Join Us for an Exciting Webinar – Back by Popular Demand!

Are you passionate about the latest advancements in RNA therapeutics? Want to stay ahead of the curve in the rapidly evolving field of bioanalysis? Due to high demand, we are excited to offer a second opportunity to attend our popular webinar, "New Frontiers in ASO and siRNA Bioanalysis – Back by Popular Demand!" This event will once again dive into cutting-edge techniques designed to streamline and accelerate the quantification of RNA therapeutics using Nucleic Acid Nanorobotics (NANs). Don’t miss this chance to catch up on the latest innovations!

🗓 Date: Tuesday, 15th October 2024
🕒 Time: 9 am (PDT) / 12 am (EDT) / 5 pm (BST)
📍 Register Now: https://form.jotform.com/242595514650358

Introduction to Challenges in Bioanalysis for ASOs and siRNA: Understand the current challenges faced in the bioanalysis of ASOs and siRNA, including sensitivity, robustness and throughput.

Nucleic Acid Nanorobotics (NANs) Unveiled: Discover our new Bioanalysis technique that can enhance accuracy, efficiency, and reliability in detecting and quantifying ASOs and siRNA at low concentrations.

Case Studies and Performance: Learn from real-world case studies demonstrating the application and performance of NAN's platform, now including direct-to-tissue homogenate data without needing extractions.

Q&A Session: A chance to engage with us and discuss your current bioanalytical challenges.

This webinar is an excellent opportunity for professionals in RNA therapeutics to expand their knowledge, learn about groundbreaking technologies, and connect with industry experts. Whether you are a researcher, scientist, or industry professional, this event will provide you with the tools and insights needed to excel in your work.

Secure your spot now by registering today!

👉 Register Here: https://form.jotform.com/242595514650358

NANOVERY WEBINAR: New Frontiers in ASO and siRNA Bioanalysis – Back by Popular Demand!

Are you passionate about the latest advancements in RNA therapeutics? Do you want to stay ahead of the curve in the ever-evolving field of bioanalysis? If so, you won't want to miss our upcoming webinar, "New Frontiers in ASO and siRNA Bioanalysis". This event promises to be an illuminating experience, diving into cutting-edge techniques designed to streamline the quantification of RNA therapeutics using Nucleic Acid Nanorobotics (NANs).


🗓 Date: Tuesday, 30th July 2024
🕒 Time: 8 am (PDT) / 11 am (EDT) / 3 pm (BST)
📍 Register Now: https://form.jotform.com/241822863438361


Understanding Bioanalysis Challenges
Gain invaluable insights into the current problems faced in the bioanalysis of ASO and siRNA. This segment will address issues such as complexity, sensitivity, throughput and robustness - key challenges in current bioanalysis workflows.

Innovative Bioanalysis Techniques
Discover how NAN technology is revolutionising the bioanalytical landscape. Learn how these advanced techniques enhance the accuracy, efficiency, and reliability of detecting and quantifying ASOs and siRNA at low concentrations, providing the way for more effective RNA therapeutics.

Real-World Applications
Our experts will share real-world case studies that highlight the practical applications and performance of the NANs platform. See firsthand how these innovations are being implemented in the field to achieve remarkable results.

Interactive Q&A
Engage directly with leading experts during our interactive Q&A session. This is your chance to discuss your specific bioanalytical challenges and get tailored advice from the pioneers in RNA therapeutics.

This webinar is an excellent opportunity for professionals in the field of RNA therapeutics to expand their knowledge, learn about groundbreaking technologies, and connect with industry experts. Whether you are a researcher, scientist, or industry professional, this event will provide you with the tools and insights needed to excel in your work.

Only 24 hours remaining! Secure your spot now by registering today!

👉 Register Here: https://form.jotform.com/241822863438361

NANOVERY WEBINAR: New Frontiers in ASO and siRNA Bioanalysis

Polymerase Chain Reaction (PCR) is a fundamental biological technique that can produce millions of copies of a target DNA sequence, enabling the analysis and engineering of these basic building blocks of life. Since its conception by the maverick Cary Mullis, potentially during his LSD travels, it has become a cornerstone of molecular biology and biotechnology, gaining widespread recognition during the COVID-19 pandemic.

