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

Rodrigo Giglioti,Márcia Cristina De Sena Oliveira,Bianca Tainá Azevedo,Henrique Nunes De Oliveira,Anibal Eugênio Vercesi Filho,Luciana Morita Katiki,Cintia Hiromi Okino

doi:10.1007/s11033-021-06808-w

Rodrigo Giglioti, Márcia Cristina De Sena Oliveira + Show 5 more

Open Access

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

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Abstract

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. 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 (< 2h), 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 3days of storage. Bovine blood RNA remained stable in K3EDTA tubes for 3days stored at common fridge and can be successfully and accurately used for gene expression studies.

Highlights

Messenger RNA levels may constitute good indicator of physiological and/or immune status of cells, addressing the host responses under different environmental conditions, targeting the better understanding of mechanisms of host-pathogen interactions.Several studies targeting the quantification of gene expression profiles have been performed to better understand human and veterinary diseases [1,2,3,4,5,6,7,8]
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
Regarding RNA concentrations and RNA integrity number (RIN) means, there were no significant differences between 2 hps to 3 dps intervals, while for A260/280 mean ratios, unaltered values were observed between 2 hps and 2 dps (Table 2)

Summary

Introduction

Several studies targeting the quantification of gene expression profiles have been performed to better understand human and veterinary diseases [1,2,3,4,5,6,7,8] In this context, blood specimens are the most common applied matrix since peripheral blood may be considered less invasive and valuable source of RNA in vertebrates [9]. Stabilizing reagents are commonly applied, such as PAXgene blood RNA tubes (PreAnalytix, Qiagen/BD, Hombrechtikon, Switzerland), RNAprotect Animal Blood Tubes (Qiagen, Venlo, Netherlands), Tempus Blood RNA tubes (Applied Biosystems, Foster City, CA) and RNAlater stabilization reagent (Thermofisher, Waltham, MA) All these reagents, except for RNAlater reagent, require proper commercial RNA extraction kits, and the costs related to these reagents may be very expensive, especially for experiments including a large sampling size. 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

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