Long-term stability of RNA in post-mortem bovine skeletal muscle, liver and subcutaneous adipose tissues

Abstract

Background: Recovering high quality intact RNA from post-mortem tissue is of major concern for gene expression studies in animals and humans. Since the availability of post-mortem tissue is often associated with substantial delay, it is important that we understand the temporal variation in the stability of total RNA and of individual gene transcripts so as to be able to appropriately interpret the data generated from such studies. Hence, the objective of this experiment was to qualitatively and quantitatively assess the integrity of total and messenger RNA extracted from bovine skeletal muscle, subcutaneous adipose tissue and liver stored at 4 degrees C at a range of time points up to 22 days post-mortem. These conditions were designed to mimic the environment prevailing during the transport of beef from the abattoir to retail outlets.

Results: The 28S and 18S rRNA molecules of total RNA were intact for up to 24 h post-mortem in liver and adipose tissues and up to 8 days post-mortem in skeletal muscle. The mRNA of housekeeping genes (GAPDH and ACTB) and two diet-related genes (RBP5 and SCD) were detectable up to 22 days post-mortem in skeletal muscle. While the mRNA stability of the two housekeeping genes was different in skeletal muscle and liver, they were similar to each other in adipose tissue. After 22 days post-mortem, the relative abundance of RBP5 gene was increased in skeletal muscle and in adipose tissue and decreased in liver. During this period, the relative abundance of SCD gene also increased in skeletal muscle whereas it decreased in both adipose tissue and liver.

Conclusion: Stability of RNA in three tissues (skeletal muscle, subcutaneous adipose tissue and liver) subjected to long-term post-mortem storage at refrigeration temperature indicated that skeletal muscle can be a suitable tissue for recovering biologically useful RNA for gene expression studies even if the tissue is subjected to post-mortem storage for weeks, whereas adipose tissue and liver should be processed within 24 hours post-mortem.

Figure 1

Stability of total RNA in bovine tissues during post-mortem storage at 4°C. The stability of total RNA was determined by agarose gel electrophoresis assay (i) and Agilent Bioanalyzer assay in (ii) skeletal muscle (a) and liver (b) (h- hours; d- days at 4°C). (For subcutaneous adipose tissue, because of the presence of high variability among the four animals studied, it was not appropriate to select an individual representative animal.)

Figure 2

Change in the 28S:18S ratio of total RNA in bovine tissues during post-mortem storage at 4°C. The 28S:18S ratio was determined by Agilent Bioanalyzer for bovine skeletal muscle (a), subcutaneous adipose tissue (b) and liver (c) (error bar = 1 SE of mean, n = 4). Note: the axis has been modified for liver to 2 d.

Figure 3

Agarose gel electrophoresis of RT-PCR products from bovine tissues subjected to post-mortem storage at 4°C up to 22 days. The amplification products from gene specific RT-PCR as assayed on 1.5% agarose gel for skeletal muscle (a), subcutaneous adipose tissue (b) and liver (c) (h- hours; d- days at 4°C). A: GAPDH, B: ACTB, C: RBP5, D: SCD. RT⁺: positive control with genomic DNA, RT^-: negative control with no template DNA.

Figure 4

Normalised relative abundance of RBP5 and SCD gene transcripts in bovine tissues during post-mortem storage at 4°C. Normalization was performed relative to the housekeeping gene/genes in skeletal muscle (a), subcutaneous adipose tissue (b) and liver (c) (error bar = 1 SE of mean (n = 4)).