Whole blood is one of the most routinely used sample types for RNA isolation and sequencing. This is largely thanks to its accessibility. New RNA-seq methods now allow researchers and clinicians to use whole blood transcriptomes for disease diagnosis, continual monitoring and prognosis (Frésard et al., 2019).
RNA quality is often measured by the RNA integrity value (RIN). However, RNA in blood can degrade when it is isolated, stored and transported. This can affect RNA quality and the ability of researchers to detect and analyze molecular biomarkers of disease (Hardwick 2020).
In this article we discuss three common methods to limit RNA degradation and maintain RNA quality in whole blood; PAXgene® blood RNA tubes (PreAnalytiX), Tempus™ blood RNA tubes (Applied Biosystems), and RNA shield blood tubes (Zymo Research).
RNA stability in whole blood
Blood plasma is high in ribonuclease (RNase) activity. RNase activity rapidly degrades RNA if whole blood samples are stored or processed incorrectly. This can change the RNA profile of whole blood samples within minutes (Rainen et al 2002; Baechler et al., 2004).
Therefore, it is critical to immediately inhibit RNase activity upon collection of whole blood. This ensures high quality RNA which is critical for downstream sequencing, biomarker identification and disease diagnosis.
How to preserve RNA quality in whole blood
To limit RNA degradation, blood is often collected using specialized storage products such as PAXgene®, Tempus™ or RNA shield blood RNA tubes. These contain reagents to ensure intracellular RNA is stabilized effectively before mRNA library preparation (Rainen et al., 2002; Skogholt et al., 2017).
These technologies allow the samples to be stored and the RNA isolated at a later time point. This is a key consideration for large, multicenter clinical studies.
Each stabilization product is similar. Between 2.5 mL and 3 mL of blood is drawn into the tube and mixed with reagents which lyse the cells and rapidly inactivate cellular RNases.
In Tempus™ blood RNA tubes, RNA is stable for five days at room temperature and at least seven days at 4oC. In PAXgene® blood RNA tubes this is reduced to three days at room temperature or five days at 4°C for. RNA stored at ambient temperature in RNA Shield blood RNA tubes are stable for one month.
For all of the approaches, RNA can be stored indefinitely when kept at -200C. For example, after six years of storage in Tempus™ blood RNA tubes or 11 years of storage in PAXgene® tubes, RNA quality was not reduced compared to the day of collection (Duale et al., 2014; PreAnalytiX, 2018).
RNA quality for ultra-high-throughput whole blood RNA sequencing
Novel ultra-high-throughput bulk RNA-seq approaches, such as Bulk RNA Barcoding and Sequencing (Blood MERCURIUS™ BRB-seq) from Alithea Genomics, are optimized for whole blood transcriptome sequencing (Alpern et al., 2019). This technology uses sample barcoding which allows researchers to pool samples for simultaneous processing.
For Blood MERCURIUS™ BRB-seq a RIN greater than 6 is recommended, although this technology can provide high-quality transcriptome data for RIN values as low as 2.2 (Alpern et al., 2019). In a representative experiment more than 75% of true differentially expressed genes were detected in samples with degraded RNA (Alpern et al., 2019).
Alithea Genomics now provides an integrated RNA extraction and sequencing pipeline for whole blood samples stored in PAXgene®, Tempus™ or RNA shield blood RNA tubes.
To find out more about whole blood RNA extraction and Blood MERCURIUS™ BRB-seq please contact us at firstname.lastname@example.org.
- Alpern, D., Gardeux, V., Russeil, J., Mangeat, B., Meireles-Filho, A.C., Breysse, R., Hacker, D. and Deplancke, B., 2019. BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing. Genome biology, 20(1), pp.1-15.
- Baechler, E.C., Batliwalla, F.M., Karypis, G., Gaffney, P.M., Moser, K., Ortmann, W.A., Espe, K.J., Balasubramanian, S., Hughes, K.M., Chan, J.P. and Begovich, A., 2004. Expression levels for many genes in human peripheral blood cells are highly sensitive to ex vivo incubation. Genes & Immunity, 5(5), pp.347-353.
- Duale, N., Lipkin, W.I., Briese, T., Aarem, J., Rønningen, K.S., Aas, K.K., Magnus, P., Harbak, K., Susser, E. and Brunborg, G., 2014. Long-term storage of blood RNA collected in RNA stabilizing Tempus tubes in a large biobank–evaluation of RNA quality and stability. BMC research notes, 7(1), pp.1-10.
- Frésard, L., Smail, C., Ferraro, N.M., Teran, N.A., Li, X., Smith, K.S., Bonner, D., Kernohan, K.D., Marwaha, S., Zappala, Z. and Balliu, B., 2019. Identification of rare-disease genes using blood transcriptome sequencing and large control cohorts. Nature medicine, 25(6), pp.911-919.
- Hardwick, J. 2020. Blood storage and transportation. ISBT Science Series, 15, pp.232-254.
- PreAnalytiX, 2018. PAXgene Blood RNA System. Technical Note. Available at: https://www.preanalytix.com/storage/download/_ProductResources_/TechnicalNotes/PROM-7266-002_BD-7969_TN_Blood_RNA_System_RNA_stability_over_11_years_storage_1118_WW_WEB.pdf
- Rainen, L., Oelmueller, U., Jurgensen, S., Wyrich, R., Ballas, C., Schram, J., Herdman, C., Bankaitis-Davis, D., Nicholls, N., Trollinger, D. and Tryon, V., 2002. Stabilization of mRNA expression in whole blood samples. Clinical chemistry, 48(11), pp.1883-1890.
- Skogholt, A.H., Ryeng, E., Erlandsen, S.E., Skorpen, F., Schønberg, S.A. and Sætrom, P., 2017. Gene expression differences between PAXgene and Tempus blood RNA tubes are highly reproducible between independent samples and biobanks. BMC research notes, 10(1), pp.1-12.