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. Examples of exclusion criteria include undergoing partial knee replacement or having a confirmed diagnosis of rheumatoid arthritis.</li>
	<li>Contact the patients to obtain informed consent prior to the surgery.</li>
</ol>
2. Sample processing (for RNA)
NOTE: Perform all tissue processing in a class II biosafety cabinet and follow sterile techniques. Always wear appropriate PPE (nitrile gloves, lab coat, safety goggles) when processing human samples. Several bone fragments are produced during TKA, a large amount of bone/cartilage will potentially be available for dissection. Due to disease progression, articular cartilage degeneration may be more severe on some bone portions, which can be factored into experimental design. Only tissues that mandate electrocautery for resection have thermal edge damage, and a concerted surgical effort is made to procure most tissues with a scalpel to minimize damage. Resected tissues must be kept hydrated at all times with sterile PBS.
<ol>
	<li>Disinfect all work surfaces and equipment with 70% ethanol, RNase decontaminant, DEPC-treated water, and again with 70% ethanol. Wipe away residual liquid with clean, lint-free tissues. Forceps, bone cutters, and scalpels are either autoclaved or soaked in 70% ethanol for at least 10 min prior to use. Keep equipment submerged in 70% ethanol when not in immediate use.</li>
	<li>Pre-label at least three cryovials with de-identified sample name and aliquot number for each tissue (e.g., TKA-1 Cartilage 1).
	<ol>
		<li>Identify each tissue type from the specimen based on the differences in size, shape, color, and texture as shown and described in Figure 2. Identify the cartilage (Figure 2A arrow), bone (Figure 2B arrow), meniscus (Figure 2C), infrapatellar fat pad (Figure 2D), anterior cruciate ligament (Figure 2E), synovium (Figure 2F), and the vastus medialis oblique muscle (Figure 2G).</li>
	</ol>
	</li>
	<li>Isolate the articular cartilage.
	<ol>
		<li>Select a bone portion with minimal cartilage degradation.</li>
		<li>Using a No.10 scalpel, cut through the cartilage depth as far as possible to dissect the full thickness of the cartilage layer. 
		NOTE: A No.10 scalpel will only penetrate cartilage, not bone.</li>
		<li>Using the full thickness of the cartilage, dissect three 5 mm cubes.</li>
	</ol>
	</li>
	<li>Isolate the subchondral bone.
	<ol>
		<li>Use the same bone section from which cartilage was collected.</li>
		<li>Using a No.10 scalpel, scrape any remaining cartilage and residual tissues from the bone surface.</li>
		<li>Hold the bone portion to be cut with forceps and use the bone cutters to cut three 5 mm cubes.</li>
	</ol>
	</li>
	<li>Isolate the meniscus.
	<ol>
		<li>Identify a relatively undamaged portion of either medial or lateral meniscus.</li>
		<li>Using a No.10 scalpel and forceps, dissect three 5 mm cubes.</li>
	</ol>
	</li>
	<li>Isolate the infrapatellar fat pad.
	<ol>
		<li>Using a No.10 scalpel and forceps, cut the yellow portion of the tissue into three equal-sized and homogenous portions (~500 mg each).</li>
	</ol>
	</li>
	<li>Isolate the anterior cruciate ligament (ACL).
	<ol>
		<li>Using a No.10 scalpel and forceps, dissect three 5 mm cubes.</li>
	</ol>
	</li>
	<li>Isolate the synovium.
	<ol>
		<li>Using a No.10 scalpel and forceps, isolate the pink cellular portion of the membrane as much as possible by scraping away fat tissue.</li>
		<li>Dissect three equal-sized and homogenous portions (~200 mg each).</li>
	</ol>
	</li>
	<li>Isolate the vastus medialis oblique muscle (VMO).
	<ol>
		<li>Using a No.10 scalpel and forceps, remove any fat tissue from the specimen, leaving only the red muscle tissue.</li>
		<li>Dissect three 5 mm cubes. 
		NOTE: The initial size of the tissue limits the size of the portions.</li>
	</ol>
	</li>
	<li>Rinse the tissue portions with sterile PBS to remove any residue or debris.</li>
	<li>To perform histology, fix each tissue portion as described in Part 3.</li>
	<li>To perform RNA extraction, cut each of the three tissue portions into smaller pieces (~1-2 mm&#160;cubes).
	<ol>
		<li>Transfer the smaller pieces to a 2 mL cryovial, secure caps tightly, flash freeze by submerging in liquid nitrogen for 30 s, and then transfer to a -80 &#176;C freezer for long-term storage (up to 4 months tested in the current protocol). Repeat this for all the aliquots of all the tissues.</li>
		<li>Continue to Tissue homogenization as described in Part 4.</li>
	</ol>
	</li>
</ol>
3. Sample processing (for histology)
CAUTION: Formalin is a hazardous chemical, only use in a chemical fume hood.
<ol>
	<li>Fill pre-labeled 15 mL conical tubes with 10% formalin solution.</li>
