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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 concentration in plasma samples from three normal control (NC), three patients with PDAC, and three patients with CP using our LSPR-based sensors. Determination of miR-10b levels in plasma, Sup-1, and Sup-2 was performed without RNA extraction, whereas total RNA was extracted from exosomes. (D–F) qRT-PCR values for miR-10b from total RNA extracted from exosomes and Sup-1 in three NC, three patients with PDAC, and three patients with CP. The LSPR-based concentration and qRT-PCR values for miR-10b in exosomes were determined from the same RNA samples from each subject, all performed in a blinded manner. Each color represents a different PDAC, CP, or control subject. MiR-10b levels in Sup-2 were below the level of detection by qRT-PCR and, hence, are not shown.
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fig8: (A–C) Determination of miR-10b concentration in plasma samples from three normal control (NC), three patients with PDAC, and three patients with CP using our LSPR-based sensors. Determination of miR-10b levels in plasma, Sup-1, and Sup-2 was performed without RNA extraction, whereas total RNA was extracted from exosomes. (D–F) qRT-PCR values for miR-10b from total RNA extracted from exosomes and Sup-1 in three NC, three patients with PDAC, and three patients with CP. The LSPR-based concentration and qRT-PCR values for miR-10b in exosomes were determined from the same RNA samples from each subject, all performed in a blinded manner. Each color represents a different PDAC, CP, or control subject. MiR-10b levels in Sup-2 were below the level of detection by qRT-PCR and, hence, are not shown.

Mentions: The concentrations of miR-10b in different biological compartments were determined using our LSPR-based assay, as shown in Figure 8A–C. All three samples from PDAC patients exhibited high levels of miR-10b in both plasma and circulating exosomes (Figure 8B). By contrast, the same LSPR-based assay revealed that miR-10b levels in plasma and exosomes from normal controls (Figure 8A) and CP patients (Figure 8C) were 50- to 60-fold lower and 4- to 10-fold lower, respectively, than those in the corresponding PDAC samples. Importantly, miR-10b levels in the CP samples (Figure 8C) were significantly higher than those in normal controls (Figure 8A). Figure 8D–F shows the relative miR-10b levels determined by qRT-PCR. Supporting Information Table S7 provides the p-values for the statistical analysis that was performed to compare PDAC, CP, and normal controls. Thus, our LSPR-based assay indicates that there are very high levels of miR-10b in the exosomes isolated from the plasma of PDAC patients (Figure 8), which is in contrast to observations in plasma from breast cancer patients where only 5% of miR-16, miR-21, and miR-24 were in the exosome compartment.73 Taken together, our data validate our hypothesis that PCCs are prone to release miR-10b as cargo within exosomes.


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 concentration in plasma samples from three normal control (NC), three patients with PDAC, and three patients with CP using our LSPR-based sensors. Determination of miR-10b levels in plasma, Sup-1, and Sup-2 was performed without RNA extraction, whereas total RNA was extracted from exosomes. (D–F) qRT-PCR values for miR-10b from total RNA extracted from exosomes and Sup-1 in three NC, three patients with PDAC, and three patients with CP. The LSPR-based concentration and qRT-PCR values for miR-10b in exosomes were determined from the same RNA samples from each subject, all performed in a blinded manner. Each color represents a different PDAC, CP, or control subject. MiR-10b levels in Sup-2 were below the level of detection by qRT-PCR and, hence, are not shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4660391&req=5

fig8: (A–C) Determination of miR-10b concentration in plasma samples from three normal control (NC), three patients with PDAC, and three patients with CP using our LSPR-based sensors. Determination of miR-10b levels in plasma, Sup-1, and Sup-2 was performed without RNA extraction, whereas total RNA was extracted from exosomes. (D–F) qRT-PCR values for miR-10b from total RNA extracted from exosomes and Sup-1 in three NC, three patients with PDAC, and three patients with CP. The LSPR-based concentration and qRT-PCR values for miR-10b in exosomes were determined from the same RNA samples from each subject, all performed in a blinded manner. Each color represents a different PDAC, CP, or control subject. MiR-10b levels in Sup-2 were below the level of detection by qRT-PCR and, hence, are not shown.
Mentions: The concentrations of miR-10b in different biological compartments were determined using our LSPR-based assay, as shown in Figure 8A–C. All three samples from PDAC patients exhibited high levels of miR-10b in both plasma and circulating exosomes (Figure 8B). By contrast, the same LSPR-based assay revealed that miR-10b levels in plasma and exosomes from normal controls (Figure 8A) and CP patients (Figure 8C) were 50- to 60-fold lower and 4- to 10-fold lower, respectively, than those in the corresponding PDAC samples. Importantly, miR-10b levels in the CP samples (Figure 8C) were significantly higher than those in normal controls (Figure 8A). Figure 8D–F shows the relative miR-10b levels determined by qRT-PCR. Supporting Information Table S7 provides the p-values for the statistical analysis that was performed to compare PDAC, CP, and normal controls. Thus, our LSPR-based assay indicates that there are very high levels of miR-10b in the exosomes isolated from the plasma of PDAC patients (Figure 8), which is in contrast to observations in plasma from breast cancer patients where only 5% of miR-16, miR-21, and miR-24 were in the exosome compartment.73 Taken together, our data validate our hypothesis that PCCs are prone to release miR-10b as cargo within exosomes.

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