In this short article, we will provide an overview of the needs and challenges associated with the quantification of miRNAs for expression analysis.

MicroRNAs (miRNAs) play a key role in regulating protein production by binding to messenger RNA (mRNA) in the cells of humans, animals, and plants. While conventionally considered repressive, miRNAs are now recognised for their dual capacity to enhance gene expression [1,2]. Despite ongoing exploration of individual miRNA roles, their significance in maintaining organism stability and health is clear.

Examining disease and homeostatic disruptions reveals significant miRNA level changes due to processes such as:

Tissue-specific release of miRNAs during cell and tissue damage and organ injury.

Hijacking of miRNA expression by cancers and viruses, or introduction of pathogen-derived miRNAs [3].

Regulation of immunity and recovery pathways in response to disease.

This heightened interest in clinical miRNA expression patterns as diagnostic biomarkers is fueled by the discovery of disease-specific miRNAs circulating in the bloodstream [4]. MiRNAs, with potential conservation across species, also emerge as promising translational biomarkers in drug development, offering early indications of drug response compared to protein changes.

In summary, miRNA biomarkers possess diagnostic potential in the clinical setting and could transform medical treatments. For drug development, they offer risk reduction during clinical trials and treatment, minimise harm to animals, and enhance the development of more effective medical treatments [6].

However, these advancements require high-quality tools for measuring miRNA expression levels.

With over 1900 human miRNA genes and 2600 mature miRNA sequences identified [7], RNA sequencing and array-based methods, though essential for extensive panels, can be cost-prohibitive for routine or high-throughput quantification. 

An alternative involves the selection of smaller miRNA panels coupled with techniques that maximise information extraction. The method of choice for quantifying smaller miRNA panels is the widely adopted Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR).


Challenges Associated with RT-qPCR for microRNAs

PCR is a very powerful technique that we owe much to. It's hard to imagine genomics or biotechnology without this laboratory workhorse.

Despite the utility of standard PCR techniques, RT-qPCR for microRNAs and other types of short RNA requires highly trained personnel due to its complex protocols, troubleshooting demands, and data analysis intricacies.


A set of critical challenges complicate the pursuit of robust data:


1.      Variability due to pre-processing steps: miRNAs are unsuitable for standard RT-primer strategies as they are too short for a primer pair to bind. This short length reduces the PCR efficiency because of low melting temperatures. Common solutions involve extension of the template through polyadenylation or step-loop RT-primers, both of which may compromise specificity and require careful primer design [8].


2.      Sample variability: Pre-analytic factors such as sample collection, processing, storage, and isolation methods can influence sample quality. Different tissue and sample types require different extraction techniques, leading to optimisation challenges. The short length of the target RNA, contamination, and low GC content in plasma hinder traditional extraction methods, resulting in sample-to-sample variability [9].


3.      Choice of Controls: As mentioned above, RT-qPCR involves various factors that can contribute to quantification errors. This includes variations in starting material, sample collection, RNA preparation, quality, and reverse transcription efficiency. As a result, a large set of controls is required, including normalisation against both endogenous and exogenous miRNAs. Selecting appropriate controls can be challenging and has resulted in a lack of standardisation across studies [10].


4.      Specificity: microRNAs exist in different forms and stages of maturity, with functions that a scientist may wish to distinguish in a PCR study:

Isomirs: These are microRNA variations that are identical in sequence but have small additions or deletions at each end which are hard for PCR primers to differentiate [11]. Depending on the scenario, a scientist may wish to quantify specific isomirs or the total related isomir content of a sample.


miRNA families: A similar challenge is seen for selectively picking up microRNA family members that have very different functions but vary by small numbers of base changes in their core sequence [12].


Mature/precursor miRNA: Active, mature miRNAs are responsible for binding to mRNA, but precursor miRNA may also be of interest. These forms both have similar sequences but differ by length and secondary structure so distinguishing them through primer-based methods poses a challenge.


In the rapidly evolving field of miRNA research, better tools for quantification are needed to support discoveries in diagnostics and drug development. At Nanovery, we see the potential of microRNAs and understand the challenges researchers face.  

If you're facing challenges in microRNA quantification, we invite you to connect with us. Let's collaborate to advance miRNA-based applications for better health.

[1] Shang, R., Lee, S., Senavirathne, G. et al. microRNAs in action: biogenesis, function and regulation. Nat Rev Genet 24, 816–833 (2023). https://doi.org/10.1038/s41576-023-00611-y

[2] Jame-Chenarboo F, Ng HH, Macdonald D, Mahal LK. High-Throughput Analysis Reveals miRNA Upregulating α-2,6-Sialic Acid through Direct miRNA-mRNA Interactions. ACS Cent Sci. 2022 Nov 23;8(11):1527-1536. doi: 10.1021/acscentsci.2c00748.