In tackling difficult-to-treat diseases, Antisense Oligonucleotide Therapies (ASOs) are short, synthetic, single-stranded oligodeoxynucleotides that can bind to a patient’s mRNA to reduce, restore, or modify protein expression through various mechanisms. While the pharmacokinetic (PK) properties of small molecules are well understood, making these larger ASO molecules more drug-like is one of the chief challenges faced by ASO developers. Techniques for PK studies, where a drug and its metabolites are tracked and quantified, also must be adapted for ASOs and often cannot reach the same power and resolution as for small molecules.

ASOs, being RNA-based, should be targetable using reverse-transcriptase quantitative PCR (RT-qPCR), which allows for the quantification of RNA sequences. Naturally, drug developers looked to this technique for quantification as part of PK bioanalysis to overcome the limited sensitivity of LC-MS. Unfortunately, the synthetic characteristics introduced to make these RNA molecules more suitable as drugs also make them challenging for PCR.

In this blog post, we will provide a summary overview of the current state of the art in bioanalytical approaches for ASOs using qPCR.

qPCR for measurement of ASOs during bioanalysis must overcome two major challenges:

Short length of ASOs:
Designing PCR primers for the available 19–25 nucleotides (nt) on an ASO is counter-productive. Typically, the optimal length of a single PCR primer lies between 18 and 24 base pairs (bp), which is comparable to the length of an ASO itself. Shorter primers are less specific during the annealing phase, leading to more non-specific binding and amplification. This necessitates an elongation step to create cDNA that can accommodate effective primer binding.

PCR enzyme compatibility with ASO chemical modifications:
ASO structures are chemically modified, via the backbone or other moieties, to improve their drug properties such as stability and tissue specificity. Unfortunately, these chemical modifications alter the sterics of enzyme binding, reducing the recognition and activity of transcriptase required to create cDNA for PCR. This causes a high degree of variability in experimental runs.

Splint ligation, originally developed for microRNA quantification, was first reported in the literature for ASO targets in 2021. Its use in drug development is presumably more established. This technique works around the aforementioned issues by translating the short, chemically modified ASO into a more suitable input for qPCR.

Rather than directly detecting the ASO, the ASO molecule hybridises and bridges two DNA template sequences. These template sequences can then be ligated together by DNA ligase to create a new single template, which is then used to create cDNA for amplification. Chemical ligation can also be used during this step but is generally a less sensitive approach.

Drawbacks of this technique include the complexity of developing a splint-ligation assay (both templates and primers must be optimised) and the skill required to run it well. Splint ligation is also less effective for advanced ASOs with 2'-O-methoxyethyl (2'-MOE) modifications, suffering from reduced sensitivity.

While developers value the sensitivity and lower switchover costs of using a qPCR assay for different ASOs, there is still a long way to go in creating easy-to-use, reagent-based assays that developers can feel confident in.

This is where Nanovery’s Nucleic Acid Nanorobotics (NANs), a completely novel, non-enzymatic approach, can help. Specially built for ASO quantification, NANs are reagent-based but enzyme-free and comparably sensitive to qPCR for a wide range of modifications and patterns.

Some advantages of using NANs are that they work directly in samples, removing extraction steps, and are incredibly simple to design and operate.

[1] Boos JA et al. "Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides." Nucleic Acids Research Aug; 41(15): e145, (2013)

[2] Marcial-Quino J et al. "Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia." Genes (Basel) Dec; 7(12): 131, (2016)

[3] Hills AC et al. "Peptide Conjugates of a 2′-O-Methoxyethyl Phosphorothioate Splice-Switching Oligonucleotide Show Increased Entrapment in Endosomes." ACS Omega 8, 43, 40463–40481 (2023)

[4] Gagnon KT et al. "Guidelines for Experiments Using Antisense Oligonucleotides and Double-Stranded RNAs." Nucleic Acid Therapeutics 29, 3 75.1, 21-30 (2023)

Overview of ASO Quantification and Bioanalysis Using qPCR

Antisense oligonucleotides (ASOs) are a growing class of therapeutic agents that modulate gene expression and promise to treat a spectrum of disorders once considered 'undruggable'.

