What Is Blood Transcriptome Sequencing?

Blood transcriptome sequencing is an RNA-seq based technology that investigates the expression level of all mRNA transcripts contained in the blood. Blood transcriptomic technologies block highly expressed hemoglobin mRNA transcripts before sequencing to provide more accurate and unbiased readouts. This sets it apart from standard RNA-seq techniques.

Blood is the pipeline of the body and blood transcriptomics provides a non-invasive gene expression profile of an organism’s physiological state at a specific timepoint. Advances in blood transcriptome sequencing now provide researchers and clinicians with novel opportunities for large-scale non-invasive diagnostics.

In this article we discuss what blood transcriptome sequencing is and the technology behind the technique.

The blood transcriptome

Whole blood contains an abundance of different cell types and other components. This includes enucleated, oxygen-carrying erythrocytes (red blood cells) and many different types of leukocytes (white blood cells) that comprise the immune system.

Each cell type has a different transcriptional state which makes whole blood a rich source of easily accessible gene expression data.

This gene expression data can detect or define physiological or pathological conditions at both local and systemic levels. Furthermore, it can even identify rare-disease genes from inaccessible tissues (Basu et al., 2021; Frésard et al., 2019).

Blood collection and RNA isolation

The first stage of a blood transcriptomic experiment is to collect whole blood by standard venipuncture. This routine procedure is beneficial for large studies thanks to its low cost and reduced variability of sample preparation compared to more complex sampling strategies.

After blood extraction, it is important to reduce RNA degradation which may occur during sample collection, processing and storage.

To limit degradation, blood is often collected using PAXgene™ blood RNA tubes (PreAnalytiX) or Tempus™ blood RNA tubes (Applied Biosystems). These contain reagents to ensure intracellular RNA is stabilized effectively before mRNA library preparation (Rainen et al., 2002; Skogholt et al., 2017).

The next step requires RNA extraction from the collected blood. Both PAXgene™ and Tempus™ blood RNA tubes have optional RNA extraction procedures which use silica-membrane based spin-column technology to isolate RNA.

The researcher or sequencing company then performs quality control on extracted RNA. During this step they determine the amount of extracted RNA and ensure that it is not degraded.

Globin blockers for blood transcriptomics

RNA extracted from whole blood samples contains high levels of hemoglobin RNAs (Mastrokolias et al., 2012). These high abundance hemoglobin transcripts use valuable sequencing space and interfere with measurements of other genes in the blood transcriptome (Krjutškov et al., 2016).

Human globin blocker reagents such as GLOBINclear™ (Invitrogen) often use specific oligonucleotides that anneal to highly abundant human globin gene transcripts. It prevents their reverse transcription and inclusion in the final sequencing library and ensures sequencing reads are used for more relevant genes in a study.

mRNA-sequencing library preparation

Previously, sequencing libraries would be prepared for each extracted blood mRNA sample individually if using Illumina TruSeq mRNA-seq kits. This approach is expensive and time-consuming for large studies.

Novel transcriptomic technologies such as Bulk RNA-Barcoding and Sequencing (MERCURIUS™ BRB-seq) now seamlessly integrate blood collection approaches and globin blockers into blood-specific mRNA-seq library preparation pipelines (Alpern et al., 2019).

Blood MERCURIUS™ BRB-seq is a highly scalable, ultra-high-throughput RNA-seq technology which relies on the early barcoding of the 3’ end of mRNA molecules for each sample. This sample barcode allows the pooling of hundreds of different samples into the same tube for simultaneous processing.

This removes the cost and technical limitations of conventional blood transcriptomic approaches. Therefore, Blood MERCURIUS™ BRB-seq will enable researchers to answer more complex biological questions using the blood transcriptome than ever before.

Please contact us at info@alitheagenomics.com to find out more about Blood MERCURIUS™ BRB-seq and its potential uses in your research.

References

• 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.
• Basu, M., Wang, K., Ruppin, E. and Hannenhalli, S., 2021. Predicting tissue-specific gene expression from whole blood transcriptome. Science Advances, 7(14), p.eabd6991.
• 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.
• Krjutškov, K., Koel, M., Roost, A.M., Katayama, S., Einarsdottir, E., Jouhilahti, E.M., Söderhäll, C., Jaakma, Ü., Plaas, M., Vesterlund, L. and Lohi, H., 2016. Globin mRNA reduction for whole-blood transcriptome sequencing. Scientific reports, 6(1), pp.1-7.
• Mastrokolias, A., den Dunnen, J.T., van Ommen, G.B., t Hoen, P.A. and van Roon-Mom, W., 2012. Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA. Bmc Genomics, 13(1), pp.1-9.
• 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.