[3] Peng, Y., Croce, C. The role of MicroRNAs in human cancer. Sig Transduct Target Ther 1, 15004 (2016). https://doi.org/10.1038/sigtrans.2015.4

[4] Pozniak, T.; Shcharbin, D.; Bryszewska, M. Circulating microRNAs in Medicine. Int. J. Mol. Sci. 2022, 23, 3996. https://doi.org/10.3390/ijms23073996

[5] Ha M, Pang M, Agarwal V, Chen ZJ. Interspecies regulation of microRNAs and their targets. Biochim Biophys Acta. 2008 Nov;1779(11):735-42. doi: 10.1016/j.bbagrm.2008.03.004.

[6] Oda S, Yokoi T. Recent progress in the use of microRNAs as biomarkers for drug-induced toxicities in contrast to traditional biomarkers: A comparative review. Drug Metab Pharmacokinet. 2021 Apr;37:100372. doi: 10.1016/j.dmpk.2020.11.007.

[7] Kozomara A, Birgaoanu M, Griffiths-Jones S. miRBase: from microRNA sequences to function. Nucleic Acids Res. 2019 Jan 8;47(D1):D155-D162. doi: 10.1093/nar/gky1141.

[8] Dellett M, Simpson DA. Considerations for optimization of microRNA PCR assays for molecular diagnosis. Expert Rev Mol Diagn. 2016;16(4):407-14. doi: 10.1586/14737159.2016.1152184.

[9] MacLellan, S.A., MacAulay, C., Lam, S. et al. Pre-profiling factors influencing serum microRNA levels. BMC Clin Pathol 14, 27 (2014). https://doi.org/10.1186/1472-6890-14-27

[10] Ban E, Song EJ. Considerations and Suggestions for the Reliable Analysis of miRNA in Plasma Using qRT-PCR. Genes (Basel). 2022 Feb 10;13(2):328. doi: 10.3390/genes13020328.

[11] Glogovitis, I.; Yahubyan, G.; Würdinger, T.; Koppers-Lalic, D.; Baev, V. isomiRs–Hidden Soldiers in the miRNA Regulatory Army, and How to Find Them? Biomolecules 2021, 11, 41. https://doi.org/10.3390/biom11010041

[12] Chirshev E, Oberg KC, Ioffe YJ, Unternaehrer JJ. Let-7 as biomarker, prognostic indicator, and therapy for precision medicine in cancer. Clin Transl Med. 2019 Aug 28;8(1):24. doi: 10.1186/s40169-019-0240-y.

Navigating the Challenges of MicroRNA Quantification

Regulating gene expression is essential to maintain the balance and functions of various systems in all living organisms. The microRNA (miRNA) molecules have emerged as significant contributors in that domain. First discovered in 1993 [1], these short, single-stranded RNA molecules (typically 21-23 bases) regulate gene expression by binding to specific regions of messenger RNA (mRNA) within the cell's cytoplasm. This binding either marks the mRNA for degradation or preserves it for future translation [2]. This way, a typical microRNA can modulate the expression and function of one or (usually) more genes.

Here, we will dive deeper into the intricate world of microRNAs, looking at microRNA-122 as a noteworthy example.

Within the family of microRNAs, miR-122 expression is tissue-specific: it is found predominantly in the liver and is virtually absent in other tissues in a healthy body. Moreover, it is the liver's most abundant miRNA, accounting for roughly 52% of its total microRNAs. But why does this specific microRNA bear such significance?

miR-122 plays a crucial role in diverse aspects of liver function:

Cellular Dynamics: miR-122 is instrumental in the differentiation of hepatocytes, the primary cells in the liver [3]. Moreover, it also modulates genes like Cyclin G1 (CCNG1) and Bcl-w, vital players in cell cycle regulation and apoptosis. Through these interactions, miR-122 can drive hepatocyte proliferation and survival.

Cholesterol and Lipid Metabolism: miR-122 governs genes such as HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase), an important enzyme in the cholesterol biosynthesis pathway, and ALDOB (aldolase B). Consequently, suppressing miR-122 results in diminished cholesterol synthesis and altered lipid metabolism [4].

Iron Homeostasis: miR-122 controls genes related to iron balance, such as HFE and HAMP (hepcidin). These genes critically influence iron absorption and storage, highlighting how alterations in their regulation can sway systemic iron levels [5].

The overarching theme is clear: miR-122 is a vital regulatory microRNA, especially considering liver metabolism and function. Notably, changes in miR-122 levels are widely recognised as a key sign of liver cell damage and have been studied extensively in various liver diseases.

