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Label-Free Nanoplasmonic-Based Short Noncoding RNA Sensing at Attomolar Concentrations Allows for Quantitative and Highly Specific Assay of MicroRNA-10b in Biological Fluids and Circulating Exosomes.

Joshi GK, Deitz-McElyea S, Liyanage T, Lawrence K, Mali S, Sardar R, Korc M - ACS Nano (2015)

Bottom Line: Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility.We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls.Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis , 402 North Blackford Street, LD 326, Indianapolis, Indiana 46202, United States.

ABSTRACT
MicroRNAs are short noncoding RNAs consisting of 18-25 nucleotides that target specific mRNA moieties for translational repression or degradation, thereby modulating numerous biological processes. Although microRNAs have the ability to behave like oncogenes or tumor suppressors in a cell-autonomous manner, their exact roles following release into the circulation are only now being unraveled and it is important to establish sensitive assays to measure their levels in different compartments in the circulation. Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility. The sensor was used to specifically detect microRNA-10b at the attomolar (10(-18) M) concentration in pancreatic cancer cell lines, derived tissue culture media, human plasma, and media and plasma exosomes. In addition, for the first time, our label-free and nondestructive sensing technique was used to quantify microRNA-10b in highly purified exosomes isolated from patients with pancreatic cancer or chronic pancreatitis, and from normal controls. We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls. Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures.

No MeSH data available.


Related in: MedlinePlus

(A–C) Determination of miR-10b concentrations in three different pancreatic cancer cell lines under two different culture conditions, normoxia (blue bars) and hypoxia (red bars), in various biological compartments using our LSPR-based sensors. Determination of miR-10b concentrations in media, Sup-1, and Sup-2 was performed without RNA extraction, whereas total RNA was extracted from cells and exosomes. All three cell lines were engineered to overexpress miR-10b. (D–F) qRT-PCR values for normoxia (purple bars) and hypoxia (green bars) using sham-transfected pancreatic cancer cells and cells engineered to overexpress miR-10b (blue and red bars). miR-10b was assayed in total RNA extracted from media, cell, exosomes, and Sup-1 under normoxia (blue bars) and hypoxia conditions (red bars). The LSPR-based concentration and qRT-PCR fold change in miR-10b levels in cells and exosomes were determined from aliquots derived from the corresponding total RNA samples. However, by qRT-PCR, miR-10b was not detectable in Sup-2. The detailed procedure for exosomes isolation and RNAs extraction procedure are provided in the Materials and Methods.
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fig5: (A–C) Determination of miR-10b concentrations in three different pancreatic cancer cell lines under two different culture conditions, normoxia (blue bars) and hypoxia (red bars), in various biological compartments using our LSPR-based sensors. Determination of miR-10b concentrations in media, Sup-1, and Sup-2 was performed without RNA extraction, whereas total RNA was extracted from cells and exosomes. All three cell lines were engineered to overexpress miR-10b. (D–F) qRT-PCR values for normoxia (purple bars) and hypoxia (green bars) using sham-transfected pancreatic cancer cells and cells engineered to overexpress miR-10b (blue and red bars). miR-10b was assayed in total RNA extracted from media, cell, exosomes, and Sup-1 under normoxia (blue bars) and hypoxia conditions (red bars). The LSPR-based concentration and qRT-PCR fold change in miR-10b levels in cells and exosomes were determined from aliquots derived from the corresponding total RNA samples. However, by qRT-PCR, miR-10b was not detectable in Sup-2. The detailed procedure for exosomes isolation and RNAs extraction procedure are provided in the Materials and Methods.

Mentions: To quantify miR-10b levels in the above PCCs, cells (4 × 105) were lysed and total RNA (including miRs) was extracted using a TRIzol kit followed by a single-step purification with the Direct-zol RNA MiniPrep kit which yielded a final elution volume of 30 μL/sample. Next, 14 μL from each sample was used for LSPR-based detection, whereas the remaining 14 μL was used for qRT-PCR. We quantified miR-10b in crude media from each cell line by incubating over the LSPR-based sensor for 12 h, as described in the Materials and Methods. Subsequently, the sensors were washed with PBS buffer, and the λLSPR was measured. Figure 5A–C illustrates the LSPR-based determination of miR-10b concentrations in two types of media from three different PCCs. RPMI medium that was collected from BxPC-3 and AsPC-1 cells that were grown under hypoxia contained ∼462 and 360 fM of miR-10b, respectively. DMEM collected from PANC-1 cells grown under hypoxia contained ∼70 fM of miR-10b. We observed a similar pattern for miR-10b concentrations that were determined following extraction of total RNA from BxPC-3, AsPC-1, and PANC-1 cells of ∼390, ∼20, and ∼5 fM, respectively. We also compared the LSPR-based values with qRT-PCR data from the same samples and they showed the same general trend (Figure 5D–F).


