Isolation of High‐Quality Total RNA from Small Animal Articular Cartilage for Next‐Generation Sequencing

Abstract

Articular cartilage is characterized by a low density of chondrocytes surrounded by an abundant extracellular matrix (ECM) consisting of a dense mixture of collagens, proteoglycans, and glycosaminoglycans. Due to its low cellularity and high proteoglycan content, it is particularly challenging to extract highquality total RNA suitable for sensitive high-throughput downstream applications such as RNA sequencing (RNA-Seq). Available protocols for high-quality RNA isolation from articular chondrocytes are inconsistent, resulting in suboptimal yield and compromised quality. This poses a significant difficulty in the application of RNA-Seq to study the cartilage transcriptome. Current protocols rely either on dissociation of cartilage ECM by collagenase digestion or pulverizing cartilage using various methods prior to RNA extraction. However, protocols for cartilage processing vary significantly depending on the species and source of cartilage within the body. Protocols for isolating RNA from human or large mammal (e.g., horse or cattle) cartilage samples are available, but this is not the case for chicken cartilage, despite the species being extensively used in cartilage research. Here, we present two improved RNA isolation protocols based on pulverization of fresh articular cartilage using a cryogenic mill or on enzymatic digestion using 1.2% (w/v) collagenase II. Our protocols optimize the collection and tissue processing steps to minimize RNA degradation and enhance RNA purity. Our results show that RNA purified from chicken articular cartilage using these methods has appropriate quality for RNA-Seq experiments. The procedure is applicable for RNA extraction from cartilage from other species such as dog, cat, sheep, and goat. The workflow for RNA-Seq analysis is also described here.

The temporal transcriptomic signature of cartilage formation

“…Full depth articular cartilage shavings were harvested from the knee joints of 35-day-old broilers as described in detail previously ( 26 ). Ethics license number: HB/15-ÉLB/03743–2/2022.…”

“…Ethics license number: HB/15-ÉLB/03743–2/2022. Shavings were minced into small chips (∼1–2 mm 3 ) using sterile scalpels, and 100 mg of cartilage chips was subjected to enzymatic digestion with 1.2% collagenase type II (Merck) in DMEM overnight at 37°C ( 26 ). Some shavings were routinely fixed, paraffin-embedded, sectioned, and stained with HE, acidic DMMB (3% acetic acid, pH 1.8), and neutral DMMB to visualise microscopic morphology.…”

“…On the designated days of culture, HDCs were washed twice with physiological NaCl and then stored at −80°C. Chondrocytes obtained from collagenase type II digested articular cartilage were collected by centrifugation at 5000 × g at 4°C for 10 min ( 26 ). For total RNA isolation, micromass cultures and pelleted articular chondrocytes were dissolved in TRI Reagent (Applied Biosystems, Foster City, CA, USA).…”

“…To obtain global transcriptome data, high-throughput mRNA sequencing analysis was performed using the Illumina sequencing platform. The quality of the total RNA samples was checked using an Agilent BioAnalyzer (Santa Clara, CA, USA) and the Eukaryotic Total RNA Nano Kit according to the manufacturer's protocol ( 26 ). For micromass cultures, samples with an RNA integrity number (RIN) value of >7 were used for library preparation.…”

The temporal transcriptomic signature of cartilage formation

“…On the designated days of culture, HDCs were washed twice with physiological NaCl and then stored at ‒80 °C. Chondrocytes obtained from collagenase type II digested articular cartilage were collected by centrifugation at 5,000× g at 4°C for 10 min (26). For total RNA isolation, micromass cultures and pelleted articular chondrocytes were dissolved in TRI Reagent (Applied Biosystems, Foster City, CA, USA).…”