RNA wasn’t always the molecule to watch.
For years, it was seen as too unstable, too messy to work with. But as the limitations of DNA panels became clear, RNA was recognised as the molecule that could reveal what the genome alone couldn’t: real-time activity, tumour dynamics, and immune signals in motion.

Back in the early 2000s, RNA was underutilised in molecular diagnostics. It was often avoided due to its inherent instability and handling challenges. Two decades on, RNA has become central to some of the most advanced diagnostic platforms in oncology, infectious disease, and immunotherapy.

The evolution, from single-gene RT-PCR assays to highly multiplexed RNA-sequencing panels, has changed the scale and sensitivity of molecular testing. The journey of RNA-based diagnostics began with focused, hypothesis-driven tests, and one of the earliest success stories came from cancer diagnostics. In chronic myeloid leukaemia (CML), a specific genetic abnormality called the BCR-ABL fusion gene became a game-changer.

Image credits: https://www.cancercouncil.com.au/chronic-myeloid-leukaemia/diagnosis/tests/ 

This fusion produces an abnormal RNA transcript, which could be detected using reverse transcription PCR (RT-PCR), allowing clinicians to directly look at the molecular footprint of the disease. Before this, doctors relied on cytogenetic tests, which could only detect cancer when large numbers of abnormal cells were present. RT-PCR, however, enabled a new era of sensitivity, able to spot even a single leukemic cell among thousands of healthy ones. This enabled minimal residual disease (MRD) monitoring, giving doctors early warning signs of relapse. The technology evolved further with real-time quantitative PCR (RT-qPCR), which didn’t just detect the cancerous transcript, it measured how much was there. By tracking the levels over time, clinicians could gauge how well treatment was working and make decisions about adjusting therapy. To this day, RT-qPCR for BCR-ABL remains a gold standard for monitoring CML and related leukaemias.

As molecular oncology evolved, the field shifted from single-gene tests to multiplexed approaches, detecting multiple gene fusions in one go. This shift was especially transformative in non-small cell lung cancer (NSCLC), where gene fusions involving ALK, ROS1, RET, and NTRK have direct therapeutic consequences. The discovery of ALK and ROS1 fusions led to targeted therapies like crizotinib and entrectinib, changing the treatment landscape. These fusions are caused by chromosomal rearrangements that create novel oncogenic transcripts—like EML4–ALK or CD74–ROS1—which are absent in normal cells, making them ideal candidates for RNA-based detection.

To keep up with the growing number of actionable fusion events, researchers turned to multiplex RT-PCR assays. These tests use pooled primers to detect many fusion variants in a single reaction, minimising RNA input and speeding up turnaround time, critical advantages in advanced cancers where sample quantity is often limited. Quality control elements like ACTB or PPIA are built into the assays to ensure RNA integrity. Compared to DNA-based panels, RNA-based tests offer a more functional readout by detecting transcripts that are actively expressed. This is especially useful for finding rearrangements with large intronic regions or cryptic breakpoints that may be missed by DNA-based next-generation sequencing (NGS).

This shift toward RNA diagnostics wasn’t just a technical upgrade - it was driven by clinical necessity. DNA testing, while foundational, often misses complex fusion events or those without known breakpoints. By contrast, RNA sequencing offers a more sensitive and direct view of what the tumour is expressing. This enables detection of fusions that might otherwise remain hidden using DNA methods alone. RNA also has practical advantages in terms of speed and sensitivity. In certain diagnostic contexts, like liquid biopsies or infectious disease surveillance, RNA can outperform DNA-based workflows. Cell-free RNA is often more abundant than cell-free DNA in circulation, and RNA-based assays can detect active viral replication or inflammatory cytokine signatures with higher precision and faster turnaround.

The field has progressed from ALK/ROS1-targeted multiplex RT‑PCR assays to comprehensive RNA‑seq platforms, marking a technological and conceptual leap that now underpins precision oncology across a broad spectrum of solid and hematologic malignancies. 

One landmark moment that cemented the clinical power of RNA diagnostics came with the development of the Oncotype DX test for breast cancer. This RNA-based assay analyses the expression of 21 genes in tumour tissue to predict the risk of recurrence and help guide decisions about chemotherapy. For thousands of patients, it has provided a clear answer to a previously uncertain question: Do I really need chemotherapy? It’s a powerful example of how RNA expression data can drive personalised treatment, reducing unnecessary toxicity while ensuring aggressive disease is properly treated. This assay analyses the expression levels of 21 genes — 16 cancer-related and 5 reference genes - using RT-qPCR performed on formalin-fixed, paraffin-embedded (FFPE) tumour samples. The test generates a Recurrence Score® ranging from 0 to 100, which helps predict the likelihood of distant recurrence within 10 years for patients with early-stage, hormone receptor-positive, HER2-negative breast cancer. Crucially, it also indicates whether a patient is likely to benefit from adjuvant chemotherapy. Those with low scores can often safely skip chemotherapy, while those with high scores may need it despite otherwise favourable clinical features. Validated in large-scale prospective trials like TAILORx and RxPONDER, this test transformed treatment planning by reducing overtreatment and personalising care based on actual tumour biology, not just tumour size or grade. 

As the field advances, the focus is shifting toward non-invasive, blood-based approaches that can offer faster, broader, and more dynamic insights, without the need for a tissue biopsy.

The next frontier lies in harnessing circulating tumour RNA (ctRNA) as a liquid biopsy tool. Unlike circulating tumour DNA (ctDNA), which captures genetic alterations, ctRNA reflects the real-time transcriptional activity of tumours, making it ideal for monitoring treatment response, detecting resistance pathways, and tracking minimal residual disease (MRD). In several cancer types, ctRNA has shown higher sensitivity than ctDNA, particularly when mutations or transcripts are present at low abundance.

Adding to this is the growing interest in exosomal RNA — RNA molecules encapsulated in tiny extracellular vesicles released by tumour cells. Protected from degradation in the bloodstream, exosomal RNA offers a stable and enriched source of fusion transcripts, oncogene expression, and pathway-level activity. Because it mirrors the active signalling landscape of the tumour, it holds enormous promise for non-invasive diagnosis, prognosis, and therapeutic monitoring.

Beyond the tumour itself, blood-based RNA profiling is now shedding light on the host immune response. By capturing RNA signatures from circulating immune cells, clinicians can assess immune activation, exhaustion markers (like LAG3, TIGIT, TOX), interferon signalling, and even PD-L1 expression — all of which are critical for predicting response to checkpoint inhibitor therapies and managing immune-related toxicities. These insights are helping stratify patients more precisely in immuno-oncology trials and practice.

With ongoing improvements in RNA extraction, stabilisation, and sequencing, the shift from tissue biopsies to fully liquid-based RNA diagnostics is accelerating. Lower input requirements, faster turnaround times, and increasing affordability are removing longstanding barriers.

At Cambrian, we’re building for this future. Our Blood RNA Extraction Kit is designed to enable high-quality RNA recovery from plasma and PBMCs, unlocking new possibilities for transcriptomic analysis in liquid biopsy applications. Whether it's fusion detection, immune profiling, or early treatment monitoring, RNA is fast becoming the most versatile and insightful molecule in the diagnostic toolkit. Explore more here.