Limits...
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

Schematic representation of the strategy for assaying miR-10b levels in various biological compartments by qRT-PCR and LSPR-based assay. Media from AsPC-1, BxPC-3, and PANC-1 cells, which were grown under normoxia and hypoxia conditions, were collected (a) and miR-10b was quantified by the LSPR-based technique and by qRT-PCR. Aliquots of media were subjected to two sequential ultracentrifugations (b) with an intervening PBS wash, and exosomes (c) and supernatant-1 (Sup-1, d) were collected separately. Sup-1 was again ultracentrifuged at 100 000g and Sup-2 (e) was collected. The LSPR-based technique was used to quantify miR-10b directly in Sup-1 and Sup-2, while qRT-PCR was used to determine the relative miR-10b levels after RNA extraction. No visible residue was detected after the second ultracentrifugation.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4660391&req=5

fig4: Schematic representation of the strategy for assaying miR-10b levels in various biological compartments by qRT-PCR and LSPR-based assay. Media from AsPC-1, BxPC-3, and PANC-1 cells, which were grown under normoxia and hypoxia conditions, were collected (a) and miR-10b was quantified by the LSPR-based technique and by qRT-PCR. Aliquots of media were subjected to two sequential ultracentrifugations (b) with an intervening PBS wash, and exosomes (c) and supernatant-1 (Sup-1, d) were collected separately. Sup-1 was again ultracentrifuged at 100 000g and Sup-2 (e) was collected. The LSPR-based technique was used to quantify miR-10b directly in Sup-1 and Sup-2, while qRT-PCR was used to determine the relative miR-10b levels after RNA extraction. No visible residue was detected after the second ultracentrifugation.

Mentions: Chemotherapy resistance occurring in conjunction with a propensity to metastasize and a lack of early stage screening procedures contributes to the high PDAC-related mortality. It has therefore been proposed that a noninvasive test for the early detection of PDAC could significantly improve screening strategies and ultimately lead to a vastly improved prognosis in this treatment-recalcitrant cancer.12,65−68 It has been suggested that miR-10b may be an ideal plasma biomarker for PDAC,13,69 and that glypican-1 carried by exosomes could serve as an early diagnostic marker for PDAC.70 To further explore the possibility that circulating miR-10b could serve as a sensitive diagnostic marker for PDAC, we sought to establish a highly sensitive and quantitative assay for miR-10b concentrations in various biological compartments that include PCC-derived conditioned media, exosomes, and plasma (Figure 1). Here, for the first time we demonstrate a sensing approach which is able to precisely quantify the concentration of (i) extracted miR-10b from human PCCs, (ii) miR-10b in Roswell Park Memorial Institute (RPMI) medium and Dulbecco’s modified Eagle’s medium (DMEM) from these cells, (iii) extracted miR-10b from exosomes from these PCCs, and (iv) miR-10b in exosome-free supernatants (Sup) generated following two sequential ultracentrifugations (Sup-1 and Sup-2) as shown in Figure 4. Our detection method overcomes the limitation of the most widely used technique, qRT-PCR, which can only provide relative miR values rather than actual miR concentrations and which require RNA extraction procedures. Because the pancreatic tumor microenvironment (TME) is hypoxic71,72 and hypoxia up-regulates miR-10b expression,13AsPC-1, BxPC-3, and PANC-1 cells engineered to overexpress miR-10b were grown under normoxia and hypoxia (1% O2) conditions. By analyzing the concentration of miRs directly in media from the above PCCs as well as in exosomes released by these PCCs, We were able to investigate the proportion of miR-10b released by PCCs directly into the culture medium by comparison to its release via 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)

Schematic representation of the strategy for assaying miR-10b levels in various biological compartments by qRT-PCR and LSPR-based assay. Media from AsPC-1, BxPC-3, and PANC-1 cells, which were grown under normoxia and hypoxia conditions, were collected (a) and miR-10b was quantified by the LSPR-based technique and by qRT-PCR. Aliquots of media were subjected to two sequential ultracentrifugations (b) with an intervening PBS wash, and exosomes (c) and supernatant-1 (Sup-1, d) were collected separately. Sup-1 was again ultracentrifuged at 100 000g and Sup-2 (e) was collected. The LSPR-based technique was used to quantify miR-10b directly in Sup-1 and Sup-2, while qRT-PCR was used to determine the relative miR-10b levels after RNA extraction. No visible residue was detected after the second ultracentrifugation.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Schematic representation of the strategy for assaying miR-10b levels in various biological compartments by qRT-PCR and LSPR-based assay. Media from AsPC-1, BxPC-3, and PANC-1 cells, which were grown under normoxia and hypoxia conditions, were collected (a) and miR-10b was quantified by the LSPR-based technique and by qRT-PCR. Aliquots of media were subjected to two sequential ultracentrifugations (b) with an intervening PBS wash, and exosomes (c) and supernatant-1 (Sup-1, d) were collected separately. Sup-1 was again ultracentrifuged at 100 000g and Sup-2 (e) was collected. The LSPR-based technique was used to quantify miR-10b directly in Sup-1 and Sup-2, while qRT-PCR was used to determine the relative miR-10b levels after RNA extraction. No visible residue was detected after the second ultracentrifugation.
Mentions: Chemotherapy resistance occurring in conjunction with a propensity to metastasize and a lack of early stage screening procedures contributes to the high PDAC-related mortality. It has therefore been proposed that a noninvasive test for the early detection of PDAC could significantly improve screening strategies and ultimately lead to a vastly improved prognosis in this treatment-recalcitrant cancer.12,65−68 It has been suggested that miR-10b may be an ideal plasma biomarker for PDAC,13,69 and that glypican-1 carried by exosomes could serve as an early diagnostic marker for PDAC.70 To further explore the possibility that circulating miR-10b could serve as a sensitive diagnostic marker for PDAC, we sought to establish a highly sensitive and quantitative assay for miR-10b concentrations in various biological compartments that include PCC-derived conditioned media, exosomes, and plasma (Figure 1). Here, for the first time we demonstrate a sensing approach which is able to precisely quantify the concentration of (i) extracted miR-10b from human PCCs, (ii) miR-10b in Roswell Park Memorial Institute (RPMI) medium and Dulbecco’s modified Eagle’s medium (DMEM) from these cells, (iii) extracted miR-10b from exosomes from these PCCs, and (iv) miR-10b in exosome-free supernatants (Sup) generated following two sequential ultracentrifugations (Sup-1 and Sup-2) as shown in Figure 4. Our detection method overcomes the limitation of the most widely used technique, qRT-PCR, which can only provide relative miR values rather than actual miR concentrations and which require RNA extraction procedures. Because the pancreatic tumor microenvironment (TME) is hypoxic71,72 and hypoxia up-regulates miR-10b expression,13AsPC-1, BxPC-3, and PANC-1 cells engineered to overexpress miR-10b were grown under normoxia and hypoxia (1% O2) conditions. By analyzing the concentration of miRs directly in media from the above PCCs as well as in exosomes released by these PCCs, We were able to investigate the proportion of miR-10b released by PCCs directly into the culture medium by comparison to its release via 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