Name: tissue collection and rna extraction from the human osteoarthritic knee joint
Uploaded: 2021-07-22T14:00:00
Description: Watch this Scientific Journal Video about Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint at JoVE.com

作者感谢使这项研究成为可能的研究参与者，并将本报告献给骨关节炎领域的新科学家。

该研究方案获得批准并遵循亨利福特卫生系统机构审查委员会（IRB #13995）制定的机构指南。
1. 患者选择
<ol><li>从计划与整形外科医生一起接受TKA的患者中确定患者。</li>
	<li>根据研究方案定义的合格标准选择患者。纳入标准的示例包括年满 18 周岁且确诊为膝关节骨关节炎。排除标准的例子包括接受部分膝关节置换术或确诊为类风湿性关节炎。</li>
	<li>在手术前联系患者以获...

<ol>
	<li>Gupton, M., Imonugo, O., Terreberry, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy, Bony Pelvis, and Lover Limb, Knee. , (2020).</li><li>Pacifici, M., Koyama, E., Iwamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Synovial+joint+and+articular+cartilage+formation:+recent+advances,+but+many+lingering+mysteries.">Mechanisms of Synovial joint and articular cartilage formation: recent advances, but many lingering mysteries.</a> Birth Defects Research Part C: Embryo Today: Reviews. 75 (3), 237-248 (2005).</li><li>Gupton, M., Munjal, A., Terreberry, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy, Hinge Joints. , (2020).</li><li>Chen, D., 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=Osteoarthritis:+Toward+a+comprehensive+understanding+of+pathological+mechanism.">Osteoarthritis: Toward a comprehensive understanding of pathological mechanism.</a> Bone Research. 5, 16044 (2017).</li><li>Murphy, L., 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=Lifetime+risk+of+symptomatic+knee+osteoarthritis.">Lifetime risk of symptomatic knee osteoarthritis.</a> Arthritis &amp; Rheumatism. 59 (9), 1207-1213 (2008).</li><li>O'Neill, T. W., McCabe, P. S., McBeth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+the+epidemiology,+risk+factors+and+disease+outcomes+of+osteoarthritis.">Update on the epidemiology, risk factors and disease outcomes of osteoarthritis.</a> Best Practice &amp; Research: Clinical Anaesthesiology. 32 (2), 312-326 (2018).</li><li>Nguyen, U. 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=Increasing+prevalence+of+knee+pain+and+symptomatic+knee+osteoarthritis:+survey+and+cohort+data.">Increasing prevalence of knee pain and symptomatic knee osteoarthritis: survey and cohort data.</a> Annals of Internal Medicine. 155 (11), 725-732 (2011).</li><li>Deshpande, B. R., 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=Number+of+persons+with+symptomatic+knee+osteoarthritis+in+the+us:+impact+of+race+and+ethnicity,+age,+sex,+and+obesity.">Number of persons with symptomatic knee osteoarthritis in the us: impact of race and ethnicity, age, sex, and obesity.</a> Arthritis Care &amp; Research. 68 (12), 1743-1750 (2016).</li><li>Loeser, R. F., Goldring, S. R., Scanzello, C. R., Goldring, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis:+a+disease+of+the+joint+as+an+organ.">Osteoarthritis: a disease of the joint as an organ.</a> Arthritis &amp; Rheumatism. 64 (6), 1697-1707 (2012).</li><li>McGonagle, D., Tan, A. L., Carey, J., Benjamin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomical+basis+for+a+novel+classification+of+osteoarthritis+and+allied+disorders.">The anatomical basis for a novel classification of osteoarthritis and allied disorders.</a> Journal of Anatomy. 216 (3), 279-291 (2010).</li><li>Bannuru, R. R., 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=OARSI+guidelines+for+the+non-surgical+management+of+knee,+hip,+and+polyarticular+osteoarthritis.">OARSI guidelines for the non-surgical management of knee, hip, and polyarticular osteoarthritis.</a> Osteoarthritis Cartilage. 27 (11), 1578-1589 (2019).</li><li>Kolasinski, S. L., 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=American+College+of+Rheumatology/Arthritis+Foundation+Guideline+for+the+Management+of+Osteoarthritis+of+the+Hand,+Hip,+and+knee.">American College of Rheumatology/Arthritis Foundation Guideline for the Management of Osteoarthritis of the Hand, Hip, and knee.</a> Arthritis Care &amp; Research. 72 (2), 220-233 (2020).