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

Author(s)

Giglioti, Rodrigo ^[1] ; Azevedo, Bianca Taina ^[1] ; de Oliveira, Henrique Nunes ^[2] ; Katiki, Luciana Morita ^[1] ; Vercesi Filho, Anibal Eugenio ^[1] ; de Sena Oliveira, Marcia Cristina ^[3] ; Okino, Cintia Hiromi ^[3]

Affiliation

^[1] Inst Zootecnia, Rua Heitor Penteado 56, BR-13380011 Nova Odessa, SP - Brazil

^[2] Univ Estadual Julio de Mesquita Filho, UNESP, BR-14884900 Jaboticabal, SP - Brazil

^[3] Embrapa Pecuaria Sudeste, BR-13560970 Sao Carlos, SP - Brazil

• Document type: Journal article
• Source: MOLECULAR BIOLOGY REPORTS; v. 49, n. 1 OCT 2021.
• Web of Science Citations: 0

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 degrees 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-gamma) 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. (AU)

• FAPESP's process: 16/07216-7 - Genomic study and immunological characterization of cattle resistant to bovine babesiosis and analysis of the genetic diversity of Babesia bovis and Babesia bigemina
• Grantee: Henrique Nunes de Oliveira
• Support type: Research Projects - Thematic Grants
• FAPESP's process: 19/22675-6 - Transcriptome analysis of resistance and/or susceptibility in beef cattle of different genetic groups to Babesia bovis infections
• Grantee: Rodrigo Giglioti
• Support type: Regular Research Grants