OncomiR detection in circulating body fluids

A PDMS microdevice perspective

Cristina Potrich, Valentina Vaghi, Lorenzo Lunelli, Laura Pasquardini, Gaia Cecilia Santini, Chiara Ottone, Marzia Quaglio, Matteo Cocuzza, Candido Fabrizio Pirri, Manuela Ferracin, Massimo Negrini, Paola Tiberio, Veronica De Sanctis, Roberto Bertorelli, Cecilia Pederzolli

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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

Original languageEnglish
Pages (from-to)4067-4075
Number of pages9
JournalLab on a Chip - Miniaturisation for Chemistry and Biology
Volume14
Issue number20
DOIs
Publication statusPublished - Oct 21 2014

Fingerprint

Body fluids
Body Fluids
Polydimethylsiloxane
MicroRNAs
Purification
Silanes
Polyethylene glycols
Lab-on-a-chip
Oncology
Polymerase chain reaction
Biomarkers
Binding sites
Transcription
RNA
Assays
baysilon
Artificial Cells
Equipment and Supplies
Fluids
Butyrates

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)
  • Bioengineering
  • Biomedical Engineering
  • Medicine(all)

Cite this

Potrich, C., Vaghi, V., Lunelli, L., Pasquardini, L., Santini, G. C., Ottone, C., ... Pederzolli, C. (2014). OncomiR detection in circulating body fluids: A PDMS microdevice perspective. Lab on a Chip - Miniaturisation for Chemistry and Biology, 14(20), 4067-4075. https://doi.org/10.1039/c4lc00630e

OncomiR detection in circulating body fluids : A PDMS microdevice perspective. / Potrich, Cristina; Vaghi, Valentina; Lunelli, Lorenzo; Pasquardini, Laura; Santini, Gaia Cecilia; Ottone, Chiara; Quaglio, Marzia; Cocuzza, Matteo; Pirri, Candido Fabrizio; Ferracin, Manuela; Negrini, Massimo; Tiberio, Paola; De Sanctis, Veronica; Bertorelli, Roberto; Pederzolli, Cecilia.

In: Lab on a Chip - Miniaturisation for Chemistry and Biology, Vol. 14, No. 20, 21.10.2014, p. 4067-4075.

Research output: Contribution to journalArticle

Potrich, C, Vaghi, V, Lunelli, L, Pasquardini, L, Santini, GC, Ottone, C, Quaglio, M, Cocuzza, M, Pirri, CF, Ferracin, M, Negrini, M, Tiberio, P, De Sanctis, V, Bertorelli, R & Pederzolli, C 2014, 'OncomiR detection in circulating body fluids: A PDMS microdevice perspective', Lab on a Chip - Miniaturisation for Chemistry and Biology, vol. 14, no. 20, pp. 4067-4075. https://doi.org/10.1039/c4lc00630e
Potrich, Cristina ; Vaghi, Valentina ; Lunelli, Lorenzo ; Pasquardini, Laura ; Santini, Gaia Cecilia ; Ottone, Chiara ; Quaglio, Marzia ; Cocuzza, Matteo ; Pirri, Candido Fabrizio ; Ferracin, Manuela ; Negrini, Massimo ; Tiberio, Paola ; De Sanctis, Veronica ; Bertorelli, Roberto ; Pederzolli, Cecilia. / OncomiR detection in circulating body fluids : A PDMS microdevice perspective. In: Lab on a Chip - Miniaturisation for Chemistry and Biology. 2014 ; Vol. 14, No. 20. pp. 4067-4075.
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