</li><li>Michael, J. W., Schluter-Brust, K. U., Eysel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology,+etiology,+diagnosis,+and+treatment+of+osteoarthritis+of+the+knee.">The epidemiology, etiology, diagnosis, and treatment of osteoarthritis of the knee.</a> Deutsches &#196;rzteblatt International. 107 (9), 152-162 (2010).</li><li>Singh, J. A., Yu, S., Chen, L., Cleveland, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+total+joint+replacement+in+the+United+States:+Future+projections+to+2020-2040+using+the+national+inpatient+sample.">Rates of total joint replacement in the United States: Future projections to 2020-2040 using the national inpatient sample.</a> Journal of Rheumatology. 46 (9), 1134-1140 (2019).</li><li>Gemayel, A. C., Varacallo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+Knee+Replacement+Techniques.">Total Knee Replacement Techniques.</a> StatPearls. , (2020).</li><li>Shan, L., Shan, B., Suzuki, A., Nouh, F., Saxena, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate+and+long-term+quality+of+life+after+total+knee+replacement:+a+systematic+review+and+meta-analysis.">Intermediate and long-term quality of life after total knee replacement: a systematic review and meta-analysis.</a> Journal of Bone and Joint Surgery (American Volume). 97 (2), 156-168 (2015).</li><li>Newton, P. T., 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=Chondrogenic+Atdc5+cells:+an+optimised+model+for+rapid+and+physiological+matrix+mineralisation.">Chondrogenic Atdc5 cells: an optimised model for rapid and physiological matrix mineralisation.</a> International Journal of Molecular Medicine. 30 (5), 1187-1193 (2012).</li><li>Chuang, Y. W., 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=Lysophosphatidic+acid+enhanced+the+angiogenic+capability+of+human+chondrocytes+by+regulating+Gi/Nf-Kb-dependent+angiogenic+factor+expression.">Lysophosphatidic acid enhanced the angiogenic capability of human chondrocytes by regulating Gi/Nf-Kb-dependent angiogenic factor expression.</a> PLoS One. 9 (5), 95180 (2014).</li><li>Johnson, C. I., Argyle, D. J., Clements, D. N. <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+models+for+the+study+of+osteoarthritis.">In vitro models for the study of osteoarthritis.</a> Veterinary Journal. 209, 40-49 (2016).</li><li>Grivel, J. C., Margolis, L. <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+human+tissue+explants+to+study+human+infectious+agents.">Use of human tissue explants to study human infectious agents.</a> Nature Protocols. 4 (2), 256-269 (2009).</li><li>Glasson, S. S., Blanchet, T. J., Morris, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surgical+destabilization+of+the+medial+meniscus+(dmm)+model+of+osteoarthritis+in+the+129/Svev+mouse.">The surgical destabilization of the medial meniscus (dmm) model of osteoarthritis in the 129/Svev mouse.</a> Osteoarthritis Cartilage. 15 (9), 1061-1069 (2007).</li><li>McIlwraith, C. W., Frisbie, D. D., Kawcak, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+horse+as+a+model+of+naturally+occurring+osteoarthritis.">The horse as a model of naturally occurring osteoarthritis.</a> Bone &amp; Joint Research. 1 (11), 297-309 (2012).</li><li>Cope, P. J., Ourradi, K., Li, Y., Sharif, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+Osteoarthritis:+The+good,+the+bad+and+the+promising.">Models of Osteoarthritis: The good, the bad and the promising.</a> Osteoarthritis Cartilage. 27 (2), 230-239 (2019).</li><li>Goldenberg, A. J., 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=IRB+practices+and+policies+regarding+the+secondary+research+use+of+biospecimens.">IRB practices and policies regarding the secondary research use of biospecimens.</a> Bmc Medical Ethics. 16, 32 (2015).</li><li>Ruettger, A., Neumann, S., Wiederanders, B., Huber, R. <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+different+methods+for+preparation+and+characterization+of+total+rna+from+cartilage+samples+to+uncover+osteoarthritis+in+vivo.">Comparison of different methods for preparation and characterization of total rna from cartilage samples to uncover osteoarthritis in vivo.