Cell-Free RNA (cfRNA) as A Liquid Biopsy Biomarker for Disease Screening and Diagnosis

What is Cell-free RNA (cfRNA)?

Cell-free RNA (cfRNA) refers to RNA molecules that are present in bodily fluids, such as blood, urine, or cerebrospinal fluid, outside of cells. It is distinct from intracellular RNA, which is confined within the cellular boundaries. cfRNA encompasses a diverse population of RNA molecules, including messenger RNA (mRNA), non-coding RNA (such as microRNA and long non-coding RNA), and fragmented RNA derived from various cellular processes.

The release of cfRNA into the extracellular space can occur through different mechanisms. For example, apoptotic or necrotic cell death can lead to the passive release of RNA molecules into the surrounding environment. Additionally, active processes like cellular secretion or exosome release can contribute to the presence of cfRNA. Once released, cfRNA can be detected and analyzed to gain insights into cellular and molecular processes occurring in the body.

Read our cfRNA Sequencing Considerations.

Cellular Origin of cfRNA

The cellular origin of cfRNA can vary depending on the specific RNA species and the physiological or pathological conditions being studied.

Cytoplasmic RNA

Many cfRNA molecules originate from the cytoplasm of cells. This includes messenger RNA (mRNA) molecules, which carry the genetic information from the DNA in the cell nucleus to the ribosomes for protein synthesis. When cells undergo normal turnover or are damaged or dying, cytoplasmic mRNA can be released into the extracellular space and subsequently detected in body fluids as cfRNA.

Mitochondrial RNA

Mitochondria, the energy-producing organelles within cells, have their own DNA and transcriptional machinery. Mitochondrial RNA (mtRNA) is generated during the transcription of mitochondrial DNA and can be released into the extracellular environment as cfRNA. Changes in mtRNA levels or mutations in mitochondrial DNA have been associated with various diseases, and the detection of mtRNA in cfRNA can provide insights into mitochondrial dysfunction.

Circulating Tumor RNA

In cancer patients, cfRNA can contain tumor-derived RNA. Tumors shed various types of RNA into the bloodstream, including tumor-specific mRNA and non-coding RNAs. These tumor-derived cfRNAs can be detected and analyzed to monitor tumor progression, identify potential therapeutic targets, and assess treatment response.

Explore The Role of Exosome RNA in Cancer.

Cell types of origin in the cell free transcriptome in human health and disease. (Vorperian et al., 2021)

Exosomal RNA

Extracellular vesicles called exosomes are secreted by cells and contain a variety of bioactive molecules, including RNA. Exosomes can carry RNA molecules from their cell of origin and transfer them to recipient cells, influencing cellular processes. The RNA cargo of exosomes can include mRNA, non-coding RNAs, and regulatory RNAs. The study of exosomal cfRNA has gained significant interest due to its potential as a source of biomarkers for various diseases.

Please refer to our article Exosomal RNA Sequencing: Introduction, Categories, and Workflow.

Immune cell-derived RNA

Immune cells, such as lymphocytes, macrophages, and dendritic cells, can release RNA molecules as part of their normal functions. These immune cell-derived cfRNAs can reflect immune responses, inflammation, or immune cell dysregulation in certain diseases.

Cell-Free RNA as A Liquid Biopsy Biomarker

cfRNA has garnered attention as a potential biomarker for various diseases due to its accessibility through non-invasive methods such as blood sampling. The analysis of cfRNA can provide valuable information about the gene expression patterns and molecular alterations associated with different physiological and pathological conditions. By profiling the cfRNA, researchers can identify specific RNA signatures or changes in gene expression that are indicative of disease presence, progression, or response to treatment.

It is worth noting that cfRNA is a dynamic and heterogeneous population of RNA molecules, originating from multiple tissues and cell types in the body. Consequently, the identification and interpretation of specific cfRNA signatures require careful analysis and validation. Advances in sequencing technologies and bioinformatics tools have facilitated the study of cfRNA, enabling researchers to explore its diagnostic, prognostic, and therapeutic potential in various diseases, including cancer, neurological disorders, and infectious diseases.

Key Benefits of cfRNA as a Liquid Biopsy Biomarker

Tissue Origin Tracing

Unlike cfDNA, which represents genomic alterations, cfRNA provides insights into the tissue of origin. By analyzing tissue-specific gene expression profiles within cfRNA, it becomes possible to identify the affected organs or cells, offering a non-invasive approach to investigate disease pathogenesis.

Early Screening for Low Tumor DNA Shedding

In cases where tumor DNA shedding into the bloodstream is limited, cfRNA can serve as a valuable alternative for early cancer detection. cfRNA analysis allows for the detection of tumor-specific transcripts, even at low levels, providing an opportunity for early intervention and improved patient outcomes.

Gene Fusion Detection

cfRNA analysis enables the identification of gene fusions, which are important drivers of tumorigenesis and therapeutic targets in various cancers. Detection of gene fusions within cfRNA can aid in accurate diagnosis, treatment selection, and monitoring of treatment response.

Learn more about Detecting Fusion Transcripts by RNA Sequencing in Screening Tumors.