Since their first market entry in the '90s, the FDA and EMA have approved at least nine ASO therapies for conditions such as Duchenne muscular dystrophy, marking significant strides in the clinical application of this technology.

ASOs modulate gene expression. At the molecular level, they enact their mechanism of action by binding to complementary mRNA sequences to promote degradation or cause exon-skipping before translation to protein. As such, ASOs offer the advantage of precise targeting of sequences with potentially fewer off-target effects compared to small molecules. ASOs can also target a sequence specific to a genotyped patient, providing a clear route to precision medicine.

A key challenge for ASOs is their large size (relative to small molecules), which makes them difficult to deliver to target cells. Therefore, understanding the journey of each molecule in the body is crucial for developing better delivery methods. To achieve this, bioanalytical pharmacokinetic (PK) studies are conducted to determine if a drug reaches its intended target by measuring the drug and its metabolites in various biological samples.

As ASOs continue to advance in clinical development for various therapeutic applications, robust bioanalytical methods are needed to evaluate them effectively at all stages of the drug pipeline and ensure the best drugs (i.e. most effective, promising, and highest-quality) make it to the clinic.

ASOs present unique challenges in PK bioanalysis, which are summarised here:

ASOs are designed to overcome degradation by nucleases and reach their site of action whilst maintaining RNA-binding affinity. To achieve this, ASOs use diverse chemical modifications, including phosphorothioate backbones, sugar modifications (e.g. OME, MOE, morpholino), or conjugates (e.g. GalNAc).

Each modification changes the ASO’s physicochemical properties, which in turn affects their performance during bioanalytical assays. This means a large degree of optimisation during the development of bioanalytical methods is often required. ASO pharmacokinetic properties are largely driven by chemistry rather than sequence, so any amendments to chemistries require independent study. Together, these factors pose issues for early-stage development, where PK screening is performed on numerous candidates. New technologies are required to facilitate rapid optimisation and validation.

ASOs are desirably administered at low doses to reduce their toxicity; therefore, highly sensitive analytical methods are required to detect and quantify them in biological matrices. Conventional detection techniques such as LC-MS or branched DNA may lack the required sensitivity. PCR, which is generally seen as the most sensitive tool for quantification of nucleic acids, suffers from poor reverse-transcriptase efficiency due to the chemical modifications in ASOs. Some workarounds, such as splint ligation PCR, alleviate these issues, but they introduce additional complexity to the protocols.

Discriminating ASOs from endogenous (natural) nucleic acids poses a significant challenge in bioanalysis. ASO sequences may overlap with homologous sequences in mRNA, leading to non-specific binding and quantification errors. Employing highly selective techniques such as combining LC-MS with sequence-specific hybridisation may yield a solution. 

Additionally, specificity towards individual metabolite sequences is also required to understand full PK. These metabolites can be one or two bases shorter or longer, which poses a large challenge for techniques other than LC-MS.

Biological matrices such as plasma and tissue contain other endogenous components, such as proteins, that can interfere with ASO quantification. Matrix effects can lead to signal suppression or enhancement in techniques such as hybridisation ELISA, which uses enzymes, compromising assay accuracy and reproducibility. Sample preparation techniques like solid-phase extraction and matrix-matched calibration standards can help to minimise matrix effects and improve assay reliability.

ASOs are susceptible to degradation by nucleases present in biological samples, leading to inaccurate quantification. To preserve ASO integrity during analysis, stabilising agents such as RNase inhibitors and chelating agents may be incorporated into sample processing protocols.

