The analysis of RNA is central to many molecular diagnostics assays, so ensuring its integrity is critical for accurate and reliable results. Unfortunately, ribonucleases (RNases) – enzymes that catalyze the degradation of RNA molecules – are ubiquitous in the environment, meaning that one misstep in the research lab can lead to the accidental introduction of RNases into samples or reagents, completely destroying days or even weeks of work. It is therefore important to use an RNase inhibitor during sample preparation and processing, providing an insurance policy to protect the RNA from unwanted degradation. An additional challenge that can occur at the point of use of molecular diagnostics is the accidental contamination of an assay with RNase during processing. Incorporating an RNase inhibitor into diagnostic kits can help make the assay more robust and guarantee the integrity of the end result.
RNase inhibitors for molecular diagnostics
The importance of RNase inhibitor for molecular diagnostics
The accidental degradation of target RNA by RNases can be prevented by the simple addition of an RNase inhibitor.
Look for an inhibitor that is formulated to meet the needs of molecular diagnostics assays. A high concentration, glycerol-free RNase inhibitor can be lyophilized and used in molecular point-of-care diagnostics kits.
Understanding RNases
Ribonucleases (RNases) are ubiquitous enzymes found across all domains of life, including bacteria, archaea and eukaryotes, as well as in some viruses.
There are several types of RNases, each with differing origins, functions and specificities, these include:1,2,3
RNase A and related family members
degrade single-stranded RNA by cleaving the phosphodiester bonds, producing 3'-phosphorylated products
RNase H
degrades the RNA strand of RNA-DNA hybrids
RNase I
degrades all types of RNA
RNase T1
specifically cleaves RNA at guanosine residues
RNases are widespread in nature, and can be found in diverse environments, from within cells to external surfaces and airborne particles. Given their omnipresence, even tiny amounts of RNases can contaminate RNA samples, leading to their rapid degradation and compromised experimental results.
Common environmental sources of RNases include:
Surfaces – RNases can adhere to surfaces such as lab benches, pipettes, and other equipment.
Regular cleaning of equipment and the use of RNase-free products are necessary to minimize contaminationAir – airborne dust particles can carry RNases,
which can settle on exposed samples and equipment.
Tissues and cells – RNases are present in almost all biological tissues and cells (including skin), performing various physiological roles. Even a brief touch can introduce RNases into a sample.
What do RNase inhibitors do?
RNase inhibitors are specialized proteins that prevent the degradation of RNA by ensuring that RNases cannot interact with RNA molecules. They function by specifically binding to RNases and blocking their enzymatic activity. These inhibitors are crucial in various RNA-related applications, as they can block RNase activity without inhibiting the other enzymes in the reaction – like the DNA polymerase and reverse transcriptase – ensuring that the integrity of RNA samples is maintained throughout experimental procedures.
When should you use an RNase inhibitor?
The role of RNase inhibitors in protecting RNA integrity cannot be overstated. Consider a scenario where a minor RNase contamination occurs during sample preparation. Even the slightest presence of RNases can lead to the degradation of RNA, rendering weeks of meticulous work futile. RNase inhibitors act as a safeguard, ensuring that the RNA remains intact and usable. The use of RNase inhibitors is therefore critical in various applications:
Sample preparation – during the extraction and purification of RNA.
cDNA synthesis – in the synthesis of complementary DNA (cDNA) from RNA templates, leading to accurate cDNA synthesis.
PCR and RT-PCR – to improve the reliability of amplification.
Factors to consider when choosing an RNase inhibitor for a molecular diagnostics workflow
There are a number of important factors to take into account when sourcing an RNase inhibitor for your molecular diagnostics kit or protocol, including the final format of your assay, possible sources of RNase contamination, and the relevant regulatory requirements. Before choosing a product, you should consider its:
Concentration – higher concentration inhibitors provide more flexibility and efficiency.
Formulation – a glycerol-free formulation might be necessary, especially if the components of the kit must be lyophilized for point-of-care testing.
Quality and purity – quality is paramount in molecular diagnostics, so choose a trusted supplier with a strong track record of meeting the expected standards in manufacturing and quality assurance, ensuring that the inhibitor will not introduce other contaminants.
Scale and filling – choose a supplier that can provide the quantity of RNase inhibitor needed for your application and stage of development. This could include custom filling to meet your specific needs.
Do you need RNase inhibitor for your molecular diagnostics assay?
At Roche CustomBiotech, we have an extensive track record of producing high quality reagents for RNA-based molecular diagnostics assays. Our new formulation of RNase inhibitor is specifically designed to meet the needs of users in this field.
High concentration advantage
Our new RNase inhibitor has a 10x concentration, the highest available on the market, enabling the use of larger sample volumes or the addition of more oligonucleotides with optimized reaction conditions. This can be particularly beneficial in high throughput settings, where precision and efficiency are critical.
Glycerol-free formulation
The glycerol-free formulation of our RNase inhibitor is another significant advantage. Glycerol, while commonly used as a stabilizer, can interfere with certain applications, particularly those requiring lyophilization. Our lyo-ready RNase inhibitor eliminates this concern, making it ideal for a wide range of molecular diagnostics applications, including those that require dried or frozen components.
High quality and impurity testing
Quality is non-negotiable in molecular diagnostics. Our RNase inhibitor undergoes extensive impurity testing to ensure that it meets the highest standards. This rigorous testing guarantees that the inhibitor itself does not introduce any contaminants that could compromise the assay, offering greater confidence and peace of mind for developers and manufacturers.
Regulatory disclaimer:
For further processing into IVD products and medical devices only.
References:
1. Sun D, Han C, Sheng J. The role of human ribonuclease A family in health and diseases: A systematic review. iScience. 2022 Oct 7;25(11):105284. doi: 10.1016/j.isci.2022.10528
2. Cerritelli SM, Crouch RJ. Ribonuclease H: the enzymes in eukaryotes. FEBS J. 2009 Mar;276(6):1494-505. doi: 10.1111/j.1742-4658.2009.06908.
3. Bechhofer DH, Deutscher MP. Bacterial ribonucleases and their roles in RNA metabolism. Crit Rev Biochem Mol Biol. 2019 Jun;54(3):242-300. doi: 10.1080/10409238.2019.1651816