Monitoring Treatment Response and Minimal Residual Disease

cfRNA-based liquid biopsy allows for real-time monitoring of treatment response and the detection of minimal residual disease (MRD). By analyzing changes in the expression levels of specific RNA molecules associated with the tumor, clinicians can assess the effectiveness of treatment and detect the presence of residual disease after therapy. This information can guide treatment adjustments and improve patient outcomes.

Dynamic Biomarker Profiling

cfRNA analysis provides a dynamic snapshot of the molecular landscape of disease. As RNA expression patterns can change over time, cfRNA-based liquid biopsy allows for the monitoring of disease progression, response to treatment, and the emergence of treatment resistance. This dynamic profiling can inform clinical decision-making and enable the timely adjustment of treatment strategies.

See how scientists use RNA Sequencing in biomarker identification.

Non-invasive and Repeatable Sampling

Liquid biopsy based on cfRNA offers a non-invasive alternative to traditional tissue biopsies. It involves a simple blood draw or collection of other bodily fluids, which is less invasive, safer, and more convenient for patients. Additionally, cfRNA-based liquid biopsy can be repeated over time to monitor disease progression and treatment response without the need for repeated tissue biopsies, reducing patient discomfort and potential complications.

Comprehensive Molecular Profiling

cfRNA carries valuable information about gene expression, alternative splicing, post-transcriptional modifications, and other RNA-based processes. This comprehensive molecular profiling provided by cfRNA analysis can reveal important insights into disease mechanisms, identify new therapeutic targets, and facilitate the development of personalized treatment strategies.

Integration with other Liquid Biopsy Approaches

cfRNA-based liquid biopsy can be combined with other liquid biopsy approaches, such as cfDNA analysis and circulating tumor cell (CTC) analysis, to provide a more comprehensive view of a patient's disease status. Integrating multiple biomarkers from different sources can enhance sensitivity, specificity, and overall diagnostic accuracy.

Sequencing-based Methods for cfRNA Detection

RNA Sequencing (RNA-seq)

RNA sequencing, a cornerstone technique in transcriptomics, is a high-throughput sequencing method that allows for an in-depth analysis of the entire transcriptome, including cfRNA molecules, within liquid biopsy samples. By converting cfRNA into complementary DNA (cDNA) and subsequently sequencing it, RNA-seq provides a wealth of information regarding gene expression levels, alternative splicing events, RNA editing, and the discovery of novel RNA species. This powerful approach enables researchers to unravel the complex transcriptional landscape and investigate disease-associated changes in gene expression.

Small RNA Sequencing

Small RNA molecules, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), play crucial roles in post-transcriptional gene regulation and have garnered significant interest in liquid biopsy studies. Small RNA sequencing focuses specifically on profiling these short RNA molecules within liquid biopsy samples. By isolating and sequencing small RNAs, researchers can gain insights into the expression patterns of miRNAs and other small regulatory RNAs. This knowledge contributes to a better understanding of their involvement in disease processes and potential diagnostic and therapeutic applications.

Cell-free long RNA biomarker discovery. (Cabús et al., 2022)

Targeted RNA Sequencing

Targeted RNA sequencing offers a focused and cost-effective approach for the profiling of specific cfRNA molecules of interest. This method utilizes customized capture probes or primers designed to selectively capture and amplify desired RNA targets within liquid biopsy samples. By concentrating sequencing efforts on a defined set of RNA molecules, such as gene fusions or known mutation hotspots, targeted RNA sequencing enables precise characterization of these specific genetic alterations. This approach is particularly valuable when investigating well-defined molecular aberrations associated with certain diseases or therapeutic responses.

Digital RNA Sequencing

Digital RNA sequencing represents a cutting-edge technique that allows for the absolute quantification of cfRNA molecules at single-molecule resolution. By partitioning individual cfRNA molecules into nanoliter-scale droplets, followed by reverse transcription and sequencing within each droplet, digital RNA sequencing achieves unparalleled sensitivity. This approach is particularly advantageous when studying rare transcripts or low-abundance cfRNA targets in liquid biopsy samples. Digital RNA sequencing provides precise quantification of individual RNA molecules, offering unprecedented insights into their expression levels and heterogeneity.

Single-Cell RNA Sequencing (scRNA-seq)

Single-cell RNA sequencing has revolutionized the analysis of cellular heterogeneity and gene expression patterns, extending its utility to liquid biopsy studies. This technique enables researchers to examine the transcriptomes of individual cells, including cfRNA molecules derived from these cells within liquid biopsy samples. By isolating and sequencing RNA from individual cells, scRNA-seq uncovers cellular diversity, identifies rare cell populations, and uncovers cell type-specific gene expression profiles. In liquid biopsy scenarios, scRNA-seq can provide valuable insights into the heterogeneity of circulating tumor cells or the characterization of specific cell types involved in disease progression or treatment response.

Please read our article Single-Cell RNA Sequencing: Introduction, Applications, and Technologies.

References:

1. Vorperian, Sevahn K., et al. "Cell types of origin in the cell free transcriptome in human health and disease." BioRxiv (2021): 2021-05.
2. Cabús, Lluc, et al. "Current challenges and best practices for cell-free long RNA biomarker discovery." Biomarker Research 10.1 (2022): 1-10.