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 language | English |
|---|---|
| Pages (from-to) | 4067-4075 |
| Number of pages | 9 |
| Journal | Lab on a Chip - Miniaturisation for Chemistry and Biology |
| Volume | 14 |
| Issue number | 20 |
| DOIs | |
| Publication status | Published - Oct 21 2014 |
Fingerprint
ASJC Scopus subject areas
- Biochemistry
- Chemistry(all)
- Bioengineering
- Biomedical Engineering
- Medicine(all)
Cite this
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 journal › Article
}
TY - JOUR
T1 - OncomiR detection in circulating body fluids
T2 - A PDMS microdevice perspective
AU - Potrich, Cristina
AU - Vaghi, Valentina
AU - Lunelli, Lorenzo
AU - Pasquardini, Laura
AU - Santini, Gaia Cecilia
AU - Ottone, Chiara
AU - Quaglio, Marzia
AU - Cocuzza, Matteo
AU - Pirri, Candido Fabrizio
AU - Ferracin, Manuela
AU - Negrini, Massimo
AU - Tiberio, Paola
AU - De Sanctis, Veronica
AU - Bertorelli, Roberto
AU - Pederzolli, Cecilia
PY - 2014/10/21
Y1 - 2014/10/21
N2 - 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
AB - 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
UR - http://www.scopus.com/inward/record.url?scp=84907855169&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84907855169&partnerID=8YFLogxK
U2 - 10.1039/c4lc00630e
DO - 10.1039/c4lc00630e
M3 - Article
VL - 14
SP - 4067
EP - 4075
JO - Lab on a Chip
JF - Lab on a Chip
SN - 1473-0197
IS - 20
ER -