miRNA的定量逆转录聚合酶链反应（qRT-PCR）。使用这种方法，我们成功地检测了HIGA小鼠肾脏中几种miRNA（miR-155-5p，miR-146a-5p和miR-21-5p）的表达水平。这种新方法可以应用于其他分析IgA肾病中miRNA的研究。

我们研究了miRNA在HIGA小鼠肾脏中的表达水平（n = 10）。该结果完全基于所描述的协议获得。选择Balb / c小鼠的肾脏作为对照（n = 10）。在两组中，均选择25周龄。供应商仅提供雌性HIGA小鼠。三种miRNA（miR-155-5p、miR-146a-5p和miR-21-5p）的表达水平;图1）被检测到，之前报道与 IgA 肾病有关15,16。采用t检验比较两组相对miRNA表达水平，P<0.05?...

Watch this Scientific Journal Video about Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice at JoVE.com

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

microRNA参与IgA肾病的发病机制。我们已经开发出一种可靠的方法来检测IgA肾病小鼠模型（HIGA小鼠）肾脏中的microRNA表达水平。这种新方法将有助于检查miRNA是否参与IgA肾病。

检测免疫球蛋白A肾病小鼠肾脏中的microRNA表达

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

detection-microrna-expression-kidneys-immunoglobulin-nephropathic

Research

JoVE Journal

Immunology and Infection

1.8K 次观看.  Jichi Medical University. microRNA参与IgA肾病的发病机制。我们已经开发出一种可靠的方法来检测IgA肾病小鼠模型（HIGA小鼠）肾脏中的microRNA表达水平。这种新方法将有助于检查miRNA是否参与IgA肾病。

视频: Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

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. ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

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 ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

重组原型泡沫病毒整合纯化过程中核酸污染的检测与去除

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

小鼠涎腺功能的测定方法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

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

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.