The relevance of miR-122 extends beyond just metabolism; it intertwines with disease pathology as well: 

Chronic Liver Disease (NASH/NAFLD): Non-alcoholic steatohepatitis (NASH) and Non-Alcoholic Fatty Liver Disease (NAFLD) are chronic liver conditions that can progress to cirrhosis and liver failure. Studies have demonstrated altered levels of miR-122 in patients with NAFLD and NASH. Specifically, elevated miR-122 levels often correlate with liver fat content and inflammation, providing insight into the disease progression and severity. Moreover, miR-122 has been proposed to regulate lipid metabolism pathways disrupted in these conditions, underscoring its potential as both a diagnostic marker and a therapeutic target.

Acute Liver Injury: One common cause of acute liver injury is paracetamol (acetaminophen) overdose. In such cases, there's a marked release of miR-122 into the bloodstream due to liver damage. Therefore, elevated serum levels of miR-122 can serve as a rapid and sensitive biomarker for paracetamol-induced liver injury, enabling timely clinical intervention.

Oncogenesis: Research has unveiled miR-122's interactions with genes and pathways linked to tumorigenesis and metastasis, especially in hepatocellular carcinoma (HCC). For instance, the Wnt/β-catenin pathway, a key pathway implicated in cancer, is one such target. Alarmingly, reduced expression of miR-122 correlates with a more aggressive tumour phenotype in HCC.

The central role miR-122 plays in various liver functions and pathologies highlights its potential in the diagnostic realm. Firstly, given its specificity to the liver and the fact that elevated levels in blood samples are indicative of liver injury or damage, it emerges as a reliable biomarker offering a glimpse into liver health and improved discrimination of disease states.

Furthermore, for conditions where miR-122 intricately intertwines with the disease mechanism, tracking its levels offers a window into the treatment's impact. By monitoring these levels, clinicians can gain insights into the efficacy of the therapeutic strategy, tweaking them if necessary for optimal patient outcomes. 

Finally, the expression patterns of miR-122 might hold prognostic insights as well. These patterns could provide early warnings, forecasting the onset or escalation of certain liver maladies. Such predictive power could be a game-changer, allowing for timely and pre-emptive interventions, potentially halting or at least mitigating the disease's progression.

miR-122 is a key diagnostic indicator of liver function and liver health. As our understanding of miR-122 deepens, so does its potential as a diagnostic biomarker and possibly a therapeutic agent in the future.

We would love to hear from you to see if we can support your work with our miRometer-122 quantification kits. These kits are simple to use, isothermal, enzyme-free and can work directly in biological samples. Connect with us today!

[1] Lee RC et al. “The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.” Cell (1993)

[2] Shang R et al. “microRNAs in action: biogenesis, function and regulation.” Nature Reviews Genetics (2023)

[3] Deng XG et al. "Overexpression of miR-122 promotes the hepatic differentiation and maturation of mouse ESCs through a miR-122/FoxA1/HNF4a-positive feedback loop" Liver International (2014)

[4] Esau C et al. "miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting" Cell Metabolism (2006)

[5] Castoldi M et al. "The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice" The Journal of Clinical Investigation (2011)

Introduction to microRNA-122

We are attending #BIOEurope in Munich, Germany, on November 6-8, 2023. We look forward to meeting potential partners and investors there.

We are attending BIOEurope 2023

For more information, please read: the full story here and here.

We at Nanovery are thrilled to announce that we have successfully raised over £1.85 million in funding to further our mission of saving lives through better testing for prostate cancer. This funding round is a significant milestone for our company, enabling us to make breakthroughs in disease detection and diagnostic technologies.

The funding includes a £500,000 investment from the North East Innovation Fund, supported by the European Regional Development Fund and managed by Northstar Ventures. Prostate Cancer Research also joined as an investor, alongside a £250,000 investment from JR Holding ASI and £305,000 from angel investors. Additionally, Innovate UK awarded Nanovery a £750,000 grant to develop our disease screening activities.

Nanovery has been at the forefront of medical innovation, developing a nanorobot platform capable of detecting chronic illnesses such as prostate cancer,  liver diseases, and severe conditions like acute liver and kidney toxicity. Our cutting-edge nanorobot advancements have applications across various sectors, including life sciences, pre-clinical drug development, agriculture bio, and food safety.

As part of our commitment to improving prostate cancer detection, we are now partnering with Prostate Cancer Research to focus on developing more reliable, simple, and accessible testing methods. Considering the mortality rates for prostate cancer have remained stagnant for 40 years, this collaboration is a timely and essential step toward improving outcomes for countless individuals.

This funding marks a new chapter for Nanovery, as we work tirelessly to revolutionise the field of disease detection and make a meaningful impact on global health. We are grateful for the support of our investors and look forward to sharing our progress with the world.

