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

The authors thank the study participants who made this research possible and dedicate this report to new scientists in the osteoarthritis field.

This study protocol was approved and followed institutional guidelines set by the Henry Ford Health System Institutional Review Board (IRB #13995).
1. Patient selection
<ol>
	<li>Identify the patients from among those scheduled to undergo TKA with an orthopedic surgeon.</li>
	<li>Select the patients based on the eligibility criteria defined by the study protocol. Examples of inclusion criteria include being 18 years of age or older and having a confirmed diagnosis of knee osteoarthritis. Exa...

<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. 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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. 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The protocol presented has proved successful for collecting seven primary human OA tissues for RNA extraction (Table 1) and histological processing (Figure 4). Prior to collecting patient samples, it is necessary to establish an IRB-approved protocol, ideally in collaboration with a surgeon or surgical team. Applying a standardized protocol for specimen collection (e.g., resection from consistent in situ locations) is essential for maximizing experimental reproducib...

The knee is the largest synovial joint in the human body, comprising the tibiofemoral joint between the tibia and the femur and the patellofemoral joint between the patella and the femur1. The bones in the knee are lined with articular cartilage and supported by various connective tissues, including menisci, fat, ligaments, and muscle, and a synovial membrane encapsulates the whole joint to create a synovial fluid-filled cavity1,2,3 (Figure 1). A healthy knee functions as a mobile hinge joint that allows frictionless motion in the frontal plane1,3. Under pathological conditions, movement can become restricted and painful. The most common degenerative knee joint disease is osteoarthritis (OA)4. A variety of risk factors are known to predispose to OA development, including older age, obesity, female sex, joint trauma, and genetics, among others5,6. There are currently an estimated 14 million people in the USA with symptomatic knee OA, with the prevalence increasing due to rising population age and rates of obesity7,8. Initially considered to be a disease of the cartilage, OA is now understood as a disease of the whole joint9. Commonly observed pathological changes in OA include articular cartilage erosion, osteophyte formation, subchondral bone thickening, and inflammation of the synovium9,10. Since there is no known cure for OA, treatments primarily focus on symptom (e.g., pain) management11,12, and once OA has progressed to end-stage, joint replacement surgery is often indicated13.
Joint replacement surgeries can either be partial or total knee replacements, with total knee arthroplasty (TKA) including replacing the entire tibiofemoral articulation and the patellofemoral joint. As of 2020, approximately 1 million TKAs are performed in the USA each year14. During TKA, an orthopedic surgeon resects the upper portion of the tibial plateau and the lower femoral condyles (Figure 2A, 2B) to be fitted with prosthetic implants. Sometimes misinterpreted by patients, in a TKA, only 8-10 mm is resected from the end of each bone, which is subsequently capped or resurfaced, with metal. An interposed polyethylene liner forms the bearing surface (i.e., padding) between the two metal implants. In addition, several soft tissue components of the joint are fully or partially excised to achieve proper joint balance. Among these tissues are the medial and lateral menisci (Figure 2C), infrapatellar fat pad (Figure 2D), anterior cruciate ligament (ACL; Figure 2E), synovium (Figure 2F), and vastus medialis oblique muscle (VMO; Figure 2G)15. Though TKAs are generally successful for OA treatment, around 20% of patients report reoccurrence of pain post-surgery16. Along with the high cost and relative invasiveness of the procedure, these limitations point to the need for further research to identify alternative treatments to mitigate the progression of OA.
To explore disease mechanisms in OA that may present new avenues for therapeutic intervention, experimental systems, including cells, tissue explants, and animal models can be used. Cells are typically cultured in monolayer and are derived from primary human or animal tissues (e.g., chondrocytes isolated from cartilage) or immortalized cells (e.g., ATDC517 and CHON-00118). While cells can be useful for manipulating experimental variables in a controlled culture environment, they do not capture conditions of the natural joint which are known to impact cell phenotypes19. To better recapitulate the complex cascade of chemical, mechanical, and cell-to-cell communication underlying OA, an alternative is found in primary human or animal tissue samples, whether used fresh or cultured ex vivo as explants, to preserve tissue structure and the cell microenvironment20. In order to study the joint in vivo, small (e.g., mouse21) and large (e.g., horse22) animal models for OA (e.g., through surgical induction, genetic alteration, or aging) are also useful. However, translation from these models to human disease can be limited by anatomical, physiological, and metabolic differences, among others23. Considering the advantages and disadvantages of experimental systems, the key strengths of being species-specific and maintaining the extracellular niche offered by the primary human OA tissues maximize the translational potential of research findings.
Primary human OA tissues can be readily obtained following TKA, making the high frequency of TKAs a valuable resource for research. Among potential experimental applications are gene expression and histological analyses. To realize the potential of primary human OA tissues for these research approaches and others, outlined are the following key considerations. First, the use of patient specimens is subject to ethical regulation, and protocols must meet Institutional Review Board (IRB) approvals24. Second, the inherent heterogeneity of human primary diseased tissues and the influence of variables such as age and sex, among others, create the need for careful patient selection (i.e., application of eligibility criteria) and data interpretation. Third, the unique biological properties of different tissues in the joint (e.g., low cellularity of cartilage and meniscus25) can present challenges during experiments (e.g., isolating high quality and quantity of RNA). This report addresses these considerations and presents a protocol for patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (i.e., assessment of RNA purity and integrity; Figure 3) to encourage the use of primary human OA tissues in the research community.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Seven unique human knee joint tissues are available for collection from patients undergoing TKA for OA (Figure 1). In this protocol, each of these tissues were identified and processed within 4 h of surgical removal (Figure 2). Following the steps outlined in Figure 3, portions of each tissue were formalin-fixed for histological assessment (Figure 4), while other portions were flash-frozen for RNA isola...

