MicroRNAs and Their Role in Personalised Medicine
Using mRNA qPCR panels as a tool to understand cancer
Circulating miRNAs are attracting interest in the burgeoning field of personalised medicine, with data supporting their diagnostic, prognostic and predictive biomarker potential. Effective miRNA profiling calls for reproducible, sensitive and specific tools with turn-around times fast enough to support investigations into what can be a rapidly changing disease progression and treatment environment. Introducing the latest miRNA RT-qPCR technology from Bioline, the EPIKTM Cancer miRNA Panel, offering sensitive SYBR® Green-based detection for 352 targets, including the most differentially expressed miRNAs and controls, for convenient, robust, and extremely specific miRNA analysis.
MicroRNAs in Cellular Processes and Disease
Since their discovery a little over 20 years ago1, miRNAs, previously overlooked within what was thought to be non-functional genome components, are now understood to be crucial regulators of important cellular functions2. The biogenesis of miRNA follows a complex path through a number of precursor forms resulting in the mature, single stranded miRNA which is about 20-25 nucleotides3. Mature miRNA interact with mRNA effecting post-translational gene regulation of cellular processes such as development, differentiation, proliferation, metabolism and apoptosis2, thus it is no surprise that aberrantly expressed miRNAs are a hallmark in many diseases, including cancer4. Recently, miRNAs were included within the traditional oncogene definition due to their vital role of controlling cell differentiation, proliferation and survival3, plus their role in the negative regulation of tumour suppressor genes is clearly evident4, and now over 12,600 publications are listed in the NCBI PubMed database relating miRNA with cancer5. Improvements in deep sequencing technology have allowed for genome-wide profiling of miRNA expression, revealing cancer-specific signatures that not only discriminate between cancer types with high accuracy, but identify tissues of origin in metastasised cancers4 – thus miRNA profiling as a cancer diagnostic became an attractive concept for development. Further, the highly regulated process of miRNA expression is sensitive to internal and external stimuli such as hormones, pharmacological molecules etc., leading to a unique miRNA profile within different tissue types, locations and time points, making them ideal candidates for prognostic and therapeutic oncology biomarkers5.
Liquid Biopsies and miRNA profiling
There is much excitement around the concept of ‘liquid biopsy’ – the ability to screen, monitor, and uniquely characterise tumours from, for example, a simple blood or plasma sample, foregoing the traditionally invasive, costly, and in many cases difficult to obtain tissue biopsy6. Although circulating cell-free tumour DNA (ctDNA) and circulating tumour cells (CTCs) are commonly the focus for these methods, circulating miRNAs are also attracting attention as viable candidates. Not only do they carry specific information about the patho-physiological state of an individual, miRNAs are remarkably stable in their protein-bound form and are present in cell-free body fluids such as plasma, serum, urine and saliva7. Correlations have been observed between specific circulating miRNAs and chemotherapy responses in a range of cancers5, and retrospective studies have also begun to identify miRNA signatures with strong predictive and prognostic potentials8. There are still many hurdles to overcome before miRNA profiling and liquid biopsy become mainstream diagnostic practice, not the least of which is establishing robust and reproducible protocols, in addition to identifying, among the growing list of candidates, which combination of targets can be linked to clinical relevance. Indeed, the very specificity with which cells express miRNA and the subsequent sensitivity to a range of stimuli, including age and gender, while attractive qualities for personalised medicine, make for a difficult moving target for applied research5.
Molecular Tools for miRNA profiling
Expression profile studies based on microarray platforms or large scale deep sequencing projects have been instrumental in the discovery and identification of miRNAs, serving to expand the current database to almost double the number of known human miRNAs in the past five years. Among the growing number of tools available for studying miRNAs, however, qPCR remains a routine favourite as it has potential to be extremely sensitive and accurate as well as being accessible to many in terms of access to instrumentation as well as overall cost. Notwithstanding the range of sample collection and extraction methods with all associated caveats, there are many challenges to overcome when applying RT-qPCR to miRNA. Targets are short, 18-22nt, as well as being highly homologous, often with as little as 1-2nt differences, in addition the mature miRNA target sequence is present in all the precursor forms9. EPIKTMmiRNA Panels are the latest release from Bioline that overcome these challenges and more, the unique assay design discriminates between even the most closely related miRNA targets, such as those within the let-7 family, with maximum sensitivity down to 10pg of total RNA, enabling low volume starting materials, such as required for blood and plasma samples and other liquid biopsy candidates10.
