Tumors contain complex mixtures of cell populations that evolve in response to treatment and host-specific factors. Tissue biopsies provide only static snapshots of a tumor’s state. But what if we could follow that evolution over time using a minimally invasive technique?
Liquid biopsies can detect tumor-derived material circulating in the bloodstream, providing a way to study treatment response, detect emerging resistance, and discover new biomarkers. Circulating tumor DNA (ctDNA), a fraction of cell-free DNA (cfDNA), is among the most widely studied analytes. But researchers are increasingly interested in adding other omic layers. Cell-free RNA (cfRNA), which includes RNA carried in extracellular vesicles, is one of them, and it helps researchers connect genotype with gene expression and activity within the tumor microenvironment.
However, cfDNA and cfRNA are often present at low concentrations, exhibit distinct stability profiles, and differ in their biological and clinical interpretation. The advantages of combining cfDNA and cfRNA in liquid biopsy can be hampered by the added complexity of the workflow.
In this article, we will cover:
- The complementary value of analyzing DNA, RNA, and proteins in liquid biopsy samples
- The technical challenges of combining multiple liquid biopsy analytes
- How to balance analyte-specific requirements with streamlined workflows
- What the future of multiomic liquid biopsy could look like
Analyzing ctDNA within the cfDNA pool provides useful information about “tumor-specific genomic alterations, such as mutations, copy-number changes, and emerging resistance-associated variants,” said Dr. Khatereh Motamedchaboki, Sr. Global Manager in Business Development, Life Science Mass Spectrometry, Omics at Thermo Fisher Scientific. This makes ctDNA a powerful tool to monitor tumor evolution and clonal selection during therapy.
But detecting a genomic alteration does not mean it is actively expressed. Adding cfRNA to liquid biopsy panels helps connect these alterations to gene expression and provides a more dynamic view of the tumor functional state, as Dr. Jonathan Shaffer, Head of NGS R&D at QIAGEN, explained.
Gene expression changes also provide information about cellular identity that driver mutations cannot. Dr. Emily Leproust, CEO and Co-founder of Twist Bioscience, highlighted lineage plasticity, in which tumor cells alter their gene expression programs to escape treatment, as seen in neuroendocrine transformation in EGFR-driven lung cancer or prostate cancer.
Combining cfDNA and cfRNA can be very useful to detect cancer-driving gene fusions and abnormal splicing events that produce oncogenic proteins, as Dr. Leproust and Dr. Shaffer highlighted. Fusion breakpoints can occur within large introns and vary significantly between individuals, making them difficult to predict based on DNA sequence alone. In contrast, as Dr. Leproust explained, the skipped transcript provides one clear, consistent signal, as seen with MET exon 14 skipping.
By bypassing these large, repetitive introns, cfRNA “compresses the target space and reduces the sequencing reads needed to detect complex structural variants,” said Dr. Shaffer. He added that each mutated DNA copy can generate 20–2,000 mRNA copies, and thus adding cfRNA could increase assay sensitivity.
However, to enjoy the benefits of working with cfDNA and cfRNA for liquid biopsy, labs need to manage a more complex workflow that preserves both analytes and delivers results within a sufficiently short turnaround time.
Combining cfDNA and cfRNA: key workflow challenges
The integration of cfDNA and cfRNA in liquid biopsy means working with analytes that show very different stability profiles. On top of that, sample material is often limited, and tumor-derived signals can be scarce, occurring at variant allele frequency (VAFs) as low as 0.1%. Labs need to take the specific characteristics of each analyte into account while combining them in a workflow that remains efficient.
Sample collection and handling to preserve both cfDNA and cfRNA
One of the main workflow challenges starts right after sample collection. As Dr. Markus Sprenger-Haussels, Vice President R&D, PreAnalytiX – A QIAGEN and BD Joint Venture, explained, stabilizing samples and processing them as soon as possible are key to preserving the analytes being studied. He stressed that delayed processing of unstabilized blood samples leads to leukocyte lysis and release of genomic DNA into the plasma, increasing background DNA, reducing the relative fraction of ctDNA, and thus affecting assay sensitivity and interpretation.
For cfRNA, the challenge is even more complex, as beyond degradation, “ongoing cellular and biological processes after blood collection can alter the expression profile of the sample,” according to Dr. Sprenger-Haussels.
Both Dr. Leproust and Dr. Sprenger-Haussels also emphasized the importance of standardized handling from collection through plasma storage, including:
- Using collection tubes and stabilization procedures validated for the intended analytes.
- Controlling collection-to-processing times and transport temperatures.
- Minimizing residual cells and platelets during plasma separation.
- Aliquoting plasma before freezing and avoiding repeated freeze–thaw cycles.
cfDNA and cfRNA extraction: together or not?
After plasma preparation, the main decision comes down to extracting cfDNA and cfRNA together or separately. Dr. Sprenger-Haussels explained that co-extraction saves sample volume and simplifies the workflow, making it advantageous when working with limited material.
However, extraction conditions optimized for one analyte can reduce the recovery of the other or bias fragment sizes, as cfDNA and cfRNA show distinct fragment and structural profiles. The workflow should also balance recovery with inhibitor elution.
QIAGEN approaches co-extraction with the QIAamp ccfDNA/RNA Kit, a two-stage silica-column workflow that can co-isolate cfDNA and cfRNA from 1–4 mL of plasma or serum, helping conserve limited sample. Protein precipitation and successive silica-column wash steps reduce contaminant carryover, and the final eluate is concentrated with a MinElute cleanup.