Stay tuned for updates on our exciting journey ahead!

Nanovery secures over £1.85 million for cancer diagnostic solution

For more information, please read: the full story at businessNEWS

Nanovery appoints new CEO Mark Chadwick to lead growth and innovation in the biotech industry. Mark brings a wealth of knowledge and expertise to the company and will be responsible for leading the company through its next phase of growth and expanding its innovative pipeline.

Nanovery Appoints Chief Executive Officer

Every second numerous biological, chemical and physical factors change at the nanoscale to maintain human health. Our systematic understanding of how these factors work and change at such a small scale has resulted in incredible advances in fundamental science and even generated various new disciplines such as nanotechnology and nanobiology. Five decades ago, Richard Feynman, in one of his famous lectures, “There is Plenty of Room at the Bottom”, foresighted that one-day atoms would be arranged individually and even used to manufacture tiny devices- this later became a famous bottom-up methodology to build microscopic items.

To date, numerous items based on nanotechnology have already become a part of our daily lives, from nanosensors that can detect salmonella in our food packages to covid-19 vaccines; mRNA encapsulated lipid nanoparticles, to save billions of lives. Echoing the words of Richard Feynman, who once famously postulated that the key feature in biology is not just about writing the information but rather what we could do with it.  At Nanovery, we are just doing that, using multiple pieces of DNA at the nanoscale as building blocks to create “nanobots, or nanorobots”, which are then programmed to detect molecular nucleic acid markers to either diagnose/monitor a specific disease or to assess the potential harm of the novel pharmaceuticals to human health.

From molecular signature to a visible signal

Besides, DNA is a much more powerful material than we are led to believe in textbooks: it can be synthesised in a lab, broken down into smaller pieces, and then “programmed and simulated” to work as a precise diagnostics tool used to diagnose the world’s deadliest disease, cancer. At Nanovery, we are experts in the engineering of the physical and chemical characteristics of DNA and translating them into novel custom-made nanorobots. Once the nanorobots are built, we add them to the liquid biopsy samples, which can be serum, urine, or cell-culture media. The result, which comes in a matter of minutes, produces a fluorescent signal detected by a conventional plate reader indicating the presence of the nucleic acid of interest.

If you’re interested in learning more about this fascinating field, we recommend reading the following paper - we find it particularly insightful!

https://dna-robotics.eu/2021/11/29/the-perspective-for-nano-sized-dna-robots

How do nanorobots fit within existing DNA technology?

We are pleased to announce that we have won an Innovate UK grant

Chronic diseases are responsible for significant incapacities, suffering, and deaths worldwide and impose a severe economic burden on the nation's health systems. They may be prevented, treated, or even cured if detected on time. While medical imaging and surgical tissue biopsies remain the gold standard in disease diagnostics, these approaches are impractical as they are associated with risks, high costs and inconvenience for patients.

Given the ease of collecting blood samples, liquid biopsy is emerging as a superior alternative to surgical tissue biopsies. Liquid biopsy is safe, non-invasive, more convenient, readily and repeatedly accessible, less expensive, and importantly provides a more detailed molecular and cellular representation of the individual patient's disease. US FDA has approved several liquid biopsy tests as companion diagnostics for targeted cancer therapies such as prostate, lung, and breast. However, current diagnostic solutions for liquid biopsies rely primarily on complex sequencing technologies that are time-consuming and require significant capital expenditure, as well as highly qualified staff to run and analyse the results. This limits the availability of liquid biopsies and causes diagnostic delays, which can increase treatment costs and decrease patient survival rates. 

Nanovery Limited was founded in 2018 to develop a novel diagnostic approach using DNA nanorobots to target specific biomarkers present in blood samples (such as DNA and RNA). This will enable clinicians to conduct the non-invasive liquid biopsies at a fraction of the time and cost while simplifying the workflow required for screening, diagnosis, and monitoring of chronic diseases. The innovative approach is based on nanorobot devices that are added to a blood sample to capture disease biomarkers. The nanorobot produces an easy-to-read fluorescent signal when it binds to the target biomarker if present. As such, it can provide rapid, accurate and sensitive diagnosis, even at an early stage of the disease.

This project objective is to further develop a nanorobot platform technology and assess the sensitivity and specificity of this new approach via preclinical evaluation in relevant environments. Nanovery aims to exemplify its nanorobot technology in liver disease and prostate cancer to address current bottlenecks in screening, diagnosis and monitoring of chronic diseases, offering 10x cheaper and 100x faster point-of-care detection.

We just got Innovate UK grant!

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We are a data driven company using DNA nanotechnology and AI to scale up testing of nucleic acids for powerful insights from valuable samples.