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How long does the mRNA remains stable in untreated whole bovine blood?

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Abstract

Background

High quality and quantity of messenger RNA (mRNA) are required for accuracy of gene expression studies and other RNA-based downstream applications. Since RNA is considered a labile macromolecular prone to degradation, which may result in falsely altered gene expression patterns, several commercial stabilizing reagents have been developed aiming to keep RNA stable for long period. However, for studies involving large number of experimental samples, the high costs related to these specific reagents may constitute a barrier.

Methods and results

In this context the present study was designed aiming to evaluate the stability of mRNA in whole bovine blood collected in EDTA tubes during storage at common fridge (4 °C). Whole blood samples were collected from six Holstein calves and submitted to RNA extraction in each different interval: immediately after blood sampling (< 2 h), at 1-day post-sampling (dps), 2 dps, 3 dps, 7 dps and 14dps intervals. RNA integrity and purity were evaluated, and RT-qPCR assays were run using seven different genes (B2M, ACTB, PPIA, GAPDH, YWHAZ, CD4 and IFN-γ) aiming to evaluate the presence of altered gene transcription during storage. All extracted RNA samples presented high purity, while optimal integrity and unaltered gene expression were observed in whole experimental group up to 3 days of storage.

Conclusion

Bovine blood RNA remained stable in K3EDTA tubes for 3 days stored at common fridge and can be successfully and accurately used for gene expression studies.

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Fig. 1

Data availability

All experimental data may be available if requested.

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Funding

This research study was supported by the São Paulo State Research Support Foundation (FAPESP) (Grant Nr. 2019/22675-6, 2016/07216-7).

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Authors and Affiliations

  1. Instituto de Zootecnia, Rua Heitor Penteado, n. 56, Nova Odessa, São Paulo, 13380 011, Brazil

    Rodrigo Giglioti, Bianca Tainá Azevedo, Luciana Morita Katiki & Anibal Eugênio Vercesi Filho

  2. Universidade Estadual Júlio de Mesquita Filho, Unesp, Jaboticabal, São Paulo, 14884‑900, Brazil

    Henrique Nunes de Oliveira

  3. Embrapa Pecuária Sudeste, São Carlos, São Paulo, 13560‑970, Brazil

    Márcia Cristina de Sena Oliveira & Cintia Hiromi Okino

Authors
  1. Rodrigo Giglioti
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  2. Bianca Tainá Azevedo
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  3. Henrique Nunes de Oliveira
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  4. Luciana Morita Katiki
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  5. Anibal Eugênio Vercesi Filho
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  6. Márcia Cristina de Sena Oliveira
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  7. Cintia Hiromi Okino
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Contributions

RG and CHO: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing; HNO, LMK, AEVF and MCSO: Conceptualization, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing. BTA: Methodology. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Cintia Hiromi Okino.

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Research involving human and animal rights

All the animals handling in accordance with the ethical principles and guidelines adopted by the Brazilian College of Experimentation. All procedures were approved by the Ethical Committee on Animal Experimentation of the Instituto de Zootecnia (Protocol Nr. 298–2020).

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Giglioti, R., Azevedo, B.T., de Oliveira, H.N. et al. How long does the mRNA remains stable in untreated whole bovine blood?. Mol Biol Rep 49, 789–795 (2022). https://doi.org/10.1007/s11033-021-06808-w

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  • DOI: https://doi.org/10.1007/s11033-021-06808-w

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