	<li>Using forceps, transfer the tissue section to the formalin-filled tubes.</li>
	<li>Fix the tissues in formalin for 1 week at room temperature while shaking/agitating if possible.</li>
	<li>After 1 week, discard the formalin into proper chemical waste disposal, rinse the tissues with PBS, and then transfer to a fresh 15 mL conical tube containing 70% ethanol for long-term storage at 4 &#176;C until samples are embedded for sectioning. 
	NOTE: For bone/cartilage only, perform decalcification as follows.</li>
	<li>After 1 week, discard the formalin into proper chemical waste disposal, and rinse the tissues with PBS.</li>
	<li>Transfer the tissue to a 50 mL conical tube with 45 mL of 10% EDTA solution (pH 7.4).</li>
	<li>If shaking/agitation is not possible, invert the tubes 10-15 times once daily.</li>
	<li>Discard and replace the EDTA solution once weekly.
	<ol>
		<li>Twice weekly, test the texture with an instrument (i.e., spatula) or gloved finger to confirm decalcification by observing deformation when pressure is applied. Time required to decalcify bone tissue will vary between samples from 4-6 weeks.</li>
	</ol>
	</li>
	<li>Once decalcified, rinse the tissue with PBS and transfer to a fresh 15 mL conical tube containing 70% ethanol for long-term storage at 4 &#176;C until samples are embedded for sectioning.</li>
</ol>
4. Tissue homogenization
CAUTION: The protocol uses the hazardous chemical phenol. Work with phenol must be performed in a chemical fume hood.
NOTE: Thoroughly clean all equipment and surfaces to be used with 70% ethanol (soak for a minimum of 10 min), followed by RNase decontaminant (soak for a minimum of 10 min), rinse with DEPC-treated water, wipe with a clean, lint-free paper towel, and then respray or soak with 70% ethanol.
<ol>
	<li>Hard tissue homogenization (articular cartilage, subchondral bone, meniscus)
	<ol>
		<li>Before homogenization, chill the mortar, pestle, and spatula using liquid nitrogen. These must be kept as cold as possible to prevent sample thaw.</li>
		<li>Process the samples one at a time, keeping the other samples at -80 &#176;C until used.</li>
		<li>Transfer the tissue sample to mortar using a chilled spatula; pour additional liquid nitrogen on top of the tissue and allow it to evaporate. Crush the tissue using the pestle. Repeatedly add more liquid nitrogen to the tissue sample, allow it to evaporate, and then continue grinding with the pestle to make a fine powder.</li>
		<li>After powdering the tissue as much as possible, transfer to a pre-chilled 1.5 mL microcentrifuge tube.
		<ol>
			<li>Pre-chill tubes by submerging in liquid nitrogen for 30 s prior to tissue transfer.</li>
		</ol>
		</li>
		<li>Add 1 mL of the acid-guanidinium-phenol solution to each tube and keep it on ice.</li>
		<li>Repeat steps 4.1.3-4.1.5 for each hard tissue sample.</li>
		<li>After each tissue, clean the mortar, pestle, and spatula with 70% ethanol, RNase decontaminant, DEPC-treated water, and an additional soak in 70% ethanol. Wipe any residual liquid with a clean, lint-free tissue.</li>
		<li>Incubate the samples on ice for an additional 20 min.</li>
	</ol>
	</li>
	<li>Soft tissue homogenization (infrapatellar fat pad, ACL, synovium, VMO)
	<ol>
		<li>Disinfect the homogenizer by running tubes of 70% ethanol, RNase decontaminant, DEPC-treated water, and an additional 70% ethanol wash, each for 30 s. Wipe any residual liquid with a clean, lint-free tissue. Repeat this between each sample.</li>
		<li>Pre-label 5 mL round bottom tubes for each sample. Add 1 mL of the acid-guanidinium-phenol solution to each tube.
		<ol>
			<li>Work on one sample at a time, keeping other samples to be processed at -80 &#176;C until use.</li>
		</ol>
		</li>
		<li>Transfer the tissue to a pre-labeled 5 mL tube with acid-guanidinium-phenol.</li>
		<li>Homogenize the tissues in 30 s pulses, keeping on ice during and between pulses. Repeat until the tissue is visually dissolved or for a maximum of five 30 s pulses. 
		NOTE: Some fibrous tissues (i.e., muscle) may not thoroughly homogenize.</li>
		<li>Incubate the dissolved tissue on ice and move to the next sample.
		<ol>
			<li>Clean the homogenizer as described in step 4.2.1. Ensure tissue chunks do not remain in the teeth of the probe; remove with sterile forceps if needed.</li>
		</ol>
		</li>
		<li>After homogenization of all the samples, incubate on ice in acid-guanidinium-phenol for an additional 20 min.</li>
		<li>Transfer the samples from round bottom tubes to pre-labeled and pre-chilled 1.5 mL microcentrifuge tubes.</li>
	</ol>
	</li>
</ol>
5. RNA extraction from tissues
CAUTION: This protocol uses hazardous chemicals such as phenol, chloroform, and isopropanol. Perform all the work in a chemical fume hood.
