For Dr. Yuri D’Alessandra and the team at Centro Cardiologico Monzino, the new chemistry in the TaqMan Advanced miRNA Assays delivers high-quality data at their fingertips in the hunt for cardiovascular disease biomarkers.
Since 2007, D’Alessandra and his team in Milan, Italy, have focused on circulating microRNAs (miRNAs), the tiny non-coding 22-nucleotide snippets that flow in the bloodstream, as potential biomarkers for cardiovascular pathology. Starting with early Applied Biosystems technology, the team is now finding success with the most up-to-date TaqMan chemistries to maximize data from extremely limited clinical research samples.
miRNA exists as extremely short segments—around 22 nucleotides—of RNA. These segments were previously dismissed as non-coding and therefore functionless, but studies have shown that these tiny genomic parcels are crucial regulators for gene transcription. Acting as RNA silencers, miRNA strands regulate post-transcriptional expression and effectively repress protein synthesis. Furthermore, they play a vital role in disease pathogenesis, where miRNA dysregulation and reduced protein production have been implicated in cancer, heart disease, kidney pathology, addiction and obesity. Once miRNA’s regulatory role was understood, clinical researchers quickly realized its potential value in future diagnosis and prognosis and as an indicator of novel therapeutic drug targets.
As D’Alessandra describes, the research at Centro Cardiologico Monzino aims to “generate a picture of all miRNAs involved in cardiovascular disease, cardiac damage and those that lead to [developing] disease.” It is a tremendous undertaking; when the team first started its research, there was no information on circulating miRNAs in cardiovascular disease. The researchers started by extracting RNA from clinical research plasma samples and then amplifying the material using polymerase chain reaction (PCR) to quantitate these tiny volumes of miRNA. Initially D’Alessandra and colleagues tried a number of extraction and quantitation methods before settling on the Applied Biosystems chemistries for speed, sensitivity and accuracy in identifying specific miRNA species.
Cardiovascular disease research
Starting with a single plate-based real-time PCR (RT-PCR) approach, Centro Cardiologico Monzino has continued to seek out newer and more advanced assays to screen plasma samples for circulating miRNA markers of cardiovascular disease. This has involved extracting RNA from clinical research samples of plasma ranging between 50 μL and 200 μL. From progressively larger cohorts, the team has identified and quantitated circulating miRNA profiles in patients with myocardial infarction and elderly patients with heart failure, comparing them with suitable control groups. The latest study involves 1,600 individuals with risk factors for heart disease. Data from these studies help define potential miRNA species for further investigation as circulating biomarkers.
As research progresses, D’Alessandra and team deal with larger sample numbers and smaller starting volumes. They have found that the TaqMan arrays are much better for the job; they are faster, more agile and cover more miRNA identifications in a shorter time. According to D’Alessandra, compared to competitors, the TaqMan assays find more miRNAs and the team is happier with the results. For this reason, they have chosen to move ahead with each new advance in TaqMan Advance miRNA chemistry and technology for quality and precision.
TaqMan chemistries deliver quality data
Centro Cardiologico Monzino is now using the ViiA 7 and QuantStudio 12K real-time PCR systems alongside the TaqMan Advanced miRNA Assay. The team has screened around 2,000 clinical research samples since starting with these systems. D’Alessandra notes that this is a big improvement in technology, with the OpenArray formats being highly suitable for screening large numbers of samples. He also suggests that the card-based approach is suitable for more limited sample numbers, describing both card and OpenArray as giving high-quality data. Immediate access to a suite of data analysis tools, specifically the Relative Quantification app via Thermo Fisher Cloud, allows the team to seamlessly analyze and extract results. The suite’s data visualization tools deliver added value.
Since achieving successful verification against the classic TaqMan miRNA chemistries, the Centro Cardiologico Monzino team now relies almost entirely on the TaqMan Advanced miRNA Assay. TaqMan delivers high-quality data, even from the small amounts of RNA in clinical research samples, with an easy workflow. D’Alessandra also notes that the TaqMan chemistries are widely used for global research into cardiovascular miRNAs.
D’Alessandra and the Centro Cardiologico Monzino team continue to look to the future and expand their cardiovascular disease biomarker research, obtaining data from larger cohort studies. In addition to quantifying miRNAs of relevance, they plan to investigate the role of long non-coding RNAs as biomarkers. In this way, they hope to identify regulators of gene expression and protein translation involved in pathogenesis. Once the data is verified, Centro Cardiologico Monzino will use the data to elucidate a mechanism for disease and generate novel therapeutic drug targets.
For research use only. Not for use in diagnostic procedures.