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

Oliveira IB, Ramos DR, Lopes KL, Souza RM, Heimann JC, Furukawa LN - Clinics (Sao Paulo) (2012)

Bottom Line: Additionally, the level of β-actin protein was preserved in both thawed intact tissue and homogenates.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Hypertension, Department of Internal Medicine, Universidade de São Paulo, São Paulo, SP, Brazil.

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.

Show MeSH
The A260/A280 ratios of the total RNA extracted from the thawed and unthawed tissue samples.
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f1-cln_67p255: The A260/A280 ratios of the total RNA extracted from the thawed and unthawed tissue samples.

Mentions: The A260/A280 ratio of the total RNA isolated from the thawed tissues was not affected relative to that of the unthawed tissues (Figure 1). However, when verifying the integrity, the 28S and 18S bands were not observed, indicating that the total RNA was degraded (Figure 2).


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

Oliveira IB, Ramos DR, Lopes KL, Souza RM, Heimann JC, Furukawa LN - Clinics (Sao Paulo) (2012)

The A260/A280 ratios of the total RNA extracted from the thawed and unthawed tissue samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3297035&req=5

f1-cln_67p255: The A260/A280 ratios of the total RNA extracted from the thawed and unthawed tissue samples.
Mentions: The A260/A280 ratio of the total RNA isolated from the thawed tissues was not affected relative to that of the unthawed tissues (Figure 1). However, when verifying the integrity, the 28S and 18S bands were not observed, indicating that the total RNA was degraded (Figure 2).

Bottom Line: Additionally, the level of β-actin protein was preserved in both thawed intact tissue and homogenates.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Hypertension, Department of Internal Medicine, Universidade de São Paulo, São Paulo, SP, Brazil.

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.

Show MeSH