NOTE: Equipment and reagents are reserved for RNA work only and must be of proper chemical grade for molecular applications (i.e., sterile, nuclease-free). This protocol succeeds both Parts 4.1 and 4.2 tissue homogenization protocols. Thoroughly clean all equipment and surfaces to be used with 70% ethanol (soak for a minimum of 10 min), followed by RNase decontaminant (soak for a minimum of 10 min). Rinse with DEPC-treated water, wipe the residual liquid with a clean, lint-free paper towel, and then respray or soak with 70% ethanol.
<ol>
	<li>Centrifuge the microcentrifuge tubes at 10000 x g for 10 min&#160;at 4 &#176;C to pellet the debris.</li>
	<li>Transfer the supernatant to a fresh 1.5 mL microcentrifuge tube. 
	NOTE: For fatty tissues, a lipid layer will sometimes be present on the top. Avoid transferring this by piercing the layer on the side of the tube with a pipette tip.</li>
	<li>Add 200 &#181;L of chloroform per 1 mL of the acid-guanidinium-phenol solution to each sample. Shake the tubes vigorously by hand for 30 s to mix. Then, incubate on ice for 2 min.</li>
	<li>Centrifuge samples at 10000 x g for 12 min at 4 &#176;C. 
	NOTE: After centrifugation, three layers will have formed: an aqueous phase containing RNA, a white DNA interphase, and a pink protein phase at the bottom.</li>
	<li>Transfer ~500 &#181;L of the upper, aqueous phase to a fresh 1.5 mL microcentrifuge tube. 
	NOTE: Do not disturb the interphase and the protein fractions. Store these phases at -80 &#176;C for future DNA or protein isolation.</li>
	<li>Add an equal volume of acid-guanidinium-phenol solution to the transferred aqueous phase, mix by inverting the tube 8-10 times. Incubate the tube on ice for 20 min.</li>
	<li>Add 200 &#181;L of chloroform per 1 mL of the acid-guanidinium-phenol solution to each sample. Shake vigorously by hand for 30 s to mix and then incubate on ice for 2 min.</li>
	<li>Centrifuge samples at 10000 x g for 12 min at 4 &#176;C.</li>
	<li>Transfer &#60;500 &#181;L of the aqueous phase to a fresh microcentrifuge tube, careful not to contaminate the sample with the other phases. 
	CAUTION: Discard the remaining phases with appropriate hazardous material disposal methods.</li>
	<li>Add an equal volume (as aqueous phase) of 100% isopropanol to each sample. Mix by inverting the tube 8-10 times. Incubate on ice for 5 min.
	<ol>
		<li>Add 1 &#181;L of glycogen coprecipitant to each sample to aid in locating the RNA pellet post-centrifugation.</li>
	</ol>
	</li>
	<li>Centrifuge the samples at 12000 x g for 25 min at 4 &#176;C.</li>
	<li>Locate the pellet in the tube (if using coprecipitant, it will appear blue). Carefully pour off the supernatant.</li>
	<li>Wash the pellet by adding 1 mL of ice-cold 75% ethanol to each sample, vortex to dislodge the pellet from the bottom of the tube. 
	NOTE: Prepare 75% ethanol using molecular grade pure ethanol and nuclease-free water.</li>
	<li>Centrifuge at 7000 x g for 5 min at 4 &#176;C. Carefully pour off the supernatant.</li>
	<li>Repeat steps 5.13 and 5.14 two more times.</li>
	<li>Quick spin (&#60;2000 x g for 5 s) to bring any residual liquid to the bottom of the tube. Use a P20 pipette to remove any residual ethanol from the bottom of the tubes.
	<ol>
		<li>Avoid touching the RNA pellet with a pipette tip. Change tips between samples.</li>
	</ol>
	</li>
	<li>With tube caps open, air dry samples at room temperature for 10 min. 
	NOTE: Pellet may become translucent as it dries. Ensuring all residual ethanol has evaporated will improve RNA purity.</li>
	<li>Add 25 &#181;L of nuclease-free water to each tube to dissolve the pellet.</li>
	<li>Incubate at room temperature for 5 min.</li>
	<li>Gently pipette up and down to mix the RNA.</li>
	<li>Aliquot 5 &#181;L of the sample into a fresh tube for quality control analysis (Part 6). Store the remaining 20 &#181;L at -80 &#176;C for gene expression assays.</li>
</ol>
6. Quality control
<ol>
	<li>Determine the concentration and purity of RNA using a spectrophotometer according to the manufacturer&#39;s instructions.</li>
	<li>Determine the RNA integrity using an electrophoresis device according to the manufacturer&#39;s instructions. 
	NOTE: Dilute the RNA to be within the range of the chip detection limits.</li>
</ol>

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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>

The authors declare no conflicts of interest.

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 ...

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

Research

JoVE Journal

Biology

5.7K Views.  Henry Ford Health System. 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.

Video: Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

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 ...