No single technology is currently considered the gold standard for oligonucleotide analysis, as study objectives vary, and bioanalytical format requirements vary according to modification and concentration range required. Each bioanalytical approach offers both positive results and related challenges. Validation procedures such as specificity, accuracy, precision, and sensitivity ensure the reliability of ASO quantification results across different laboratories and studies

____

In conclusion, effective bioanalysis of ASOs requires overcoming various inherent challenges from their structural complexity, low concentrations, and interactions with biological matrices. 

Existing techniques can be customised to address ASO-specific quantification. However, there is still a need for sensitive and robust techniques that can be rapidly optimised for new ASOs to drive the pipeline forward and see better drugs helping patients. 

Better tools for ASO quantification are something we are addressing here at Nanovery. For more information, please visit our pages at:

Or simply get in touch to find out more. 

[1] Roberts TC et al. "Advances in oligonucleotide drug delivery." Nature Reviews Drug Discovery 19, 673–694 (2020)

[2] Shin M et al. "Quantification of antisense oligonucleotides by splint ligation and quantitative polymerase chain reaction." Nucleic Acid Therapeutics 32.1, 66-73 (2022)

[3] Guimaraes G et al. "Antisense oligonucleotide in vitro protein binding determination in plasma, brain, and cerebral spinal fluid using hybridization LC-MS/MS." Drug Metabolism and Disposition, 50(3), 268-276 (2022)

[4] Osawa M et al. "Sensitive and specific quantification of antisense oligonucleotides using probe alteration-linked self-assembly reaction technology." BioTechniques 75.1, 21-30 (2023)

Challenges in Antisense Oligonucleotide PK Bioanalysis

Spring has brought with it not only new blooms and good weather but also the most impressive quarter we have ever experienced at Nanovery. This period has been marked by incredible growth, thanks to the remarkable efforts and skills of our team. Their dedication and expert work are the driving forces behind our success.

The flourishing progress we've made this quarter is a source of immense pride and a beacon of our collective potential. We are immensely proud of what we have achieved together, reflecting the energy and passion that every member brings to Nanovery.

1. Demystifying DNA Nanorobots: A Visual Journey

We recently launched an animated video that illuminates the sophisticated science behind our NAN technology. For users, the process is incredibly simple: add nanorobots to a sample you're testing, and that's it. This straightforward action hides the sophisticated and complex interactions happening on a molecular level - the aspects that have traditionally been challenging to explain.

Nucleic Acid Detection with Nanorobotics

The video opens a window into the world of nanorobots, offering insights into the advanced DNA nanotechnology powering their operations within the test tube. Our goal was to make the detailed science behind these robots easy for everyone to understand. We were happy to see that many people loved the video. They told us it helped them grasp complex ideas and showed them the exciting science behind what we do.

We're excited to create more content that explains the science behind our work and welcome your ideas on topics that would be helpful for our future videos. In the meantime, enjoy the animation!

2. Expanding Horizons: Advancing ASO Detection in RNA Therapeutics

We're excited to announce a significant expansion of our business operations into the dynamic field of RNA Therapeutics, with a particular focus on the bioanalysis of antisense oligonucleotides (ASOs). This strategic move marks a pivotal moment in our growth, as we deepen our commitment to advancing healthcare through cutting-edge biotechnology.

Key highlights of this initiative are our collaborations with biotech companies across the UK, US, and Europe. These collaborations have allowed us to deploy our nanorobot technology directly in customer laboratories, where it has shown impressive results. The nanorobots are functioning exceptionally well, proving to be effective with minimal training and compatible with existing laboratory equipment. This achievement underscores our technology's versatility and its potential to revolutionise the bioanalysis of ASOs and other modalities.

If you are engaged in ASO research and facing challenges with bioanalysis for pharmacokinetic (PK) studies, we encourage you to reach out to us. Our nanorobot technology provides the solution you need, streamlining your research and offering unparalleled analytical performance within biological samples. Join us in this journey towards innovative solutions in RNA Therapeutics.

