Postmortem stability of total RNA isolated from rabbit ligament, tendon and cartilage

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Abstract

The stability of RNA, particularly mRNA, in tissues is under complex regulation. Most studies to date have focused on very cellular tissues and not connective tissues such as ligaments, tendons and cartilage. As the availability of such tissues for transplantation or research purposes is frequently delayed following death, it is important to determine whether RNA stability in such tissues is influenced by time postmortem. To approach this question, skeletally mature NZW rabbits were used to investigate RNA integrity over time in dense, hypocellular connective tissues and in several hypercellular organ tissues such as brain, kidney, liver and lung. Samples were analyzed at varying intervals postmortem with respect to rRNA integrity by agarose gel electrophoresis and ethidium bromide staining and mRNA integrity by Northern blot analysis and RT–PCR. No degradation of rRNA or loss in integrity of mRNA for genes of low and high copy number was observed up to 96 h postmortem. These findings confirm that it is likely appropriate to use properly stored postmortem dense connective tissues for molecular biological investigations.

Introduction

The regulation of RNA turnover, particularly mRNA expression, is a complex process which is influenced by many factors. These include induction or synthesis factors, as well as other factors which control the degradation rates of the RNA 1, 2, 3, 4. These latter include modification of polyA regions, presence of nuclease specific sequences and the presence of RNA-binding proteins which stabilize specific mRNA species [1]. These factors, as well as other potential influences, contribute to the heterogeneity observed in mRNA turnover, with mRNA for some molecules turning over rapidly and others exhibiting a more stable phenotype. As pointed out by Ross [4], much of our understanding regarding RNA turnover has been derived from in vitro cell studies and the study of RNA turnover in animals is more restricted. Such studies have focused on very cellular tissues such as the liver, which respond to a variety of stimuli for the rapid induction and turnover of a number of mRNA species (i.e. acute phase reactants) [5]. Some studies have addressed the issue of postmortem RNA stability in cellular human tissues such as lung and pancreas [6], brain, lung, liver, heart and kidney [7]or heart, lung, liver and kidney [8]. However, the study of RNA stability in very hypocellular tissues such as ligament, tendon and cartilage has not been addressed to any degree. This deficiency in information is of interest for three reasons. First, it is of interest to determine whether RNA stability in cells of hypocellular, and relatively hypoxic, environments such as in ligaments, regulate turnover differently than more cellular and vascularized tissues. The second reason is of a more practical nature and relates to the postmortem availability of human tissues. The third reason is that joint tissues do not heal well for reasons which remain obscure. For example, ruptured human anterior cruciate ligaments (ACLs) tend to heal poorly 9, 10, 11and abnormalities in matrix protein production and/or degradation at the gene level may be implicated 12, 13, 14, 15. Molecular analysis of ACL tissues may help to clarify this issue.

To determine whether postmortem material of connective tissues could provide intact RNA, we undertook a controlled study of animal tissue stored at 4°C versus others maintained at room temperature. In order to compare hypocellular connective tissues to more cellular organs, the stability of RNA in brain, kidney, liver and lung was also investigated.

Section snippets

Tissue preparation

Skeletally mature female NZW rabbits were euthanized by an overdose of sodium pentobarbital (Euthanyl, MTC Pharmaceuticals, Cambridge, Ontario). Tissues from the right knee joint (medial collateral, lateral collateral and anterior cruciate ligaments, patellar tendon, and cartilage) were collected immediately, while the same tissues from the left knee joint were stored at 4°C until collection at 24, 48, 72 and 96 h after death. Small samples of brain, kidney, liver and lung were also collected at

RNA yields from connective tissues

The yield of total RNA did not change significantly over time in any of the connective tissues collected (Table 2). The mean yield at time zero for MCL, LCL and ACL ranged from 0.128 to 0.079 μg/mg wet weight. The mean yield for patellar tendon (PT) was 0.043 μg/mg wet weight. This finding is in keeping with the total DNA concentration differences for these tissues where normal rabbit patellar tendon has less DNA than the knee ligaments [21]. Such yields of RNA are considerably lower than yields

Discussion

The results presented in this report demonstrate that rRNA and mRNA from hypocellular connective tissue is not overtly degraded for up to 96 h postmortem at 4°C and up to 48 h postmortem at room temperature. This was confirmed using ethidium bromide stained agarose gels, Northern blotting and RT–PCR. In all cases, the results are consistent for both rRNA and mRNA. Furthermore, the mRNA results for all of the transcripts investigated yielded similar outcomes. Thus, RNA in these hypocellular

Acknowledgements

The authors thank Drs. G.H.R. deMaat and J.W. Valk, University of Utrecht, The Netherlands, for their participation in preliminary aspects of this study and Judy Crawford for excellent secretarial assistance in the preparation of the manuscript. This investigation was supported by grants from the Medical Research Council of Canada and the Arthritis Society. DAH is the Calgary Foundation–Grace Glaum Professor in Arthritis Research and CBF is an AHFMR Scientist.

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