Low-Input RNA-Sequencing in Patients with Cartilage Lesions, Osteoarthritis, and Healthy Cartilage

Abstract

Objective To analyze and compare cartilage samples from 3 groups of patients utilizing low-input RNA-sequencing. Design Cartilage biopsies were collected from patients in 3 groups ( n = 48): Cartilage lesion (CL) patients had at least ICRS grade 2, osteoarthritis (OA) samples were taken from patients undergoing knee replacement, and healthy cartilage (HC) was taken from ACL-reconstruction patients without CLs. RNA was isolated using an optimized protocol. RNA samples were assessed for quality and sequenced with a low-input SmartSeq2 protocol. Results RNA isolation yielded 48 samples with sufficient quality for sequencing. After quality control, 13 samples in the OA group, 9 in the HC group, and 9 in the CL group were included in the analysis. There was a high degree of co-clustering between the HC and CL groups with only 6 genes significantly up- or downregulated. OA and the combined HC/CL group clustered significantly separate from each other, yielding 659 significantly upregulated and 1,369 downregulated genes. GO-term analysis revealed that genes matched to cartilage and connective tissue development terms. Conclusion The gene expression profiles from the 3 groups suggest that there are no major differences in gene expression between cartilage from knees with a cartilage injury and knees without an apparent cartilage injury. OA cartilage, as expected, showed markedly different gene expression from the other 2 groups. The gene expression profiles resulting from this low-input RNA-sequencing study offer opportunities to discover new pathways not previously recognized that may be explored in future studies.