Next-generation–omics technologies are increasingly being used in biomedical research, and Thermo Fisher Scientific is at the forefront of this revolution. In this interview, Dr. Nicoletta Nuzziello discusses how these cutting-edge methods, in particular Applied Biosystems™ TaqMan® Advanced miRNA Assays for qPCR, help her study the molecular basis of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). She also discusses how she and her team customized these technologies to better serve their research program.
Tell us who you are and the details of your academic background?
My name is Nicoletta Nuzziello. I earned my master’s degree in cellular and molecular biology in 2012 and doctoral degree (PhD) in biomolecular, pharmaceutical, and medical sciences (curriculum in neuroscience) from the University of Bari, Italy. Currently, I am working at the Institute of Biomedical Technologies (ITB), National Research Council (CNR) of Bari as postdoctoral research fellow under the supervision of Dr. Maria Liguori, neurologist and researcher at ITB-CNR.
What type of research are you doing?
The main focus of my research is to discover circulating molecular markers associated with neurological diseases such as multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (SMA). The ultimate goal is to identify suggestive biomarkers for clinical phenotypes and response to treatment of these disease conditions, as well as to add new insight into the pathogenesis of these disorders and possibly suggest novel targets for potential therapeutic strategies.
At present, I am involved in several projects that aim to investigate the expression profiles of miRNome and its potential targetome in different biological matrices (e.g., whole blood, serum, and cerebrospinal fluid) of selected patients using next-generation “–omics” technologies. The data obtained from these extensive methodologies are analyzed by an integrated team of experts in bioinformatics and biostatistics at the ITB-CNR with whom I actively collaborate.
Furthermore, I am conducting a comprehensive analysis of small noncoding RNAs packaged in human extracellular vesicles to shed light on the complex role of these vesicles as shuttles of circulating biomarkers in the studied diseases.
What is the main research question that you are trying to address?
To date, there is an urgent need to identify possible molecular signatures that allow us to decipher the pathogenetic mechanism in multifactorial neurodegenerative diseases, which could potentially provide critical information for the selection of those circulating biomarkers (e.g., miRNAs) best suited for therapeutic efforts and/or monitoring the progression of these diseases.
How long have you been studying this research question?
I have been studying this research question for the last 5 years. In 2014, a multidisciplinary two-year research project grant was awarded by the Italian Federation for Multiple Sclerosis (FISM), which enabled us to identify patterns of circulating markers (miRNAs) associated with a selected population of pediatric MS. Furthermore, several significant collaborations were achieved in order to collect a large number of patients with MS, ALS, and other neurodegenerative diseases. The research results have been published in several peer-reviewed international scientific journals and presented at various international conferences.
What are the challenges of studying this system?
In the last decade, the rapid development of high-throughput next-generation sequencing (HT-NGS) technologies together with emergence of bioinformatics tools have led to the discovery of many new miRNAs as well as the generation of a large amount of sequencing data (from several targets in a large population of patients) that need to be validated. In my view, this is the main challenge of this type of research.
Why did you choose Applied Biosystems™ TaqMan® Advanced miRNA Assays?
The main advantage of TaqMan Advanced miRNA Assays is that all mature miRNAs are converted into cDNA in the same tube, employing universal reverse transcription and miRNA amplification steps. We opted for a 384-well microfluidic array card configuration, which presented the ideal solution for profiling large numbers of miRNAs in a considerable number of samples.
What has your experience with the TaqMan Advanced miRNA Assays been like?
The miRNA expression analysis was performed with custom-designed TaqMan Advanced miRNA Assays by fitting the microfluidic array card configuration based on our experimental design. This approach proved to be more cost-effective than the conventional 96- and 384-well real-time PCR (qPCR), required limited hands-on time, delivered high-quality data, and was highly specific because of the innovative sample-loading ports that significantly reduced operator errors (e.g., pipetting).
What are the next steps in your research?
We are planning to investigate the expression profile of validated miRNAs and their potential target genes in larger cohort studies by using nanofluidic Applied Biosystems™ TaqMan® OpenArray™ Plates. This nanoliter-scale, high-throughput qPCR platform will help process a larger number of samples and perform a large number of assays per sample simultaneously. Indeed, the card-based approach is suitable for more limited sample and /or target numbers, compared with the TaqMan OpenArray Plates.
What is the future for this type of research?
In the era of precision medicine, the cost-effectiveness, user-friendly interfaces, high processivity, and specificity of the above-mentioned approaches may make them suitable platforms for point-of-care tests in the diagnosis and monitoring of disease (e.g., the disease progression and response to treatment).