</a> BMC Research Notes. 3, 7 (2010).</li><li>Reno, C., Marchuk, L., Sciore, P., Frank, C. B., Hart, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+total+RNA+from+small+samples+of+hypocellular,+dense+connective+tissues.">Rapid isolation of total RNA from small samples of hypocellular, dense connective tissues.</a> BioTechniques. 22 (6), 1082-1086 (1997).</li><li>Fox, A. J., Bedi, A., Rodeo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basic+science+of+human+knee+menisci:+structure,+composition,+and+function.">The basic science of human knee menisci: structure, composition, and function.</a> Sports Health. 4 (4), 340-351 (2012).</li><li>Carballo, C. B., Nakagawa, Y., Sekiya, I., Rodeo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+science+of+articular+cartilage.">Basic science of articular cartilage.</a> Clinics in Sports Medicine. 36 (3), 413-425 (2017).</li><li>Le Bleu, H. 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=Extraction+of+high-quality+RNA+from+human+articular+cartilage.">Extraction of high-quality RNA from human articular cartilage.</a> Analytical Biochemistry. 518, 134-138 (2017).</li><li>Ali, S. A., Alman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+extraction+from+human+articular+cartilage+by+chondrocyte+isolation.">RNA extraction from human articular cartilage by chondrocyte isolation.</a> Analytical Biochemistry. 429 (1), 39-41 (2012).</li><li>Schroeder, 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+Rin:+An+RNA+integrity+number+for+assigning+integrity+values+to+rna+measurements.">The Rin: An RNA integrity number for assigning integrity values to rna measurements.</a> BMC Molecular Biology. 7, 3 (2006).</li><li>Li, 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=Multi-platform+assessment+of+transcriptome+profiling+using+RNA-seq+in+the+abrf+next-generation+sequencing+study.">Multi-platform assessment of transcriptome profiling using RNA-seq in the abrf next-generation sequencing study.</a> Nature Biotechnology. 32 (9), 915-925 (2014).</li><li>Nazarov, P. V., 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=RNA+sequencing+and+transcriptome+arrays+analyses+show+opposing+results+for+alternative+splicing+in+patient+derived+samples.">RNA sequencing and transcriptome arrays analyses show opposing results for alternative splicing in patient derived samples.</a> BMC Genomics. 18 (1), 443 (2017).</li><li>Madissoon, 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=scRNA-seq+assessment+of+the+human+lung,+spleen,+and+esophagus+tissue+stability+after+cold+preservation.">scRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation.</a> Genome Biology. 21 (1), (2019).</li><li>Scholes, A. N., Lewis, 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=Comparison+of+RNA+isolation+methods+on+RNA-seq:+implications+for+differential+expression+and+meta-analyses.">Comparison of RNA isolation methods on RNA-seq: implications for differential expression and meta-analyses.</a> BMC Genomics. 21 (1), 249 (2020).</li><li>Smith, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+normal+synovium.">The normal synovium.</a> Open Rheumatology Journal. 5, 100-106 (2011).</li><li>Kukurba, K. R., Montgomery, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+sequencing+and+analysis.">RNA sequencing and analysis.</a> Cold Spring Harbor Protocols. 2015 (11), 951-969 (2015).</li><li>Mobasheri, A., Kapoor, M., Ali, S. A., Lang, A., Madry, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+deep+phenotyping+in+osteoarthritis:+how+can+high+throughput+omics+technologies+advance+our+understanding+of+the+cellular+and+molecular+taxonomy+of+the+disease.">The future of deep phenotyping in osteoarthritis: how can high throughput omics technologies advance our understanding of the cellular and molecular taxonomy of the disease.</a> Osteoarthritis and Cartilage Open. 3 (2), 100144 (2021).</li></ol>

