In 1960, two researchers in Philadelphia spotted a strange anomaly in the blood cells of leukaemia patients. A mysteriously short chromosome that no one had seen before. It would go on to change the course of cancer biology. This was the Philadelphia chromosome, the first genetic abnormality linked directly to cancer.

What caused it?

A reciprocal translocation between chromosomes 9 and 22. This event fuses two genes, BCR from chromosome 22 and ABL from chromosome 9.

The result?

A rogue tyrosine kinase that’s always switched on, pushing white blood cells into unchecked growth. This single genetic glitch is the hallmark of Chronic Myeloid Leukaemia (CML) and is also found in some cases of Acute Lymphoblastic Leukaemia (ALL). Because of its specificity, BCR-ABL is both a diagnostic and prognostic marker. Detecting its presence confirms the diagnosis and helps classify disease subtypes. The most widely used approach involves detecting the BCR-ABL fusion transcript using quantitative RT-PCR (qRT-PCR). This method not only confirms the diagnosis but also helps monitor minimal residual disease (MRD) and track response over time. Transcript levels are measured at diagnosis and then at regular intervals to see how the disease is behaving. qRT-PCR can detect extremely low levels of residual disease, down to one leukemic cell among 100,000 normal cells, making it ideal for MRD monitoring. However, the assay is highly sensitive to RNA quality. Poor integrity or contamination can lead to inaccurate quantification or false negatives, affecting treatment decisions.

The diagnostic pitfalls in BCR-ABL testing

Despite significant advances in downstream molecular detection, the quality of RNA input remains a critical bottleneck, particularly in real-world clinical settings where pre-analytical variables are harder to control. RNA is inherently unstable and prone to degradation due to delayed sample processing, suboptimal storage conditions, or repeated freeze-thaw cycles, all of which compromise transcript integrity. 

Contamination with genomic DNA is also a frequent issue. If not removed properly, it can lead to false positives in RT-PCR assays or interfere with accurate read mapping in RNA sequencing, complicating data interpretation.

Another common challenge is heme contamination, resulting from incomplete lysis of red blood cells, which can inhibit enzymatic reactions such as reverse transcription and PCR. Patients undergoing chemotherapy or immunosuppressive therapy add another layer of complexity. Their samples may have markedly low white blood cell counts, limiting the total RNA yield and increasing the risk of assay dropout. In some cases, even when RNA is successfully extracted, residual salts, proteins, or alcohols from incomplete purification can inhibit enzymatic reactions and reduce efficiency.

Each of these pitfalls, alone or in combination, can erode the sensitivity and specificity of BCR-ABL detection. And when transcript levels are already low, there’s no room for error.

How does Cambrian’s RNA extraction protocol provide intact RNA?

The Cambrian Blood RNA Extraction Kit, validated on Manta, our automation platform, is engineered to preserve RNA integrity through chaotrope-driven lysis, enzymatic cleanup, and stabilising buffers. Together, they form a tightly controlled sequence that keeps your RNA stable, clean, and ready for downstream use.

<include in the blood rna workflow overview image that pia is creating for the app note>

1. Selective RBC lysis and debris clearance
The workflow begins with a two-step red blood cell (RBC) lysis. A dedicated hypotonic lysis buffer selectively disrupts RBCs while preserving the white blood cells (WBCs), which are the primary source of RNA. This is followed by PBS washing, which removes residual RBC contaminants and helps release RNases inherently present within red blood cells. Clearing these early prevents potential RNA degradation downstream. Additionally, the wash step eliminates residual haemoglobin, which can inhibit enzymatic reactions, ensuring optimal RNA yield and performance in sensitive assays.

2. Complete WBC lysis and pellet solubilisation
WBCs are lysed using a potent chaotropic lysis/binding buffer containing guanidinium isothiocyanate (GITC), a well-established chaotrope that denatures proteins, inactivates RNases, and solubilises cellular components. Alongside this, an anionic surfactant disrupts the lipid bilayers of the cell membrane, facilitating efficient cell lysis. Crucially, the anionic surfactant also enhances the dissolution of any residual or clotted WBC pellets, often a source of yield loss in challenging blood samples. This action ensures that even small or fibrous pellets are fully solubilised and RNA is not trapped or degraded.

3. RNase inactivation during lysis
The lysis buffer is supplemented with ‘Reagent R’. This targets disulfide bonds in RNases, the structural backbone critical for their enzymatic activity. By disrupting these bonds during lysis, Reagent R effectively inactivates RNases before they can degrade RNA. This is essential for preserving the full spectrum of RNA transcripts, including long and low-abundance species.

4. On-bead DNase digestion with chaotrope wash
The protocol includes a dedicated on-bead DNase digestion step using RNase-free DNase to remove contaminating genomic DNA. This is followed by a wash containing a chaotropic agent, which simultaneously inactivates residual DNase and maintains a denaturing environment that prevents nuclease reactivation. This step enhances RNA purity without compromising integrity or yield, critical for applications requiring precise transcript-level resolution.

5. RNA elution in stabilising buffer
The final elution is performed in an RNA-stabilising buffer designed to protect the structural integrity of RNA during storage and handling. This buffer helps maintain the quality of both short and long transcripts, supports reliable quantification, and ensures high reproducibility across samples.

Find the complete RNA extraction protocol here.

If your lab works with BCR-ABL detection, get in touch. Cambrian’s extraction workflow is built to deliver the sensitivity you need. Learn more here.