Abstract
Evaluating RNA quality and transcriptomic profile of beef muscle over time post-mortem may provide insight into RNA degradation and underlying biological and functional mechanisms that accompany biochemical changes occurring post-mortem during transformation of muscle to meat. RNA was extracted from longissimus thoracis (LT) sampled from British Continental crossbred heifer carcasses (n = 7) stored at 4°C in an abattoir drip cooler at 5 time points post-mortem, i.e., 45 min (0 h), 6 h, 24 h, 48 h, and 72 h. Following RNA-Sequencing, processed reads were aligned to the ARS-UCD1.2 bovine genome assembly. Subsequent differential expression (DE) analysis identified from 51 to 1434 upregulated and 27 to 2256 downregulated DE genes at individual time points compared to time 0 h, showing a trend for increasing counts of both upregulated and downregulated genes over time. Gene ontology and biological pathway term enrichment analyses on sets of DE genes revealed several processes and their timelines of activation/deactivation that accompanied or were involved with muscle transformation to meat. Although the quality of RNA in refrigerated LT remained high for several days post-mortem, the expression levels of several known biomarker genes for meat quality began to change from 24 h onwards. Therefore, to ensure accuracy of predictions on meat quality traits based on the expression levels of those biomarker genes in refrigerated beef muscle tissue, it is crucial that those expression measurements be made on RNA sampled within 24 h post-mortem. The present study also highlighted the need for more research on the roles of mitochondrial genes and non-coding genes in orchestrating muscle tissue processes after death, and how pre-mortem immune status might influence post-mortem meat quality.
Highlights
IntroductionRNA quality and functional transcriptome of beef muscle over time post-mortem and novel transcript discovery
14,630 genes (52.99% of the total) were identified as expressed in the longissimus thoracis (LT) samples used in this experiment, following filtering out of low expressed genes
A principal component analysis (PCA) plot based on the normalised read counts showed samples separated by time point along the first principal component (PC1) with separation becoming more distinct towards later time points, indicative of substantial and progressively increasing changes in gene expression with time post-mortem (S2 Fig)
Summary
RNA quality and functional transcriptome of beef muscle over time post-mortem and novel transcript discovery. The use of RNA-Seq for gene expression analysis relies on careful selection of experimental conditions, including sampling time, physiological condition of individuals sampled, environmental factors, biological tissue, tissue sampling location, as well as sample storage conditions. Obtaining high quality RNA is especially challenging from postmortem tissue where RNA extraction may either not be possible immediately after organismal death or where RNA samples are required at extended time points post-mortem. Initial RNA quality and RNA degradation rates are tissue type dependent It has been found in beef carcasses stored at 4 ̊C that RNA sampled from skeletal muscle remained at high quality for longer durations post-mortem than RNA from liver and adipose tissue [2]. Differences in RNA degradation rates have been attributed in part to the concentration of ribonucleases already present in cells and/or originating from bacteria or other environmental contamination, with ribonuclease-rich organs such as pancreas and liver exhibiting quicker RNA fragmentation than others [4]
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