所提出的方案已被证明可以成功地收集七种主要的人类OA组织进行RNA提取（表1）和组织学处理（图4）。在收集患者样本之前，有必要建立IRB批准的方案，最好是与外科医生或手术团队合作。应用标准化的实验方案进行标本采集（例如，从一致的原位位置切除）对于最大限度地提高实验再现性至关重要。组织样本应装在无菌容器中运输到实验室，并在手术?...

膝关节是人体最大的滑膜关节，包括胫骨和股骨之间的胫股关节以及髌骨和股骨之间的髌股关节1。膝盖中的骨骼衬有关节软骨，并由各种结缔组织支撑，包括半月板，脂肪，韧带和肌肉，滑膜封装整个关节以形成充满滑液的腔1，2，3（图1）。健康的膝盖作为一个移动的铰链关节，允许在额平面1，3中无摩擦运动。在病理条件下，运动会受到限制和疼痛。最常见的退行性膝关节疾病是骨关节炎（OA）4。已知各种危险因素易发生OA发展，包括年龄较大，肥胖，女性，关节创伤和遗传学，除其他外5，6。目前，美国估计有1400万人患有有症状的膝关节OA，由于人口年龄和肥胖率上升，患病率增加7，8。OA最初被认为是软骨的疾病，现在被理解为整个关节的疾病9。OA中通常观察到的病理变化包括关节软骨侵蚀，骨赘形成，软骨下骨增厚和滑膜炎症9，10。由于OA没有已知的治愈方法，治疗主要集中在症状（例如，疼痛）管理11，12，并且一旦OA进展到终末期，通常需要关节置换手术13。
关节置换手术可以是部分或全膝关节置换术，全膝关节置换术（TKA）包括置换整个胫股关节和髌股关节。截至2020年，美国每年约有100万个TKA进行14。在TKA期间，整形外科医生切除胫骨平台的上部和下股骨髁（图2A，2B）以安装假体植入物。有时被患者误解，在TKA中，只有8-10毫米从每块骨头的末端切除，随后用金属覆盖或重新表面。插入的聚乙烯衬垫在两个金属植入物之间形成轴承表面（即衬垫）。此外，关节的几个软组织成分被完全或部分切除，以实现适当的关节平衡。这些组织包括内侧和外侧半月板（图2C），髌下脂肪垫（图2D），前交叉韧带（ACL;图2E）、滑膜（图2F）和内侧斜肌（VMO;图2G）虽然TKA通常对OA治疗是成功的，但大约20%的患者报告手术后疼痛复发16。除了手术的高成本和相对侵入性外，这些局限性表明需要进一步研究以确定替代疗法以减轻OA的进展。
为了探索OA中的疾病机制，这些机制可能为治疗干预提供新的途径，可以使用实验系统，包括细胞，组织外植体和动物模型。细胞通常在单层中培养，并且来源于原代人或动物组织（例如，从软骨中分离的软骨细胞）或永生化细胞（例如，ATDC517 和CHON-00118）。虽然细胞可用于在受控培养环境中操纵实验变量，但它们不能捕获已知影响细胞表型的自然关节条件19。为了更好地概括OA背后的化学，机械和细胞间通讯的复杂级联反应，在原代人或动物组织样品中发现了一种替代方案，无论是使用新鲜还是 培养的离体 作为外植体，以保留组织结构和细胞微环境20。为了研究 体内关节，小（如小鼠21）和大（如马22）动物模型对OA（如手术诱导、遗传改变或衰老）也是有用的。然而，从这些模型到人类疾病的转化可能受到解剖学，生理学和代谢差异的限制，其中包括23。考虑到实验系统的优缺点，物种特异性和维持主要人类OA组织提供的细胞外生态位的关键优势最大限度地发挥了研究结果的转化潜力。
原代人类OA组织在TKA之后可以很容易地获得，这使得TKA的高频率成为研究的宝贵资源。潜在的实验应用包括基因表达和组织学分析。为了实现初级人类OA组织在这些研究方法和其他方面的潜力，概述了以下关键考虑因素。首先，患者标本的使用受道德规范的约束，并且协议必须符合机构审查委员会（IRB）的批准24。其次，人类原发性病变组织固有的异质性以及年龄和性别等变量的影响，使得需要仔细选择患者（即，应用资格标准）和数据解释。第三，关节中不同组织的独特生物学特性（例如，软骨和半月板25的低细胞性）在实验过程中可能会带来挑战（例如，分离高质量和数量的RNA）。本报告解决了这些考虑因素，并提出了患者选择，样品处理，组织均质化，RNA提取和质量控制（即评估RNA纯度和完整性; 图 3）鼓励在研究界使用初级人类OA组织。

