Journal
JOURNAL OF ANIMAL SCIENCE
Volume 90, Issue -, Pages 22-24Publisher
OXFORD UNIV PRESS INC
DOI: 10.2527/jas.53935
Keywords
colon; gene expression; RNA integrity number value; RNA stability
Categories
Funding
- Marine Institute
- Department of Agriculture, Food and the Marine
- National Development Plan
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Surgical removal of porcine intestinal tissue followed by an ex vivo challenge is an alternative technique of testing the anti-inflammatory effect of bioactive compounds in the intestine of live pigs. We investigated the effects of ex vivo incubation of porcine colonic tissue on the quantity and quality of total RNA over a 12-h time period. Colonic tissue of pig (n = 6) was surgically removed immediately postslaughter and incubated for 0, 3, 6, and 12 h in a humidified cell culture incubator with 5% CO2 at 37 degrees C. Tissue samples were processed for RNA extraction. The quantity and quality of total RNA were assessed on a NanoDrop Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. Ex vivo incubation had an effect on both the quantity (P < 0.001) and quality (P < 0.001) of total RNA. Relative to the RNA yield at 0 h (505.0 +/- 48.64 mu g/mg), the yield was significantly reduced after 6 h (227.6 +/- 25.52 mu g/mg; P < 0.001) and 12 h (159.3 +/- 24.19 mu g/mg; P < 0.001) of incubation. The 28S and 18S rRNA bands were visibly intact after 0, 3, and 6 h of incubation. However, after 12 h of incubation, a degraded RNA profile was evident. The RNA integrity number (RIN) values for the 0, 3, 6, and 12 h of incubation were 9.4 +/- 0.10, 9.0 +/- 0.10, 6.7 +/- 0.17 (P < 0.001), and 3.3 +/- 0.24 (P < 0.001), respectively. The transcript abundances of 4 constitutively expressed genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), beta 2-microglobulin (B2M), and peptidylprolyl isomerase A (PPIA) were reduced at both 6 and 12 h of incubation. It is concluded that ex vivo incubation of porcine colonic tissue up to 3 h postmortem generates good quality total RNA suitable for gene expression studies.
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