3. Advancing Clinical Studies: The miRometer-122 Journey

Our journey with the miRometer-122 has reached a pivotal milestone. The first phase of our key study with clinical samples, focusing on the detection of microRNA-122 in blood plasma samples from patients, has been completed with encouraging outcomes. Our technology was compared to both ALT blood tests (the current standard of care) and qPCR (the gold standard for microRNA quantification), and the results were positive, demonstrating our system's effectiveness and precision.

Excited by this success, we're now transitioning to the second phase of the study. This next step is designed to challenge our technology even further by testing it without any form of sample purification or biomarker extraction. Direct-to-sample measurement represents a significant hurdle for current technologies, but we are confident our system is up to the task. This ability could mark a substantial leap forward in simplifying and expediting biomarker analysis.

As we embark on this next phase, the anticipation is high. Our approach could significantly impact molecular diagnostics by offering a simpler, quicker, and equally accurate alternative to conventional methods. Stay tuned for further updates as we continue to explore the potential of our technology in transforming the field.

4. Welcoming a New Expert to Our Team: Cheryl Arrowsmith

Cheryl Arrowsmith is the newest member of our crew. She joins us as an assay validation scientist after spending 15 years in diagnostics, a journey that began with her studies in Biomedical Sciences at the University of Sunderland. She's skilled in progressing products from the lab bench to full-scale production, navigating the demands of quality management systems with ease—and always ready with a clever joke to lighten the mood.

Now stepping into DNA nanorobot validation, Cheryl is embracing the new challenges of DNA nanotechnology at Nanovery. It's a bold move, blending her deep-rooted experience with a curiosity for cutting-edge science. Cheryl's ability to see both the forest and the trees makes her perfectly suited for the precision and pioneering innovation we value in our lab.

Her approach? Think Sherlock Holmes in a lab coat, solving each challenge with sharp analysis and a sense of humour that keeps even the most daunting tasks light-hearted. Cheryl's arrival marks not just the addition of a seasoned scientist to our team but a vibrant personality ready to make the intricacies of nanorobot validation engaging and, dare we say, fun! Welcome aboard, Cheryl.

5. Securing Innovations and Sharing Knowledge

We're proud to announce that Nanovery has filed a new patent, safeguarding our latest innovations in dynamic DNA nanotechnology. This strategic move ensures the protection of our unique developments, reinforcing our commitment to leading the edge of scientific discovery. This patent not only secures our intellectual property but also strengthens our position in the biotechnology industry, promising exciting possibilities ahead.

In addition to protecting our advancements, we've published a study on using AI and machine learning to predict the rate constants of toehold-mediated strand displacement. This publication reflects our efforts to deepen the scientific community's understanding of molecular interactions, providing a practical tool for researchers working with DNA nanotechnology. It's a testament to our team's hard work in bridging the gap between complex biochemical processes and computational prediction methods. You can read it here: [LINK].

6. Forging Connections at RNA Leaders and BioEurope Spring

This month, Nanovery made a strong impression at two key events, with Mark attending the RNA Leaders forum in Basel for 15 meetings, and Jurek engaging in 25 meetings at BioEurope Spring in Barcelona. Both events were centred around forging strategic partnerships and deepening our understanding of the RNA therapeutics landscape.

Both forums consistently emphasised the pressing need for new bioanalysis solutions. Our technology’s potential to advance RNA-based therapies, especially in the ASO and siRNA fields, was clear. This was a pivotal opportunity to showcase how our solutions can meet the industry's challenges.

Jurek’s experience at BioEurope Spring was enriched by attending company presentations at the drug discovery and development session. This platform offered valuable insights into the current landscape and future directions of RNA therapeutics, further emphasising the relevance of our bioanalysis technology in this evolving field.

A highlight of the event was the evening reception held at the Museu Nacional d'Art de Catalunya. This stunning venue provided a perfect setting for informal discussions and networking, fostering connections that could shape the future of RNA therapeutics.

