Blood RNA Sequencing: Introduction, Challenges, Workflow, and Applications
What is Blood Transcriptomics?
Considering that disease-causing and disease-protecting immune cells need to be transferred from lymphatic and secondary lymphoid organs through the peripheral circulation to their sites of action in disease, a clearer picture of the cells and their action gene expression is provided by identifying the components in whole blood. Blood transcriptome sequencing technologies are currently applied to rare disease analysis, immune disease research, and pathogenic infections.
Challenges of Blood RNA Sequencing
Sequencing the transcriptome of whole blood RNA samples has always been challenging. Blood consists of three major cell types, the main component being erythrocytes (95%), including progenitor erythrocytes called reticulocytes, followed by platelets (5%) and leukocytes (<1%), with leukocytes in the minority, which provide the most information and are the primary subject of study. However, the globin transcripts produced by reticulocytes in whole blood samples account for about 70% of the total whole blood mRNA, and it generates almost worthless data. Therefore, failure to remove all globin mRNA increases the multiplex rate, reduces the amount of valid data for transcriptome analysis, and has a serious impact on the analysis of low-abundance transcripts. Moreover, RNA molecules in blood are also susceptible to degradation, which can affect the quality of the sequencing results.
Blood Transcriptomics Workflow and Protocols
The whole blood RNA-seq workflow involves several key steps, including sample preparation, RNA extraction, library preparation, sequencing and data analysis.
- Blood Sample Sampling
Whole blood mRNA contains a high abundance of globin mRNA transcripts. These transcripts can account for up to 70% of the total mRNA fraction. The presence of these transcripts, which are conserved in sequence and of extremely limited research value, results in wasted data and significantly reduces the sensitivity of detection of other low-abundance transcripts. Since globin mRNA is mainly derived from mature erythrocytes, we strongly recommend whole-blood extraction with leukocyte isolation before RNA extraction.
- Extraction of total RNA from blood samples
It involves breaking open the red blood cells and removing the leukocytes, which are the main source of RNA in whole blood samples. A variety of extraction methods can be utilized, such as column-based purification, magnetic bead-based purification, or organic extraction methods. You can refer to our RNA sequencing sample submission and preparation guidelines.
- RNA quality control using capillary gel electrophoresis
- Construction of a strand-specific RNA-seq library selected by polyA
- Data analysis
Process the resulting raw sequencing data with bioinformatics tools to remove low-quality reads and align the remaining reads to the reference genome. Once reads are aligned, the next step is to quantify gene expression levels and identify differentially expressed genes using specialized software tools.
Applications of Blood RNA Sequencing
Blood is widely used in clinical research due to its ease of sampling, transport from the body to the whole body, contact with almost every tissue and organ and rapid dynamic changes. And the use of blood for transcriptome sequencing has long been studied in a variety of blood disorders such as acute myeloid leukemia, oncology and even rare diseases. Interrogation of the whole blood transcriptome can identify disease-specific alterations in gene expression, gene expression pathways and cell subpopulation representation. It can also be used to assess response to therapeutic interventions, to monitor disease progression, and to define molecular phenotypes that may respond to specific clinical management and pharmacological agents.