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

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

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

This protocol encourages the use of primary human tissues as a clinically relevant model for osteoarthritis research. Performing gene expression and histological analyses in these tissues may provide novel mechanistic insights. This technique standardizes methods for the identification and processing of human knee joint components.It enables the extraction of high quality RNA that meets the requirements for downstream gene expression assays. It is challenging to obtain high quality RNA from diseased joint tissues, so the importance of seemingly insignificant steps, such as keeping everything clean and cool should not be underestimated. Demonstrating the procedure will be Thomas Wilson, a research associate and Navdeep Kaur, a postdoctoral fellow from my laboratory.Begin by identifying the hard tissues, including cartilage, bone, and meniscus and soft tissues, including infrapatellar fat pad, anterior cruciate ligament, synovium and vastus medialis oblique muscle from the specimen based on the differences in the size, shape, color and texture. Cut each of the tissues into three sections. Rinse the tissue sections with sterile PBS to remove any residue or debris.For RNA extraction, cut each of the three tissue sections into approximately one to two millimeter cubes. Transfer the smaller pieces to a two milliliter cryo vial. After securing the caps tightly, flash freeze the vials by submerging in liquid nitrogen for 30 seconds, then transfer the vial to the minus 80 degrees Celsius freezer for long-term storage, and repeat this procedure for all the tissues.Homogenize the hard tissue using a mortar and pestle. Before homogenization, chill the mortar, pestle and spatula using liquid nitrogen to prevent the sample from thawing. Process only one sample at a time.Transfer the tissue sample to mortar using a chilled spatula. Pour liquid nitrogen on top of the tissue. After the liquid nitrogen evaporates, crush the tissue with a pestle.Repeatedly add liquid nitrogen while grinding the tissue to obtain fine tissue powder. Transfer the fine tissue powder in a 1.5 milliliter micro centrifuge tube pre-chilled by submerging in liquid nitrogen for 30 seconds. Add one milliliter of the ice-cold acid guanidinium phenol solution to each tube and incubate the tubes on ice for 20 minutes.Before homogenizing different hard tissue, thoroughly clean the mortar, pestle and spatula with 70%ethanol, then with RNase decontaminant, followed by DEPC treated water. Soak the mortar, pestle and spatula in 70%ethanol, then wipe any residual liquid with a clean, lint-free tissue. Homogenize the soft tissue using the tissue homogenizer.For disinfecting the homogenizer, wash the probe by running tubes of 70%ethanol and RNase decontaminant for 30 seconds per wash, then wash with DEPC treated water, followed by an additional 70%ethanol, each for 30 seconds. Wipe any residual liquid with a clean, lint-free tissue. Add one milliliter of acid guanidinium phenol solution to pre-labeled, five milliliter round bottom tubes, and transfer the tissue sample into the tube.Place the tube on ice throughout the procedure and homogenize the tissue for a maximum of five 32nd pulses, or until the tissue is visually dissolved. Incubate the homogenized tissue on ice until all the tissue samples are processed. Disinfect and clean the homogenizer before processing the next tissue sample.Using sterile forceps, remove any tissue chunk present in the teeth of the probe. Incubate all the homogenized tissue on the ice for additional 20 minutes and then transfer the tissue sample to a pre-labeled, pre-chilled 1.5 milliliter micro centrifuge tube. Seven unique human knee joint tissues obtained from osteoarthritis patients were processed within four hours of surgical removal.The tissues were identified visually and confirmed by H&E histological staining. The H&E stained hard tissue sections, articular cartilage, subchondral bone and meniscus were visualized at six times magnification and 40 times magnification. Similarly, the H&E stained soft tissue sections, infrapatellar fat pad, anterior cruciate ligament, synovium and vastus medialis oblique muscle were visualized at six times magnification and then at 40 times magnification.The quality assessment of extracted RNA indicated the presence of high quality samples based on integrity, yield and purity, as well as low quality samples, suggesting that despite using an optimized method, external factors like disease severity may impact the RNA quality across different tissues from the same patient. Correct identification of each tissue type is necessary to enable intra-and inter-tissue comparisons within and across patient samples, especially given the considerable heterogeneity of primary disease tissues. Sequencing technologies are rapidly evolving, and isolation of high quality RNA is a critical prerequisite to using these technologies to unravel underlying tissue-specific mechanisms in complex, chronic diseases like osteoarthritis.

tissue-collection-rna-extraction-from-human-osteoarthritic-knee

Research

JoVE Journal

Biology

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

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

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

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

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

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

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

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

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

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

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

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 in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

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

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

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

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

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

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

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

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...