Label-Free Nanoplasmonic-Based Short Noncoding RNA Sensing at Attomolar Concentrations Allows for Quantitative and Highly Specific Assay of MicroRNA-10b in Biological Fluids and Circulating Exosomes.

Joshi GK, Deitz-McElyea S, Liyanage T, Lawrence K, Mali S, Sardar R, Korc M - ACS Nano (2015)

(A–C) Determination of miR-10b concentrations in three different pancreatic cancer cell lines under two different culture conditions, normoxia (blue bars) and hypoxia (red bars), in various biological compartments using our LSPR-based sensors. Determination of miR-10b concentrations in media, Sup-1, and Sup-2 was performed without RNA extraction, whereas total RNA was extracted from cells and exosomes. All three cell lines were engineered to overexpress miR-10b. (D–F) qRT-PCR values for normoxia (purple bars) and hypoxia (green bars) using sham-transfected pancreatic cancer cells and cells engineered to overexpress miR-10b (blue and red bars). miR-10b was assayed in total RNA extracted from media, cell, exosomes, and Sup-1 under normoxia (blue bars) and hypoxia conditions (red bars). The LSPR-based concentration and qRT-PCR fold change in miR-10b levels in cells and exosomes were determined from aliquots derived from the corresponding total RNA samples. However, by qRT-PCR, miR-10b was not detectable in Sup-2. The detailed procedure for exosomes isolation and RNAs extraction procedure are provided in the Materials and Methods.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4660391&req=5

fig5: (A–C) Determination of miR-10b concentrations in three different pancreatic cancer cell lines under two different culture conditions, normoxia (blue bars) and hypoxia (red bars), in various biological compartments using our LSPR-based sensors. Determination of miR-10b concentrations in media, Sup-1, and Sup-2 was performed without RNA extraction, whereas total RNA was extracted from cells and exosomes. All three cell lines were engineered to overexpress miR-10b. (D–F) qRT-PCR values for normoxia (purple bars) and hypoxia (green bars) using sham-transfected pancreatic cancer cells and cells engineered to overexpress miR-10b (blue and red bars). miR-10b was assayed in total RNA extracted from media, cell, exosomes, and Sup-1 under normoxia (blue bars) and hypoxia conditions (red bars). The LSPR-based concentration and qRT-PCR fold change in miR-10b levels in cells and exosomes were determined from aliquots derived from the corresponding total RNA samples. However, by qRT-PCR, miR-10b was not detectable in Sup-2. The detailed procedure for exosomes isolation and RNAs extraction procedure are provided in the Materials and Methods.
Mentions: To quantify miR-10b levels in the above PCCs, cells (4 × 105) were lysed and total RNA (including miRs) was extracted using a TRIzol kit followed by a single-step purification with the Direct-zol RNA MiniPrep kit which yielded a final elution volume of 30 μL/sample. Next, 14 μL from each sample was used for LSPR-based detection, whereas the remaining 14 μL was used for qRT-PCR. We quantified miR-10b in crude media from each cell line by incubating over the LSPR-based sensor for 12 h, as described in the Materials and Methods. Subsequently, the sensors were washed with PBS buffer, and the λLSPR was measured. Figure 5A–C illustrates the LSPR-based determination of miR-10b concentrations in two types of media from three different PCCs. RPMI medium that was collected from BxPC-3 and AsPC-1 cells that were grown under hypoxia contained ∼462 and 360 fM of miR-10b, respectively. DMEM collected from PANC-1 cells grown under hypoxia contained ∼70 fM of miR-10b. We observed a similar pattern for miR-10b concentrations that were determined following extraction of total RNA from BxPC-3, AsPC-1, and PANC-1 cells of ∼390, ∼20, and ∼5 fM, respectively. We also compared the LSPR-based values with qRT-PCR data from the same samples and they showed the same general trend (Figure 5D–F).

Bottom Line: Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility.We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls.Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis , 402 North Blackford Street, LD 326, Indianapolis, Indiana 46202, United States.

ABSTRACT
MicroRNAs are short noncoding RNAs consisting of 18-25 nucleotides that target specific mRNA moieties for translational repression or degradation, thereby modulating numerous biological processes. Although microRNAs have the ability to behave like oncogenes or tumor suppressors in a cell-autonomous manner, their exact roles following release into the circulation are only now being unraveled and it is important to establish sensitive assays to measure their levels in different compartments in the circulation. Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility. The sensor was used to specifically detect microRNA-10b at the attomolar (10(-18) M) concentration in pancreatic cancer cell lines, derived tissue culture media, human plasma, and media and plasma exosomes. In addition, for the first time, our label-free and nondestructive sensing technique was used to quantify microRNA-10b in highly purified exosomes isolated from patients with pancreatic cancer or chronic pancreatitis, and from normal controls. We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls. Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures.

No MeSH data available.


Related in: MedlinePlus