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	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>05 402</td>
			<td>Sterile, nuclease-free. Reserved for RNA work only.</td>
		</tr>
		<tr>
			<td>10% Formalin</td>
			<td>Cardinal Health</td>
			<td>C4320-101</td>
			<td>Store in chemical cabinet when not in use.</td>
		</tr>
		<tr>
			<td>100% Chloroform (Molecular Biology Grade)</td>
			<td>Fisher Scientific</td>
			<td>ICN19400290</td>
			<td>Sterile, nuclease-free. Reserved for RNA work only, store in chemical cabinet when not in use.</td>
		</tr>
		<tr>
			<td>100% Ethanol (Molecular Biology Grade)</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td>Sterile, nuclease-free. Reserved for RNA work only, when diluting use DEPC/nuclease-free water.</td>
		</tr>
		<tr>
			<td>100% Isopropanol (Molecular Biology Grade)</td>
			<td>Fisher Scientific</td>
			<td>AC327272500</td>
			<td>Sterile, nuclease-free. Reserved for RNA work only, store in chemical cabinet when not in use.</td>
		</tr>
		<tr>
			<td>100% Reagent Alcohol</td>
			<td>Cardinal Health</td>
			<td>C4305</td>
			<td>Diluted to 70% with dH2O for cleaning purposes.</td>
		</tr>
		<tr>
			<td>15 cm sterile culture dishes</td>
			<td>Thermo Scientific</td>
			<td>12-556-003</td>
			<td>Sterile, nuclease-free.</td>
		</tr>
		<tr>
			<td>15 mL polypropylene (Falcon) tubes</td>
			<td>Fisher Scientific</td>
			<td>14 959 53A</td>
			<td>Sterile, nuclease-free.</td>
		</tr>
		<tr>
			<td>2 mL cryovials (externally threaded)</td>
			<td>Fisher Scientific</td>
			<td>10 500 26</td>
			<td>Sterile, nuclease-free.</td>
		</tr>
		<tr>
			<td>5 mL round-bottom tubes</td>
			<td>Corning</td>
			<td>352052</td>
			<td>Sterile, nuclease-free. Reserved for RNA work only.</td>
		</tr>
		<tr>
			<td>50 mL polypropylene (Falcon) tubes</td>
			<td>Fisher Scientific</td>
			<td>12 565 271</td>
			<td>Sterile, nuclease-free.</td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td>For RNA integrity measurement.</td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td colspan="2">General lab equipment</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Cutters</td>
			<td>Fisher Scientific</td>
			<td>08 990</td>
			<td>Sterilized with 70% EtOH.</td>
		</tr>
		<tr>
			<td>Chemical Fume Hood</td>
			<td colspan="2">General lab equipment</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpels (No.10)</td>
			<td>Thermo Scientific</td>
			<td>3120032</td>
			<td>Sterile, nuclease-free.</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Life Technologies</td>
			<td>15-576-028</td>
			<td>10% solution with dH2O.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Any vendor</td>
			<td></td>
			<td>Sterilized with 70% EtOH.</td>
		</tr>
		<tr>
			<td>Glycoblue Coprecipitant</td>
			<td>Fisher Scientific</td>
			<td>AM9516</td>
			<td>Reserved for RNA work only, store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Any vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen Dewar</td>
			<td colspan="2">General lab equipment</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar and Pestle</td>
			<td>Any vendor</td>
			<td></td>
			<td>Reserved for RNA work only, sterilzed per protocol.</td>
		</tr>
		<tr>
			<td>Nanodrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>For RNA purity and yield measurements.</td>
		</tr>
		<tr>
			<td>Nuclease-free/DEPC-treated water</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Sterile, nuclease-free. Reserved for RNA work only.</td>
		</tr>
		<tr>
			<td>PBS (Sterile)</td>
			<td>Gibco</td>
			<td>20 012 050</td>
			<td>Sterile, nuclease-free.</td>
		</tr>
		<tr>
			<td>Pipettes (2 &#181;L, 20 &#181;L, 200 &#181;L, 1000 &#181;L) &#38; tips</td>
			<td>Any vendor</td>
			<td></td>
			<td>Sterile, nuclease-free.</td>
		</tr>
		<tr>
			<td>Plasma/Serum Advanced miRNA kit</td>
			<td>Qiagen</td>
			<td>217204</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td>22625101</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Thermo Scientific</td>
			<td>50 197 8158</td>
			<td>Sterile, nuclease-free.</td>
		</tr>
		<tr>
			<td>RNAse Away/RNAseZap</td>
			<td>Fisher Scientific</td>
			<td> 
			7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula (semimicro size)</td>
			<td>Any vendor</td>
			<td></td>
			<td>Reserved for RNA work only.</td>
		</tr>
		<tr>
			<td>Tissue homogenizer</td>
			<td>Pro Scientific</td>
			<td>01-01200</td>
			<td>Reserved for RNA work only, sterilzed per protocol.</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Fisher Scientific</td>
			<td>15 596 026</td>
			<td>Sterile, nuclease-free. Reserved for RNA work only.</td>
		</tr>
	</tbody>
</table>

