RNA expression profiling from the liquid fraction of synovial fluid in knee joint osteoarthritis patients

Abstract

Objective: To investigate the RNA profile of synovial fluid (SF) from osteoarthritis (OA) patients and carry out cluster analysis of OA-related genes.

Methods: RNA of SF from OA patients was isolated using RNA-specific Trizol. A cDNA library was built and subjected to the second-generation sequencing using HisSeq4000 with a data size of 8G. The sequencing reads were aligned to the UCSC human reference genome (hg38) using Tophat with default parameters. Gene function enrichment was generated using DAVID.

Results: The minimum weight 0.096 µg RNA of SF sample was used for sequencing analysis, which produced 66,154,562 clean reads, 91.28% of which were matched to the reference with 2,682 genes identified. Some of the unmatchable reads matched RNAs of bacteria, mainly Pseudomonas. The detected human RNAs in samples fell into different categories of genes, including protein-coding ones, processed and unprocessed pseudogenes, and long noncoding, antisense and miscellaneous RNAs that mediate various biological functions. Interestingly, 80% of the expressed genes belonged to the mitochondrial genome.

Conclusion: These results suggest that less than 0.1 µg RNA is sufficient for establishing a cDNA library and deep sequencing, and that the liquid fraction of SF contains a whole RNA repertoire that may reflect a history of previous microorganism infections.

Keywords: Osteoarthritis; knee joint; second-generation sequencing; synovial fluid; transcriptome.

Figure 1

Procedure of establishment of cDNA library.

Figure 2

Distribution of detected RNA genes into different gene categories in samples. A: Different RNA gene categories in Sample Li; B: Distribution of RNA genes in Sample Niu. Note: LincRNA: long intervening noncoding RNA; misc_RNA: miscellaneous RNA; TEC: to be experimentally confirmed; IG_V_pseudogene: immunoglobulin_V_pseudogene; IG_V_gene: immunoglobulin_V_gene; Mt_rRNA: mitochondrial rRNA; TR_V_pseudogene: T cell receptor_V_pseudogene; snoRNA: small nuclear RNA; IG_C_gene: immunoglobulin_C_gene.

Figure 3

Distribution of detected RNA genes for different subcellular or functional categories, arranging in the percentage occupied by this number in the total identified genes from samples. A: Sample Li; B: Sample Niu.