Malila Yuwares, Srimarut Yanee, U-Chupaj Juthawut, Strasburg Gale, Visessanguan Wonnop
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.
Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824, USA.
Asian-Australas J Anim Sci. 2015 Nov;28(11):1649-56. doi: 10.5713/ajas.15.0167.
Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major) and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR) as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6) immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem). The delayed postmortem duration did not significantly affect A260/A280 and A260/A230 (p≥0.05), suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05), the value was not statistically different between 20 min and 6 h samples (p≥0.05), indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), peptidylprolylisomerase A (PPIA) and TATA box-binding protein (TBP) as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and peroxisome proliferator-activated receptor delta (PPARD) were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05). Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05). The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken skeletal muscle. For the best RNA quality, chicken skeletal muscle should be immediately collected after evisceration or within 20 min postmortem, and rapidly preserved by deep freezing.
基因表达谱分析为与肉质相关的死后分子变化提供了新的见解。为了获得可靠的转录本定量结果，需要高质量的RNA。本研究的目的是分析从鸡骨骼肌（胸大肌）中分离的RNA的完整性及其作为模板用于定量实时聚合酶链反应（qPCR）的能力，该能力是屠宰后间隔时间的函数，这些间隔时间代表了鸡屠宰场中去内脏、胴体冷却和成熟阶段的终点。去内脏后立即从胴体（n = 6）中解剖鸡胸肉，每个样品的三分之一立即速冻并标记为死后20分钟。其余肌肉保存在冰上，直到下一轮样品采集（死后1.5小时和6小时）。死后延迟时间对A260/A280和A260/A230没有显著影响（p≥0.05），表明总RNA的纯度没有改变。除了1.5小时样品中28s:18s核糖体RNA比值略有下降（p<0.05）外，20分钟和6小时样品之间该值无统计学差异（p≥0.05），表明总RNA在6小时内保持完整。使用qPCR研究了编码β-肌动蛋白（ACTB）、甘油醛-3-磷酸脱氢酶（GAPDH）、次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HPRT）、肽基脯氨酰异构酶A（PPIA）和TATA盒结合蛋白（TBP）的参考基因以及与肉质相关基因（胰岛素样生长因子1（IGF1）、丙酮酸脱氢酶激酶同工酶4（PDK4）和过氧化物酶体增殖物激活受体δ（PPARD））的丰度。ACTB、GAPDH、HPRT和PPIA的转录本丰度在所有死后时间点之间存在显著差异（p<0.05）。PDK4和PPARD的转录水平在6小时样品中显著降低（p<0.05）。研究结果表明，死后时间延长对鸡骨骼肌转录本定量的可靠性有不利影响。为了获得最佳的RNA质量，鸡骨骼肌应在去内脏后或死后20分钟内立即采集，并通过深度冷冻快速保存。