As we reflect on the recent months, it's clear that a lot has been happening at Nanovery, driven by the exceptional efforts of our team. Expanding into new areas, achieving key milestones, and engaging with the broader scientific community have been highlights of our journey. None of this would have been possible without the dedication and expertise of our team members, whose hard work continues to propel us forward!

Stay updated by following Nanovery on LinkedIn for the latest news and developments.

Quarterly Update: March 2024 Highlights

This November has been a busy month for Nanovery, filled with significant advancements and promising developments as we round off the year.

Reflecting on our journey, we're excited to share these key highlights with our community, highlighting our relentless pursuit of innovation in biotechnology.

1. Technological Breakthrough in Serum Analysis

A major breakthrough this month was our success in using our technology to analyse crude serum samples directly from patients. This milestone, a first for our technology, is a significant leap from previous tests, proving our technology's capability in real-world application and towards intended use in clinical settings. Our team is tirelessly working towards the final tests and controls in preparation for our first clinical study. Watch this space for the full clinical results coming soon!

2. Insights from the BIO-Europe Conference in Munich

Jurek (our founder) and Mark (CEO) represented Nanovery at the BIO-Europe conference in Munich. With over 30 productive meetings, our focus at BIO-Europe was twofold: engaging with developers in the microRNA biomarker space and exploring the emerging field of RNA therapeutics, identifying potential challenges and opportunities in bioanalysis. These conversations have been useful in shaping our strategic direction and understanding of the biotech landscape.

Our participation in the LSX Inv€$tival Showcase in London has been a cornerstone in kick-starting our next investment round. We engaged with a diverse group of investors, from diagnostics and therapeutics to deep tech and biotech tools. Mark also gave a presentation at the event, highlighting our journey and potential to impact the growing nucleic acid analysis market.

It was interesting to learn about the recent successful exits in the industry, particularly those involving platform startups and scaleups, and gain investors' insights and feedback. For more information or to explore investment opportunities, please reach out to Mark (mark@nanovery.co.uk) or Jurek (jurek@nanovery.co.uk).

4. Supporting Career Development in the Local Biotech Industry

Tarin, one of our talented junior scientists at Nanovery, spoke this month at Durham University. Her presentation was part of the 'Biological Enterprise' module. She introduced Nanovery and explained our work in developing a DNA nanotechnology platform. Her talk focused on her role in this exciting field. Tarin shared her journey, starting with making reagents for pharma and life sciences. She then explored DNA nanotechnology's practical applications during her studies.

Her presentation gave students a firsthand account of her journey from academia to a role in biotechnology. This event not only highlights Tarin's personal and professional growth but also signifies her dedication to giving back to the community that shaped her. Through initiatives like these, where our team members share their experiences and insights, we believe we can effectively guide and inspire the next generation of scientists and biotech entrepreneurs.

Further strengthening our academic ties, Jurek was invited by Dr Emanuela Torelli, an expert in NanoBio Technology at the Interdisciplinary Computing and Complex Biosystems group (ICOS2), to give a guest lecture at Newcastle University. His talk, aimed at Master’s students, delved into entrepreneurial skills essential in the biotech industry. This event also provided an opportunity to discuss potential collaborations in bio-detection with Dr. Torelli, a testament to Nanovery’s commitment to pioneering research and cutting-edge science.

In addition, Nanovery currently supports three industrial PhD studentships in collaboration with Newcastle University and Durham University, underscoring our commitment to nurturing future talents in biotechnology.

5. Celebrating Nanovery’s 5th Anniversary

This November, we celebrated a remarkable milestone – Nanovery's 5th anniversary. The occasion was a reflection of our journey's highs and lows and a testament to our team's resilience and pioneering spirit. Our team of ten, instrumental in shaping our company culture and trajectory, enjoyed a well-deserved afternoon off to celebrate this achievement.

Thank you to everyone who has been part of our journey so far. Your support and enthusiasm continue to drive us forward. Stay updated by following Nanovery on LinkedIn for the latest news and developments.

Quarterly Update: November 2023 Highlights

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

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