tissue collection and rna extraction from the human osteoarthritic knee joint

骨关节炎（OA）是一种慢性退行性关节疾病，最常影响膝盖。由于目前尚无治愈方法，全膝关节置换术（TKA）是一种常见的手术干预。使用从TKA获得的原代人OA组织的实验提供了研究 离体疾病机制的能力。虽然OA以前被认为主要影响软骨，但现在已知它会影响关节中的多个组织。该协议描述了来自七个独特组织的患者选择，样品处理，组织均质化，RNA提取和质量控制（基于RNA纯度，完整性和产量），以支持膝关节的疾病机制研究。经知情同意，从接受TKA治疗OA的患者处获得样本。在手术后4小时内通过闪光冷冻RNA或福尔马林固定进行组织学检查，洗涤和储存组织组织。收集的组织包括关节软骨、软骨下骨、半月板、髌下脂肪垫、前交叉韧带、滑膜和内侧斜肌。针对每种组织类型测试RNA提取方案。最显著的修饰涉及用于低细胞、高基质、硬组织（被视为软骨、骨骼和半月板）与相对高细胞、低基质、软组织（被视为脂肪垫、韧带、滑膜和肌肉）的崩解方法。研究发现，粉碎适用于硬组织，均质化适用于软组织。观察到一些受试者倾向于在多个组织中产生比其他受试者更高的RNA完整性数（RIN）值，这表明疾病严重程度等潜在因素可能会影响RNA质量。从原代人类OA组织中分离高质量RNA的能力为复杂的基因表达实验（包括测序）提供了生理相关模型，可以导致更容易转化为患者的临床见解。

七种独特的人类膝关节组织可供接受TKA治疗OA的患者收集（图1）。在该协议中，这些组织中的每一个都被鉴定并在手术切除后的4小时内进行处理（图2）。按照 图3中概述的步骤，每个组织的一部分被福尔马林固定用于组织学评估（图4），而其他部分则被速冻以进行RNA分离。通过崩解方法（分别为粉碎与均?...

Watch this Scientific Journal Video about Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint at JoVE.com

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

从全膝关节置换术后的患者获得的原代组织为骨关节炎研究提供了一个实验模型，具有最大的临床可转化性。该协议描述了如何从七个独特的膝关节组织中鉴定，处理和分离RNA，以支持人类骨关节炎的机制研究。

