High-quality RNA is essential for high-throughput RNA-seq. It ensures transcriptomic data is accurate, and reliable, and can drive novel findings and conclusions with confidence. Researchers can now choose from many RNA preservation methods to improve RNA quality and quantity for downstream sequencing applications.
In this article, we discuss the various RNA preservation methods available to improve both the quality and quantity of extracted RNA.
The importance of RNA preservation
The first step of any high-throughput RNA-seq experiment is to isolate high-quality RNA from your samples of choice.
Degradation of RNA occurs due to cellular ribonuclease (RNase) activity and is influenced by temperature, tissue processing protocols, and storage conditions (Salehi and Najafi 2014). Researchers should manage each of these factors when they collect samples to preserve RNA quality and maximize the quantity they retrieve.
RNA preservation is a vital step before RNA-seq library preparation. Good-quality RNA ensures maximum library complexities for accurate quantification of gene expression (Gallego Romero et al., 2014). In comparative studies, this is key to detecting reliable gene expression differences between samples.
With poor-quality RNA comes an increase in technical artifacts and misleading results. For example, one study assessed the effect of different levels of RNA degradation on RNA-seq results. They found that the variation in gene expression levels in their study was closely linked with RNA sample quality (Gallego Romero et al., 2014).
Therefore, wherever possible, researchers should preserve samples immediately upon collection with the appropriate method.
Methods to preserve RNA
The exact method you should choose to preserve RNA depends on the sample type you have. For example, RNA preservation methods optimized for blood samples are different from those for cell or tissue samples.
RNA in whole blood can be preserved with PAXgene® blood RNA tubes (PreAnalytiX), Tempus™ blood RNA tubes (Applied Biosystems), and RNA shield blood tubes (Zymo Research). These blood collection tubes contain reagents to stabilize RNA before mRNA library preparation (Rainen et al., 2002; Skogholt et al., 2017).
However, for cell and tissue samples, different toxic and non-toxic reagents are available.
Chemical preservation reagents, such as TRIzol™ Reagent (ThermoFisherScientific), inhibit RNase activity and disrupt cells during sample homogenization but are toxic and require special handling environments (Rio et al., 2010).
In contrast, RNAlater™ stabilization solution (Invitrogen) is non-toxic. It rapidly permeates unfrozen cells and tissue samples and precipitates RNases into an aqueous sulfate salt solution. One major advantage is that it minimizes the need for immediate freezing or processing of tissue samples. This may be a concern in human studies or in the field. This method preserves RNA equally well as flash freezing (Mutter et al., 2004; Hentze et al., 2019).
Flash freezing is the process of immediately freezing samples to limit any RNA degradation by RNases. In multi-center studies with big sample sizes, this is often laborious and requires extensive freezer capacity.
Immediate preservation of RNA may not always be possible (Hentze et al., 2019). Sometimes researchers have no option but to sequence low-quality or low quantities of RNA. This is often due to poor RNA preservation, low starting cell numbers, or precious samples.
RNA-seq for low-quality and quantity RNA samples
Advances in sequencing technologies now allow transcriptomic analyses of even low-quality and quantities of RNA samples, with equal performance to high-quality RNA.
Bulk RNA Barcoding and Sequencing (MERCURIUS™ BRB-seq) from Alithea Genomics uses sample barcoding at the 3’ end of an mRNA transcript (Alpern et al., 2019). Because only the 3’ end of mRNA is sequenced, reliable accurate sequencing data is generated, even if a transcript is degraded (Alpern et al., 2019).
RNA quality is often measured by the RNA integrity value (RIN). For MERCURIUS™ BRB-seq a RIN greater than 6 is recommended, although this technology provides high-quality transcriptome data for RIN values as low as 2.2 (Alpern et al., 2019). Alpern et al., (2019) detected 75% of true differentially expressed genes in samples with degraded RNA sequenced by MERCURIUS™ BRB-seq (Alpern et al., 2019).
Furthermore, standard MERCURIUS™ BRB-seq requires 50ng-1μg of RNA but a high sensitivity low-input MERCURIUS™ BRB-seq is also available for as little as 100pg RNA.
- 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.
- Gallego Romero, I., Pai, A.A., Tung, J. and Gilad, Y., 2014. RNA-seq: impact of RNA degradation on transcript quantification. BMC biology, 12(1), pp.1-13.
- Hentze, J.L., Kringelbach, T.M., Novotny, G.W., Hamid, B.H., Ravn, V., Christensen, I.J., Høgdall, C. and Høgdall, E., 2019. Optimized biobanking procedures for preservation of RNA in tissue: comparison of snap-freezing and RNAlater-fixation methods. Biopreservation and Biobanking, 17(6), pp.562-569.
- Mutter, G.L., Zahrieh, D., Liu, C., Neuberg, D., Finkelstein, D., Baker, H.E. and Warrington, J.A., 2004. Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays. BMC genomics, 5(1), pp.1-7.
- 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.
- Rio, D.C., Ares, M., Hannon, G.J. and Nilsen, T.W., 2010. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harbor Protocols, 2010(6), pp.pdb-prot5439.
- Salehi, Z. and Najafi, M., 2014. RNA preservation and stabilization. Biochem Physiol, 3(126), p.2.
- 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.