title: Transcriptome analysis of ageing in uninjured human Achilles tendon.
abstract: BACKGROUND
  The risk of tendon injury and disease increases significantly with increasing age. The aim of the study was to characterise transcriptional changes in human Achilles tendon during the ageing process in order to identify molecular signatures that might contribute to age-related degeneration.
  
  
  METHODS
  RNA for gene expression analysis using RNA-Seq and quantitative real-time polymerase chain reaction analysis was isolated from young and old macroscopically normal human Achilles tendon. RNA sequence libraries were prepared following ribosomal RNA depletion, and sequencing was undertaken by using the Illumina HiSeq 2000 platform. Expression levels among genes were compared by using fragments per kilobase of exon per million fragments mapped. Differentially expressed genes were defined by using Benjamini-Hochberg false discovery rate approach (P<0.05, expression ratios 1.4 log2 fold change). Alternative splicing of exon variants were also examined by using Cufflinks. The functional significance of genes that showed differential expression between young and old tendon was determined by using ingenuity pathway analysis.
  
  
  RESULTS
  In total, the expression of 325 transcribed elements, including protein-coding transcripts and non-coding transcripts (small non-coding RNAs, pseudogenes, long non-coding RNAs and a single microRNA), was significantly different in old compared with young tendon (±1.4 log2 fold change, P<0.05). Of these, 191 were at higher levels in older tendon and 134 were at lower levels in older tendon. The top networks for genes differentially expressed with tendon age were from cellular function, cellular growth, and cellular cycling pathways. Notable differential transcriptome changes were also observed in alternative splicing patterns. Several of the top gene ontology terms identified in downregulated isoforms in old tendon related to collagen and post-translational modification of collagen.
  
  
  CONCLUSIONS
  This study demonstrates dynamic alterations in RNA with age at numerous genomic levels, indicating changes in the regulation of transcriptional networks. The results suggest that ageing is not primarily associated with loss of ability to synthesise matrix proteins and matrix-degrading enzymes. In addition, we have identified non-coding RNA genes and differentially expressed transcript isoforms of known matrix components with ageing which require further investigation.
authors.affiliation: Comparative Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst, Chester High Road, Neston, Wirral, CH64 7TE, UK. peffs@liv.ac.uk.
authors.forename: Mandy Jayne
authors.initials: MJ
authors.lastname: Peffers
authors.affiliation: Centre for Genomic Research, Institute of Integrative Biology, University of Liverpool, Biosciences Building, Crown Street, Liverpool, L69 7ZB, UK. fangy@liverpool.ac.uk.
authors.forename: Yongxiang
authors.initials: Y
authors.lastname: Fang
authors.affiliation: Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK. k.cheung@newcastle.ac.uk.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Cheung
authors.affiliation: School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, 535 Clementi Road, Singapore, 599489, Singapore. timxum@gmail.com.
authors.forename: Tim Koh Jia
authors.initials: TK
authors.lastname: Wei
authors.affiliation: Comparative Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst, Chester High Road, Neston, Wirral, CH64 7TE, UK. pclegg@liverpool.ac.uk.
authors.forename: Peter David
authors.initials: PD
authors.lastname: Clegg
authors.affiliation: Institute of Orthopaedics and Musculoskeletal Science, University College London, Stanmore Campus, Royal National Orthopaedic Hospital, Brockley Hill, Stanmore, HA7 4LP, UK. h.birch@ucl.ac.uk.
authors.forename: Helen Lucy
authors.initials: HL
authors.lastname: Birch
mesh_terms: D000125:Achilles Tendon / Q000378:metabolism*; D000375:Aging / Q000235:genetics*; D020869:Gene Expression Profiling; D006801:Humans; D012313:RNA / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D012313:RNA