Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Summary

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.

Abstract

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.

Introduction

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 cavity^1,2,3 (Figure 1). A healthy knee functions as a mobile hinge joint that allows frictionless motio....

Protocol

This study protocol was approved and followed institutional guidelines set by the Henry Ford Health System Institutional Review Board (IRB #13995).

1. Patient selection

1. Identify the patients from among those scheduled to undergo TKA with an orthopedic surgeon.
2. 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.
3. Contact the....

Discussion

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.......

Materials

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