Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012;67(3):255-9.
doi: 10.6061/clinics/2012(03)09.

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

Ivone Braga de Oliveira  1 , Débora Rothstein Ramos, Karen Lucasechi Lopes, Regiane Machado de Souza, Joel Claudio Heimann, Luzia Naôko Shinohara Furukawa
Affiliations

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

Ivone Braga de Oliveira et al. Clinics (Sao Paulo). 2012.

Abstract

Objective: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours.

Methods: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure.

Results: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of β-actin protein was preserved in both thawed intact tissue and homogenates.

Conclusion: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues.

PubMed Disclaimer

Conflict of interest statement

No potential conflict of interest was reported.

Figures

Figure 1
Figure 1
The A260/A280 ratios of the total RNA extracted from the thawed and unthawed tissue samples.
Figure 2
Figure 2
The 28S and 18S bands of the isolated total RNA from the control (unthawed) and thawed tissues. 1 and 2 = control liver; 3 and 4 = liver; 5 and 6 = kidney; 7 and 8 = heart; 9 and 10 = white adipose tissue; 11 = pancreas; 12, 13 and 14 = brown adipose tissue; 15 = neonate heart.
Figure 3
Figure 3
A The A260/A280 ratios of the total RNA in unthawed (control) and thawed isolated total RNA samples. Figure 3B - RNA concentration in unthawed (control) and thawed isolated total RNA samples.
Figure 4
Figure 4
A Integrity of the thawed and unthawed isolated total RNA samples. 1 = ladder; 2 and 3 = unthawed kidney (control); 4 to 18 =  thawed isolated total kidney RNA. Figure 4B - Renin mRNA level in thawed isolated total kidney RNA. Figure 4C - β-Actin) mRNA level in thawed isolated total kidney RNA.
Figure 5
Figure 5
Protein level of β-actin in the thawed homogenates (samples: 1 = kidney, 2 = heart and 3 = BAT), thawed tissues (samples: 4 = brown adipose tissue; 5, 6 and 7 = kidney; 8 = brown adipose tissue) and unthawed tissues (samples: 9 = kidney).
Figure 6
Figure 6
Microphotographs of thawed and unthawed tissue. (A) kidney, (B) thawed kidney, (C) heart, (D) thawed heart, (E) BAT, (F) thawed BAT, (G) liver, (H) thawed liver, (I) white adipose tissue, (J) thawed white adipose tissue (400x).
See this image and copyright information in PMC

References

    1. Kisand K, Kerna I, Kumm J, Jonsson H, Tamm A. Impact of cryopreservation on serum concentration of matrix metalloproteinases (MMP)-7, TIMP-1, vascular growth factors (VEGF) and VEGF-R2 in biobank samples. Clin Chem Lab Med. 2010;49:229–35. - PubMed
    1. Yokomuro H, Shiono N, Ozawa T, Fujii T, Watanabe Y, Koyama N, et al. Effect of cryopreservation on cell proliferation and immunogenicity of transplanted human heart cells. Ann Thorac Cardiovasc Surg. 2010;16:105–12. - PubMed
    1. Venkatasubramanian RT, Wolkers WF, Shenoi MM, Barocas VH, Lafontaine D, Soule CL, et al. Freeze-thaw induced biomechanical changes in arteries: role of collagen matrix and smooth muscle cells. Ann Biomed Eng. 2010;38:694–706. - PubMed
    1. Evans PJ, Mackinnon SE, Levi ADO, Wade JA, Hunter DA, Nakao Y, et al. Cold Preserved Nerve Allografts: Changes in basement membrane, viability, immunogenicity, and regeneration. Muscle Nerve. 1998;21:1507–22. - PubMed
    1. Jewell SD, Srinivasan M, McCart LM, Williams N, Grizzle WH, LiVolsi V, et al. Analysis of the molecular quality of human tissues. Am J Clin Pathol. 2002;118:733–41. - PubMed