Still, Dr. Sprenger-Haussels recommends monitoring DNA and RNA separately. Key controls include:
- Recovery: DNA and RNA spike-ins added before extraction, spanning relevant fragment sizes.
- Contamination: a full-process negative control and a characterized positive sample.
- cfDNA: quantify with a sensitive method such as qPCR or dPCR, check fragment sizes and high-molecular-weight DNA contamination by capillary electrophoresis, and assess amplifiability.
- cfRNA: check reverse-transcription performance and detection of both short and longer targets. Include a no-RT control to identify DNA-derived signal.
- Downstream suitability: monitor sequencing performance; include low-frequency variant, fusion or expression controls where relevant.
The decision ultimately comes down to the biological question, the available sample volume, the specific DNA and RNA populations to study, and the downstream assay requirements.
Library preparation: balancing sensitivity and workflow efficiency
At the library preparation stage, labs again face a choice: prepare cfDNA and cfRNA libraries together or separately.
Dr. Leproust explained that separate library preparation offers better control over each analyte’s particularities, which can be especially useful when working with very low-input samples or when end repair and adapter ligation are needed to capture short cfDNA fragments. Some applications that benefit from independent library preparation are those where sensitivity is key, such as minimal residual disease (MRD) or low-VAF detection, or when adding cfRNA to an already optimized cfDNA workflow.
The trade-off is extra processing and sample requirements. To limit this, Dr. Leproust recommends workflows optimized for each analyte, so laboratories can meet DNA/RNA sensitivity needs with less input and preserve material for other omic analysis. “Twist’s cfDNA Library Preparation Kit with TrueAmp™ Polymerase Mix maximizes molecular conversion efficiency and high-fidelity amplification, supporting reliable variant detection even in sample inputs down to 1 ng,” she explained.
cfDNA library prep can be run in parallel with Twist’s RNA Library Prep Kit. According to Dr. Leproust, they take about two and five hours, respectively, meaning both libraries can be ready within a day.
Combined library preparation to simplify workflows
An alternative is to combine DNA and RNA library preparation in a single workflow. Dr. Shaffer reported that the QIAseq Multimodal DNA/RNA Library Kit can be used with cfDNA and cfRNA, as long as cfRNA is adequately preserved during preanalytical processing. Combining both analytes in the same workflow reduces sample splitting, transfers, and handling steps and can produce sequencing-ready libraries in about six hours.
The QIAGEN kit also adds Unique Molecular Indices (UMIs) to both DNA and RNA, helping distinguish biological signals from technical noise. This approach can help conserve limited sample material and simplify the workflow, but as Dr. Shaffer highlighted, labs still need to validate DNA and RNA recovery and sensitivity for their specific application.
The future of multiomic liquid biopsy beyond DNA and RNA
Combining DNA and RNA in liquid biopsies helps connect tumor genetics with gene expression, but as Dr. Motamedchaboki noted, “genomic and transcriptomic alterations do not always correlate with functional cellular states or therapeutic response.”
Thus, researchers are increasingly exploring additional omic layers, including proteomics, protein post-translational modifications like glycoproteomics and phosphoproteomics, as well as metabolomics and lipidomics, to characterize dysregulated signaling pathways, adaptive resistance mechanisms, and clinically relevant patient subtypes. Extracellular vesicles are attracting attention in this field as “they carry molecular information that reflects active biological processes within tumor cells and their microenvironment,” added Dr. Motamedchaboki.
But detecting circulating proteins and lipids of interest in plasma can be difficult, as they show low abundance against a much larger background. Advances in mass spectrometry are improving sensitivity and throughput. This is achieved through advances like the Thermo Scientific Orbitrap Astral Zoom Mass Spectrometer, which uses ion pre-accumulation and a dedicated low-input detector mode. Also, the targeted affinity-based methods like Olink Assays can be combined with mass spectrometry to obtain broader protein coverage when working with very low-abundance targets.
Dr. Motamedchaboki shared with us her visions on the future of omics in liquid biopsies: “In the future, as these technologies mature and become more broadly accessible, clinicians may be able to leverage an integrated clinical multiomics workflow that combines genomic, transcriptomic, proteomic, phosphoproteomic, lipidomic, and metabolomic data.” This more comprehensive view will help improve patient stratification, more accurately predict therapeutic response, and support more dynamic treatment decisions in precision oncology.
* Unless otherwise indicated, QIAGEN and PreAnalytix products mentioned are for research use only. Not for use in diagnostic procedures. Products and product claims may differ from country to country based on regulations, clearances and approvals and may not be available in certain country. Please visit www.preanalytix.com, www.qiagen.com or contact your local supplier or country representative for further details.
About the author: Nuria M. Wentzien got her Ph.D. from the University of Granada in 2023. After finishing her Ph.D., she discovered that science communication brings together her two favorite worlds: science and writing. Her career reflects her passion for learning and exploring new topics, from a Bachelor's degree in Biochemistry (with a strong focus on human sciences) to a Ph.D. in sustainable agriculture. She brings this same curiosity to every project, making readers feel as inspired by science as she does. Now dedicated to helping life-science companies communicate rigorous science in an engaging way, she is the co-founder of Helixa Communications.