Assessment of RNA Stability in Postmortem Tissue from New-Born Lambs
The recovery of high quality RNA from postmortem tissue is crucial to gene expression analyses. The acquisition of postmortem tissue has inherent time delays and, hence, understanding the temporal variation in the stability of total RNA is imperative. This experiment aimed: ( 1 ) to qualitatively and quantitatively assess the integrity of total RNA derived from a range of new-born ovine tissues (liver, spleen, thyroid, skeletal muscle, ileum, and perirenal adipose tissue) which were stored at ambient temperature until extraction at 0, 3, 6, and 9 h postmortem; and ( 2 ) to analyze the stability of the reference gene(s) and expression of specific target genes in these tissues. Postmortem sampling time resulted in variable reductions in the relative integrity number (RIN) values across the tissues, ranging from 0.9 to 1.8% in liver, spleen, skeletal muscle, and ileum to 5.7-11.1% in the thyroid and perirenal adipose tissues, respectively (P < 0.05). In conclusion, tissues with small reductions in RIN value can exhibit disproportionately large differences in the normalization factor used to calculate the target gene expression. Hence, changes in transcript abundance due to RNA degradation are not always sufficiently buffered through normalization with reference genes. The normalization factor should be presented alongside the RIN value in postmortem tissue studies.