Skip to main content
Log in

Long-term stability of RNA isolated from muscle of red seabream (Pagrus major) during ice storage

  • Published:
Fish Physiology and Biochemistry Aims and scope Submit manuscript

Abstract

Recovering high-quality intact RNA from postmortem tissue is of major concern for gene expression studies. However, it is difficult to perform RNA extraction from aquacultured fish immediately after death, as rapid and accurate skills are needed for the procedure. The objective of this study was to quantitatively assess the integrity of total RNA extracted from muscle, liver, and digestive tract tissues of red seabream stored in ice as whole bodies, at a range of time points up to 10 days postmortem, using RNA integrity number (RIN) and quantitative PCR (qPCR). The RIN of total RNA in muscle remained over 8.0 for 5 days postmortem. The RINs in the liver and digestive tract were under 5.0 at 2 days postmortem. The mRNA levels of tissue inhibitor metalloproteinase 2 (TIMP-2) and β-actin, measured using qPCR in muscle, decreased to 87.8% at 1 day postmortem and to 45.5% at 2 days postmortem, from that at 0 days postmortem. In the liver and digestive tract, the mRNA levels were not significantly changed until 1 day postmortem. These results indicate that RNA, especially from fish muscle, can be maintained at high quality for several days postmortem solely by storing the fish body in ice.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Bahar B, Monahan FJ, Moloney AP, Schmidt O, MacHugh DE, Sweeney T (2007) Long-term stability of RNA in post-mortem bovine skeletal muscle, liver and subcutaneous adipose tissues. BMC Mol Biol 8:108–120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Catts VS, Catts SV, Fernandez HR, Taylor JM, Coulson EJ, Lutze-Mann LH (2005) A microarray study of post-mortem mRNA degradation in mouse brain tissue. Mol Brain Res 138:164–177

    Article  CAS  PubMed  Google Scholar 

  • Fitzpatrick R, Casey OM, Morris D, Smith T, Powell R, Sreenan JM (2002) Postmortem stability of RNA isolated from bovine reproductive tissues. Biochim Biophys Acta 1574:10–14

    Article  CAS  PubMed  Google Scholar 

  • Fontanesi L, Colombo M, Beretti F, Russo V (2008) Evaluation of post mortem stability of porcine skeletal muscle RNA. Meat Science 80:1345–1351

    Article  CAS  PubMed  Google Scholar 

  • Fleige S, Pfaffl MW (2006) RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med 27:126–139

    Article  CAS  PubMed  Google Scholar 

  • Finger JM, Mercer JF, Cotton RG, Danks DM (1987) Stability of protein and mRNA in human postmortem liver-analysis by two-dimensional gel electrophoresis. Clinica Chimica Acta 170:209–218

    Article  CAS  Google Scholar 

  • Gingrich J (2008) RNA quality indicator is new measure of RNA integrity reported by the Experion automated electrophoresis system. Bioradiations 126:22–25

    Google Scholar 

  • Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, fourth ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press

  • Heumuller-Klug S, Sticht C, Kaiser K, Wink E, Hagl C, Wessel L (2015) Degradation of intestinal mRNA: a matter of treatment. World J Gastroenterol 21(12):3499–3508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hossain MS, Koshio S, Ishikawa M, Yokoyama S, Sony NM, MAO D, Kader MA, Bulbul M, Fujieda T (2016) Efficacy of nucleotide related products on growth, blood chemistry, oxidative stress and growth factor gene expression of juvenile red sea bream, Pagrus major. Aquaculture 464:8–16

    Article  CAS  Google Scholar 

  • Humphreys-Beher MG, King FK, Bunnel B, Brody B (1986) Isolation of biologically active RNA from human autopsy for the study of cystic fibrosis. Biotechnol Appl Biochem 8:392–403

    CAS  PubMed  Google Scholar 

  • Inoue H, Kimura A, Tuji T (2002) Degradation profile of mRNA in a dead rat body: basic semi-quantification study. Forensic Sci Int 130:127–132

    Article  CAS  PubMed  Google Scholar 

  • Kaneko G, Shirakami H, Yamada T, Ide S, Haga Y, Satoh S, Ushio H (2016) Short-term fasting increases skeletal muscle lipid content in association with enhanced mRNA levels of lipoprotein lipase 1 in lean juvenile red seabream (Pagrus major). Aquaculture 452:160–168

    Article  CAS  Google Scholar 

  • Larionov A, Krause A, Miller W (2005) A standard curve based method for relative real time PCR data processing. BMC Bioinformatics 6, 62

  • Marchuk L, Sciore P, Reno C, Frank CB, Hart DA (1998) Postmortem stability of total RNA isolated from rabbit ligament, tendon and cartilage. Biochim Biophys Acta 1379:171–177

    Article  CAS  PubMed  Google Scholar 

  • Ponce M, Infante C, Funes V, Manchado M (2008) Molecular characterization and gene expression analysis of insulin-like growth factors I and II in the red banded seabream, Pagrus auriga: transcriptional regulation by growth hormone. Comp Biochem Physiol B 150:418–426

    Article  CAS  PubMed  Google Scholar 

  • Reyes-Becerril M, Salinas I, Cuesta A, Meseguer J, Tovar-Ramirez D, Ascencio-Valle F, Esteban MA (2008) Oral delivery of live yeast Debaryomyces hansenii modulates the main innate immune parameters and the expression of immune-relevant genes in the gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol 25:731–739

    Article  CAS  PubMed  Google Scholar 

  • Seear PJ, Sweeney GE (2008) Stability of RNA isolated from post-mortem tissues of Atlantic salmon (Salmo salar L.). Fish Physiol Biochem 34:19–24

    Article  CAS  PubMed  Google Scholar 

  • Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 7:3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Terova G, Preziosa E, Marelli S, Gornati R, Bernardini G, Saroglia M (2011) Applying transcriptomics to better understand the molecular mechanisms underlying fish filet quality. Food Chemistry 124(3):1268–1276

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank Ms. Azusa Setoguchi and Mr. Shirou Kanno for sampling help. We are also grateful to Dr. Haruhisa Fukada, Mr. Junpei Shinagawa, and the staff of the Laboratory of Fish Nutrition, Kochi University, for rearing the experimental fish.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Katsuji Morioka.

Additional information

Highlights

• Just by storing samples in ice, the total RNA integrity in aquacultured red seabream muscle was maintained at higher than the recommended value for gene expression analysis for up to 5 days.

• The integrity of RNA in the liver and digestive tract was more rapidly degraded, dipping below the criteria for gene expression analysis within 2 days, under the same conditions as muscle.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nakatsuji, N., Adachi, K. & Morioka, K. Long-term stability of RNA isolated from muscle of red seabream (Pagrus major) during ice storage. Fish Physiol Biochem 45, 819–828 (2019). https://doi.org/10.1007/s10695-018-0588-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10695-018-0588-8

Keywords

Navigation