Assessment of the use of RNA quality metrics for the screening of articular cartilage specimens from clinically normal dogs and dogs with osteoarthritis.
OBJECTIVE
To assess 2 methods of RNA purification by use of different quality metrics and identify the most useful metric for quality assessment of RNA extracted from articular cartilage from dogs with osteoarthritis.


METHODS
40 articular cartilage specimens from the femoral heads of 3 clinically normal dogs and 37 dogs with osteoarthritis.


METHODS
RNA was extracted from articular cartilage by 2 purification methods. Quality metrics of each sample were determined and recorded by use of a UV spectrophotometer (Spec I; to determine the 260 to 280 nm absorbance ratio [A(260):A(280) ratio]), a second UV spectrophotometer (Spec II; to determine A(260):A(280) and A(260):A(230) absorbance ratios), and a microfluidic capillary electrophoresis analyzer (to determine the ribosomal peak ratio [RR], degradation factor [DF], and RNA integrity number [RIN]). The RNA was extracted from affected (osteoarthritic) articular cartilage and assessed with the same quality metrics. Metric results were compared with visual analysis of the electropherogram to determine the most useful RNA quality metric.


RESULTS
No differences in methods of RNA purification were determined by use of quality metrics. The RNA extracted from unaffected (normal) cartilage was of higher quality than that extracted from affected (osteoarthritic) cartilage, as determined by the RIN and Spec II A(260):A(230) ratio. The RIN and RR were the most sensitive metrics for determining RNA quality, whereas the DF was most specific. A significant proportion (32%) of RNA extracted from osteoarthritic articular cartilage specimens was determined as being of low quality.


CONCLUSIONS
No single metric provided a completely sensitive and specific assessment of the quality of RNA recovered from articular cartilage.
Clements
Dylan N
DN
Connective Tissue Research Group, Faculty of Veterinary Science, University of Liverpool, Liverpool, L69 3BX, UK.
Vaughan-Thomas
Anne
A
Peansukmanee
Siriporn
S
Carter
Stuart D
SD
Innes
John F
JF
Ollier
William E R
WE
Clegg
Peter D
PD
D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism*; D004283:Dog Diseases / Q000175:diagnosis* / Q000235:genetics*; D004285:Dogs / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D010003:Osteoarthritis / Q000175:diagnosis / Q000235:genetics / Q000662:veterinary*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*
D012313:RNA