RNA Integrity Is Not Affected When Tissue Sampling Is Performed Post-Scald in Market Weight Yorkshire Pigs
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Tissue samples are collected as quickly as possible following exsanguination to minimize the risk of RNA degradation and facilitate gene expression assays. However, collecting subcutaneous adipose tissue and longissimus muscle requires cutting through the hide which leaves the underlying tissue exposed during scalding-dehairing which is counter to best practice for collecting meat quality data and poses possible food safety issues. In order to test the effect of sampling pre- or post-scalding on RNA-based assays and indices of meat quality, subcutaneous adipose and longissimus dorsi tissues were harvested from the right tenth rib of market weight Yorkshire hogs (n=8) immediately following exsanguination and again immediately following scalding. Total RNA was extracted from all samples and RNA quality was assessed both visually by gel electrophoresis and by determining an RNA Integrity Number (RIN). The expression of adipose and muscle marker genes were then measured using real-time PCR. Ultimate pH, visual color score, and objective Hunter color scores were compared between carcasses that had been sampled prior to scalding and those carcasses that had not been sampled at all, (n=8). All RNA samples exhibited sharp ribosomal bands with a 28S to 18S ratio greater than one when visualized on a denaturing gel. All RIN values were greater than 8.8 while no differences in OD 260/280 ratio (P > 0.71) or RIN values (P > 0.21) existed between sampling times indicating that scalding did not negatively affect RNA integrity in either adipose tissue of longissimus muscle. There were no differences in the mRNA expression levels of the ADIPOQ, LEP, GLUT4 or PPAR genes in adipose tissue or CKM, MYOG, GLUT4 or TNNT1 in longissimus muscle sampled pre- or post-scalding as determined by real-time PCR. However, sampling tissue prior to scalding resulted in greater visual color score (P < 0.001) and lesser L* (P < 0.001) and b* (P < 0.001) values while neither a* values (P > 0.53) nor 24h pH (P > 0.41) was affected. These data indicate that sampling post-scalding did not impair RNA quality or the ability to measure gene expression via RNA-based assays such as real-time PCR. However, sampling tissue prior to scalding and post scalding did result in darker color of the underlying muscle 24 h postmortem. Thus, if both RNA-based assays and meat quality endpoints are to be performed using the same animal, the best choice would be that tissue sampling should occur at a time point immediately following scalding.