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Milano, Italy

Potrich C.,Fondazione Bruno Kessler | Potrich C.,CNR Institute of Biophysics | Vaghi V.,Fondazione Bruno Kessler | Lunelli L.,Fondazione Bruno Kessler | And 15 more authors.
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2014

There is an increasing interest in circulating microRNAs (miRNAs) as potential minimally invasive diagnostic biomarkers in oncology. Considerable efforts are being made in the development of lab-on-a-chip devices for biomedical applications to purify and detect miRNAs from biological fluids. Here, we report the development of an innovative polydimethylsiloxane (PDMS)-based parallel device whose internal surface can opportunely be functionalized with positively charged 3-aminopropyltriethoxysilane (APTES) alone or mixed with two different neutral poly(ethylene glycol) silanes (PEG-s). The differently functionalized internal surfaces of the PDMS chip were characterized with s-SDTB (sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate) and the portion of the surface able to adsorb a synthetic fluorescently labeled miRNA was determined. Interestingly, the adsorbed miRNA (both synthetic and cell supernatant-derived) was found mainly on the bottom surface of the chip and could be reverse transcribed into cDNA directly on the same PDMS chip used for its purification, saving hours with respect to the use of standard purification kits. We identified 0.1% APTES/0.9% PEG-silane as the most efficient PDMS functionalization to capture both synthetic and extracellular miRNA. Moreover, the amount of captured miRNA was increased by treating the cell supernatant with a commercially available lysis buffer for RNA extraction. We assessed that the available miRNA binding sites on the functionalized surface were efficiently saturated with only one incubation, shortening the time and greatly simplifying the protocol for miRNA purification from biological samples. Finally, the extracellular miRNA purification efficiency of the PDMS functionalized multichip determined via real-time quantitative polymerase chain reaction (RT-qPCR) was confirmed by droplet digital PCR (ddPCR) quantification. This work shows an innovative, rapid and easy to use microdevice for the purification and reverse transcription of circulating miRNAs, approaching the realization of diagnostic and prognostic oncomiR-based assays. This journal is © the Partner Organisations 2014.

De Cecco L.,Fondazione Istituto Nazionale Dei Tumori | Dugo M.,Fondazione Istituto Nazionale Dei Tumori | Canevari S.,Fondazione Istituto Nazionale Dei Tumori | Daidone M.G.,Unit of Biomarkers | Callari M.,Fondazione Istituto Nazionale Dei Tumori
Critical Reviews in Oncogenesis | Year: 2013

The relevance of microRNAs (miRNAs) in mammalian species was highlighted in the past decade. Inherent to the rapid advancements in the miRNA field was the growing need for applicable and validated experimental tools that enabled rapid progress from discovery to development of diagnostic and therapeutic applications. In the last few years, different technologies have been developed to measure miRNA expression, and one important challenge is making sense of miRNA profiles. In fact, technical and analytical aspects have substantial impact on subsequent biological interpretations and their translational applications. In the context of the workflow of the entire process of miRNA analysis, we will herein (i) consider some of the more relevant pre-analytical aspects such as study design, sample processing, and miRNA extraction, (ii) describe the available analytical tools for miRNA profiling (reverse transcription quantitative PCR, microarray hybridization, next-generation sequencing) and discuss their strengths and weaknesses, (iii) highlight the available postanalytical strategies for quality control, normalization, and statistical and bioinformatic analyses of miRNA profiling data, and (iv) summarize cross-platform comparison studies. © 2013 by Begell House, Inc.

Callari M.,Functional Genomics Core Facility | Dugo M.,Functional Genomics Core Facility | Musella V.,Unit of Biomarkers | Marchesi E.,Functional Genomics Core Facility | And 6 more authors.
PLoS ONE | Year: 2012

Background: Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples. Methodology/Principal Findings: We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport. Conclusions: Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs - gene expression integration approach. © 2012 Callari et al.

Callari M.,Unit of Biomarkers | Callari M.,Core Oncology | Tiberio P.,Unit of Biomarkers | De Cecco L.,Core Oncology | And 6 more authors.
Analytical Biochemistry | Year: 2013

MicroRNAs have been found to be deregulated in several diseases and, due to their high stability in body fluids, represent promising noninvasively detectable biomarkers. However, numerous technical variables can affect accurate measurement of circulating miRNAs. Using a microarray-based method we assessed the: (i) adequate intra- and inter-array reproducibility of miRNA profiling; (ii) feasibility of using archival plasma samples stored for an extended period of time and available in limited amounts; (iii) good correlation between different batches; and (iv) time-dependent increase of background signals close to the chip expiration date. © 2013 Elsevier Inc. All rights reserved.

Appierto V.,Unit of Biomarkers | Callari M.,Unit of Biomarkers | Cavadini E.,Unit of Biomarkers | Morelli D.,Unit of Laboratory Medicine | And 2 more authors.
Bioanalysis | Year: 2014

Background: The identification and management of hemolyzed samples are crucial issues in the development of new blood-based biomarkers. Results: Using experiments of controlled hemolysis and lipemia and two plasma series from cancer patients, we developed and validated a lipemia-independent hemolysis score (HS). HS resulted strictly associated with the amount of lysed erythrocytes and with serum index measurement (reference method), highly reproducible, and able to identify as hemolyzed plasma/serum samples containing ≥6.1 mg/dl of free hemoglobin. Conclusion: We developed a simple, robust, sensitive, cost-effective, spectrophotometrically-based system to identify hemolyzed plasma/serum specimens. The procedure requires only 2 μl of sample, thus representing a useful tool for research studies and an essential pre-analytical quality control for an optimal biobanking of liquid biopsies. © 2014 Future Science Ltd.

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