What Is High-Throughput Detection of Gene Expression?
The high-throughput detection of gene expression relies on next generation sequencing technologies to assess the level of expression of all genes in the genome. These technologies have revolutionized transcriptomic research and we are now entering the ultra-high-throughput era of gene expression studies.
In this article, we discuss the differences between low- and high-throughput detection of gene expression, and how novel advances in the throughput of sequencing technologies may replace traditional low-throughput methods in every day experiments.
Low-throughput qRT-PCR
The classical method to investigate gene expression is quantitative reverse transcription polymerase chain reaction (qRT-PCR) (Gibson, Heid and Williams, 1996; Bustin, 2000).
Firstly, mRNA is reverse transcribed into complementary DNA (cDNA). Gene-specific primers amplify 100 to 300 base pair fragments of cDNA by PCR which is monitored by fluorescent probes.
It is a routine laboratory technique but qRT-PCR is labour-intensive and costly for exploratory studies where a convincing target gene is not yet determined. This is because a researcher must select genes of interest and design specific primers for each of these. This gene-by-gene approach makes qRT-PCR a low-throughput method to detect gene expression.
It is a highly sensitive technique which has revolutionized our understanding of many aspects of gene regulation, development, disease, alongside countless other areas. It remains inherently low-throughput but other higher-throughput techniques are becoming more accessible.
High-throughput RNA-seq
Next generation sequencing technologies such as RNA-sequencing (RNA-seq) have dramatically increased the throughput of gene expression experiments (Costa et al., 2013; Stark, Grzelak and Hadfield, 2019). Researchers can now investigate the expression of all genes in the genome simultaneously with high sensitivity.
An RNA-seq experiment begins with extraction and enrichment of mRNA from biological samples. mRNA is then reverse transcribed into cDNA, like in the low-throughput qRT-PCR approach. cDNA is fragmented and adapters are added to the cDNA molecules to prepare a library which is amplified before sequencing.
The most recent sequencing machines, such as the NextSeq 2000 from Illumina, can generate up to 1.2 billion reads per run. These continual advances allow many more samples to be multiplexed and sequenced at the same time.
RNA-seq now provides gene expression information on hundreds of thousands of genes making this approach truly high-throughput.
Ultra-high-throughput RNA-seq approaches
New ultra-high-throughput RNA-seq techniques now allow more samples to be multiplexed than ever before, with no loss in data quality compared to standard RNA-seq techniques at the same read depth (Alpern et al., 2019).
Technologies such as Bulk RNA Barcoding and sequencing (MERCURIUS™ BRB-seq) tag the 3’ end of each mRNA molecule in a sample with a sample barcode and unique molecular identifier (UMI). This barcode allows researchers to pool thousands of samples and identify each sample and PCR duplicates during data analysis after sequencing.
Thanks to its ultra-high-throughput capacity, MERCURIUS™ BRB-seq generates genome-wide gene expression data at a similar cost as profiling four genes with qRT-PCR (Alpern et al., 2019). This approach is anticipated to transform basic laboratory practice and drive future large-scale transcriptomic studies and discoveries.
Please contact us at info@alitheagenomics.com to see how MERCURIUS™ BRB-seq can help in your next gene expression study.
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.
Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of molecular endocrinology, 25(2), pp.169-193.
Costa, C., Giménez-Capitán, A., Karachaliou, N. and Rosell, R., 2013. Comprehensive molecular screening: from the RT-PCR to the RNA-seq. Translational lung cancer research, 2(2), p.87.
Gibson, U.E., Heid, C.A. and Williams, P.M., 1996. A novel method for real time quantitative RT-PCR. Genome research, 6(10), pp.995-1001.
Stark, R., Grzelak, M. and Hadfield, J., 2019. RNA sequencing: the teenage years. Nature Reviews Genetics, 20(11), pp.631-656.