Advanced Design Concepts for miRNA qPCR Panels
There are many ways to overcome the difficulties of miRNA priming for RT-qPCR, many of which involve universal priming steps and additions of long tail nucleotides, resulting in a reduction of qPCR efficiency and target specificity9. EPIKTM miRNA Panel assays forego the universal priming approach and incorporate a unique 3-primer system, all of which are miRNA specific, including the initial RT stem-loop primer, allowing for clear discrimination of mature miRNAs as well as superior specificity to identify very closely related targets10. Despite the use of specific primer sets, the overall protocol is efficiently streamlined, less than 2 hours of instrument run-time leads to interpretation of profile changes in less than half a day, capitalising on the speed advantage that qPCR holds over sequencing or microarray approaches, and answering the turn-around time requirements of personalised medicine. To focus study efforts on the most differentially expressed miRNA candidates, panel design draws on a bioinformatics mega-study in collaboration with MiRXes, screening thousands of miRNA and cancer related articles and incorporating the top 340 targets. Additionally, assay performance is enhanced to ensure robust quantification over a 7-log linear dynamic range, covering low and high expressed targets within a single run10. In deference to the rising costs of laboratory technology, the RT step is linked to SYBR® Green detection, negating the use of sequence specific probes while maintaining specificity with the three specific primer approach.
As efforts continue towards the transition of miRNA profiling from bench to bedside, RT-qPCR panels offer an ideal tool to support current applied research and the resulting clinical processes. The latest technology in the field is represented by the strong assay design of the EPIKTM Cancer miRNA Panel, covering the most useful targets to monitor disease states, and offering the performance and sensitivity required for low level starting materials or liquid biopsy applications.
How time flies! It's 2012 and this year Bioline: The PCR Company celebrates its 20 year anniversary.
The world has changed rapidly and dramatically since Bioline was first established in 1992 by Marco Calzavara, president of Bioline, but we are still manufacturing and supplying a range of consistently high-quality products to life science researchers and institutions around the world.
From our base in London, UK, we have rapidly expanded to become a leading primary manufacturer and distributor of PCR enzymes and reagents for molecular biology; providing complete solutions to researchers in universities, major research institutions, hospitals, biotech firms, diagnostics and pharmaceutical firms and laboratories globally.
Bioline’s products have been cited thousands of times in leading journals by researchers all over the world and, as part of our 20 year anniversary celebrations, we've started to document some of these publications in our Bioline Scholar Monthly series of blog posts.
When asked about the milestone of two decades of service provision to life science, Marco Calzavara, founder and president of Bioline, commented:
"Our aim at Bioline for the past 20 years has been to develop high-quality and reliable products that really make the difference, delivering consistent results time after time. I am excited and proud to continue Bioline´s great work as a cutting edge molecular biology company, ensuring that we maintain our outstanding reputation as a high-quality and reliable firm."
Richard L. Eberly, Chief Commercial Officer of Meridian Bioscience, Inc., added:
"Congratulations to the Bioline team on its 20th anniversary of successful operations throughout the world. We are enthusiastic about the success of the merger and impressed by the scientific capabilities of Bioline, as well as its well-deserved reputation for innovation and quality. We remain committed to expanding the rapidly growing portfolio of highly specialized molecular biology products from Bioline that enable the development of genomic tests utilized by researchers, clinical diagnostic laboratories, diagnostic test manufacturers and biotechnology companies."
As part of the 20 year celebrations, we are running a monthly Mystery Prize Giveaway for the rest of 2012. Lucky customers will find a golden ticket hiding in their delivery, so keep a close look out when you receive your delivery because you may find one included with your next order!
We'd like all our customers over the years to join in the celebrations and you can do so by visiting the special Bioline 20 Year Anniversary microsite, where we have a timeline showing the major milestones over the last 20 years and an exclusive interview with our president, as well as other Bioline employees. You can also keep up to date with all the latest 20 year anniversary news and updates by following @ThePCRCompany and @BiolineUSA on Twitter.
It just remains to thank all our customers, both old and new alike, for your custom and we look forward to supplying you with more high-quality enzymes and reagents for the next 20 years!