Insulin-like growth factor-I in growing horses and RNA isolation from small articular cartilage samples
Cosden, Rebekah Stacey
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A longitudinal study was designed to characterize developmental patterns of plasma (PL) and synovial fluid (SF) total insulin-like growth factor-I (IGF-I) concentrations, as well as their association with measurements of skeletal growth in Thoroughbred horses. Horses were randomly assigned to one of two dietary treatment groups and fed diets with either a high or low starch content to examine the effects of dietary energy source on PL and SF IGF-I. At 3, 6, 9, 12 and 15 mo of age, PL and carpal SF samples were collected for analysis of total IGF-I. Body weight gain, wither height gain and forearm length gain were calculated for the 90 day periods between SF and PL sampling. No influence of diet on PL or SF IGF-I was detected (P > 0.05). Average SF IGF-I concentrations were 30.1 Â± 1.8% of that found in PL, and PL and SF IGF-I were positively correlated (r = 0.48, P = 0.0003) There was an effect of month of age on both PL and SF IGF-I concentrations (P < 0.05). There was a positive correlation between all measures of gain except forearm length gain with PL and SF IGF-I (r = 0.41 to 0.55, P < 0.05). In our second study, we evaluated the use of a liquid-nitrogen cooled mortar and pestle, motorized freezer mill and rotor-stator homogenizer for homogenization of small (<50mg) articular cartilage samples. The rotor-stator homogenizer produced quanitfiable RNA yields, and was used to evaluate three different RNA isolation protocols. Two of the protocols were commercially available RNA extraction kits, with the third a modified guanidinium isothiocyanate/acid-phenol extraction procedure. The combined average yield for all protocols was 91.9 ng RNA/mg of cartilage. All protocols yielded a sufficient quantity of quality RNA suitable for gene expression analysis.
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