When it comes to transcriptomics and gene expression studies, an intact RNA is crucial. But it’s challenging. RNA’s notorious fragility and inherent instability stem from both enzymatic and chemical vulnerabilities. RNases - enzymes that degrade RNA - are abundant inside WBCs. Even more challenging is their widespread presence in the environment: on human skin, lab surfaces, gloves, and even in the air. RNase A, in particular, is especially problematic because it remains active without any cofactors and is highly resistant to denaturation, making it extremely difficult to inactivate once introduced.

Beyond enzymatic threats, RNA’s chemical structure also contributes to its fragility. Unlike DNA, RNA contains a reactive hydroxyl group on the 2′ carbon of its ribose sugar. This 2′-OH can initiate intramolecular cleavage of the phosphodiester bond, especially under alkaline conditions, leading to spontaneous degradation. This, coupled with its single-helix structure, makes RNA far more prone to breakdown than DNA.

The other enemy is temperature. RNA begins to degrade within minutes at room temperature, especially in whole blood, where RNase activity is high. Even delays of 1–2 hours between collection and lysis can significantly reduce RNA integrity. So, how long is too long? Most protocols suggest that processing within 2 hours is ideal, but real-world constraints often stretch this window. That’s where robust stabilisation strategies become essential.

RNA degradation can also occur at multiple stages throughout the extraction workflow, from collection and storage to lysis, binding, washing, and elution, each introducing risks that can compromise RNA integrity if not tightly controlled. Lysis is a critical inflexion point. If it doesn’t immediately and effectively inactivate RNases, degradation accelerates. Suboptimal lysis buffers or delays between sample addition and buffer action can undermine everything downstream. Even after lysis, RNA remains vulnerable. Harsh wash buffers can fragment RNA, especially during prolonged bead drying. Mechanical stress, such as vortexing, can shear RNA, particularly longer transcripts.

With so many threats at every step, preserving RNA integrity demands more than just careful handling; it calls for a robust workflow.

How does Cambrian’s extraction protocol keep RNA intact?

The protocol kicks off by inactivating RNases. Reagent R targets their Achilles’ heel, the disulfide bonds that hold RNases together. By breaking these bonds right at the point of lysis, Reagent R quickly renders RNases inactive and stops degradation before it starts. In tandem, the lysis buffer contains guanidinium isothiocyanate (GITC), a powerful chaotropic agent. GITC denatures proteins, including nucleases, by disrupting their tertiary structure, and it helps to solubilise cellular components efficiently. The use of GITC is a proven strategy in molecular biology for protecting RNA.

Fun fact: Before the development of more advanced preservation reagents, researchers commonly stored RNA directly in the GITC solution. A 1998 study published in BioTechniques demonstrated that RNA—whether purified or from crude lysates—remained stable for up to 18 months at −20°C when stored in GITC. This long-standing practice underscores just how effective this compound is at maintaining RNA integrity during extended storage periods.

To remove any residual genomic DNA that might interfere with downstream applications, our protocol includes a DNase treatment step after lysis. This ensures that the final RNA preparation is free from DNA contamination, which is critical for accurate expression analysis.

Following digestion, we incorporate a third wash step with a chaotrope. This serves a dual purpose: it removes DNase and any remaining protein contaminants, and it re-establishes a chaotropic environment to prevent reactivation of any residual nucleases. This strategic wash enhances RNA purity without compromising yield.

The final elution is carried out in an RNA-stabilising buffer, optimised to maintain the structural integrity of long transcripts. This buffer not only supports downstream applications like qPCR and RNA-seq but also helps ensure high reproducibility across samples.

Read the full protocol here.

Performance preview

RNA was extracted from whole blood using the Cambrian Blood RNA Extraction Kit on the Manta automation platform. The integrity of the isolated RNA was assessed using the Agilent TapeStation system, which separates RNA based on nucleotide size and resolves distinct 18S and 28S rRNA peaks. The sample showed a RIN score of 9.3 and a 28S/18S ratio of ~2.0, both indicative of minimal degradation. These metrics confirm that the RNA is of high integrity and suitable for sensitive downstream applications such as qPCR and RNA-seq, including fusion transcript detection like BCR-ABL.

Figure 1: RNA Integrity (RIN) assessment using the Agilent RNA ScreenTape system

Table 1: RNA Integrity Numbers (RIN) across 10 whole blood *samples

Working with RNA extractions? We’d love to hear how you’re approaching it. And if degradation has been a pain point, let’s talk. Let’s connect.