从人骨关节炎膝关节收集和RNA提取

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

该协议鼓励使用原代人体组织作为骨关节炎研究的临床相关模型。在这些组织中进行基因表达和组织学分析可以提供新的机制见解。该技术标准化了识别和处理人类膝关节成分的方法。它能够提取满足下游基因表达测定要求的高质量RNA。从患病的关节组织获得高质量的RNA具有挑战性，因此不应低估看似微不足道的步骤的重要性，例如保持一切清洁和凉爽。演示该程序的将是研究助理Thomas Wilson和我实验室的博士后研究员Navdeep Kaur。首先根据大小、形状、颜色和质地的差异，从标本中识别硬组织，包括软骨、骨骼和半月板以及软组织，包括髌下脂肪垫、前交叉韧带、滑膜和内侧蓬斜肌。将每个组织切成三个部分。用无菌PBS冲洗组织切片，以除去任何残留物或碎屑。对于RNA提取，将三个组织切片中的每一个切成大约一到两毫米的立方体。将较小的碎片转移到两毫升冷冻小瓶中。紧紧固定盖子后，通过浸没在液氮中30秒来快速冷冻小瓶，然后将小瓶转移到零下80摄氏度的冰箱中进行长期储存，并对所有组织重复此过程。使用研钵和研杵使硬组织均质化。在均质化之前，使用液氮冷却研钵，杵和刮刀，以防止样品解冻。一次只处理一个样品。使用冷刮刀将组织样品转移到砂浆中。将液氮倒在组织顶部。液氮蒸发后，用研杵压碎组织。在研磨组织的同时反复加入液氮，得到细小的组织粉。将细组织粉末转移到1.5毫升微量离心管中，通过浸没在液氮中预冷30秒。向每个管中加入一毫升冰冷的酸性胍苯酚溶液，并将管在冰上孵育20分钟。在均质化不同的硬组织之前，用70%乙醇彻底清洁研钵，杵和刮刀，然后用RNase去污剂，然后用DEPC处理的水。将研钵，研杵和刮刀浸泡在70%乙醇中，然后用干净的无绒纸巾擦拭任何残留的液体。使用组织均质器使软组织均质化。为了对均质器进行消毒，每次洗涤通过运行70%乙醇和RNase去污剂的管子洗涤探针30秒，然后用DEPC处理过的水洗涤，然后另外加70%乙醇，每次30秒。用干净、无绒毛的纸巾擦拭任何残留液体。将一毫升酸性胍苯酚溶液加入预先标记的五毫升圆底管中，并将组织样品转移到管中。在整个过程中将管子放在冰上，并使组织匀浆最多五次32次脉冲，或直到组织在视觉上溶解。将均质组织孵育在冰上，直到所有组织样品都得到处理。在处理下一个组织样品之前，对均质器进行消毒和清洁。使用无菌镊子，去除探针牙齿中存在的任何组织块。将所有均质组织在冰上再孵育20分钟，然后将组织样品转移到预先标记的，预冷的1.5毫升微量离心管中。从骨关节炎患者身上获得的七个独特的人类膝关节组织在手术切除后四小时内得到处理。通过视觉鉴定组织并通过H&E组织学染色确认组织组织学。H&E染色的硬组织切片，关节软骨，软骨下骨和半月板以六倍放大倍率和40倍放大倍率可视化。同样，H&E染色的软组织切片，髌下脂肪垫，前交叉韧带，滑膜和内侧腹斜肌在六倍放大镜下可视化，然后在40倍放大镜下可视化。提取的RNA的质量评估表明，基于完整性，产量和纯度的高质量样品以及低质量样品的存在，这表明尽管使用了优化的方法，但疾病严重程度等外部因素可能会影响同一患者不同组织的RNA质量。正确鉴定每种组织类型对于在患者样本内和患者样本之间进行组织内和组织间比较是必要的，特别是考虑到原发性疾病组织具有相当大的异质性。测序技术正在迅速发展，高质量RNA的分离是使用这些技术解开骨关节炎等复杂慢性疾病中潜在组织特异性机制的关键先决条件。

Research

JoVE Journal

Biology

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

从特定的大脑区域的RNA或蛋白质提取大鼠脑微解剖

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

科研

生物学

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

使用Bio - Rad公司的总RNA试剂盒从Neuroprecursor细胞的RNA提取

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

人类胰岛的分离：第一部分：收集和胰腺组织消化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

人死后脑组织中的单细胞群获得高品质的RNA

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

从环境中自然产生的趋磁细菌的采集，分离和富集

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

提取与分析皮质醇从人与猴的毛

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

从小鼠胰腺RNA提取：核糖核酸酶丰富的组织

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

福尔马林固定石蜡包埋的组织核心的制备RNA和DNA提取

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

Y-27632 丰富成人皮肤组织中人类黑色素细胞的产量

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

从人类肌肉提比利斯前部高级隔间收集骨骼肌肉活检，用于机械评估

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...