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Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor.

Chen KI, Pan CY, Li KH, Huang YC, Lu CW, Tang CY, Su YW, Tseng LW, Tseng KC, Lin CY, Chen CD, Lin SS, Chen YT - Sci Rep (2015)

Bottom Line: In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET.Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET.After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, National Taiwan University, Taipei 106, Taiwan.

ABSTRACT
Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3'-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.

No MeSH data available.


Related in: MedlinePlus

Detection of the endogenous miRNA by SiNW-FET.(A) A flow diagram of a reusable DNAprobe/SiNW-FET device. The MPTMS-modified SiNW-FET (SH/SiNW-FET) provides reversible disulfide bonding sites for the DNAprobe tagged with a thiol group at the 3′ end (DNAprobe/SiNW-FET). After the targeted miRNAs bind to the DNAprobe/SiNW-FET, the bound DNAprobe-miRNA complex can be eluted by flushing dithiothreitol (DTT) to break the disulfide bond, returning the device surface to SH/SiNW-FET. (B) The electrical conductance changes (ΔGs) of SH/SiNW-FET during repeated cycles of DTT-buffer washing, miR159probe modification, and RNA binding (0.3 μg/μL total RNA extracted from Arabidopsis). (C) miRNA159 in the Input and Eluted mixtures. After using miR159probe/SiNW-FET, the relative amounts of miR159 (upper panel) and miR168 (lower panel) in the bound fraction (Eluted) to the total RNA (Input) extracted from Arabidopsis were analyzed by RT-qPCR. (D,E) Comparison of the detection limits between RT-qPCR and SiNW-FET. We determined the amounts of miR21 expressed in different concentrations of total RNA extracted from cancer cell lines, MCF-7 and M10, by (D) RT-qPCR with specific primers or (E) miR21probe/SiNW-FET.
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f1: Detection of the endogenous miRNA by SiNW-FET.(A) A flow diagram of a reusable DNAprobe/SiNW-FET device. The MPTMS-modified SiNW-FET (SH/SiNW-FET) provides reversible disulfide bonding sites for the DNAprobe tagged with a thiol group at the 3′ end (DNAprobe/SiNW-FET). After the targeted miRNAs bind to the DNAprobe/SiNW-FET, the bound DNAprobe-miRNA complex can be eluted by flushing dithiothreitol (DTT) to break the disulfide bond, returning the device surface to SH/SiNW-FET. (B) The electrical conductance changes (ΔGs) of SH/SiNW-FET during repeated cycles of DTT-buffer washing, miR159probe modification, and RNA binding (0.3 μg/μL total RNA extracted from Arabidopsis). (C) miRNA159 in the Input and Eluted mixtures. After using miR159probe/SiNW-FET, the relative amounts of miR159 (upper panel) and miR168 (lower panel) in the bound fraction (Eluted) to the total RNA (Input) extracted from Arabidopsis were analyzed by RT-qPCR. (D,E) Comparison of the detection limits between RT-qPCR and SiNW-FET. We determined the amounts of miR21 expressed in different concentrations of total RNA extracted from cancer cell lines, MCF-7 and M10, by (D) RT-qPCR with specific primers or (E) miR21probe/SiNW-FET.

Mentions: The expressions of RNAs are under strict regulation and usually in a small quantity except those house-keeping genes. RT-qPCR is the most sensitive technique to identify the expression of a specific RNA from a mixture owing to its precise amplification procedure. To verify the detection sensitivity and target selectivity of a SiNW-FET in probing the miRNAs of interest from total extracted RNA, we anchored the single-strand DNAs (ss-DNAs) onto a 3-mercaptopropyl-trimethoxysilane (MPTMS)-modified SiNW-FET (referred to as SH/SiNW-FET) via disulfide bonding (referred to as DNAprobe/SiNW-FET) (Fig. 1A) and perfused the DNAprobe/SiNW-FET with extracted RNA for the selective binding of the complementary miRNA to the DNAprobe. We then eluted the bound DNAprobe-miRNA complexes using dithiothreitol (DTT) to reduce the disulfide bonds and returned the SiNW-FET surface to its original state for device reusability.


Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor.

Chen KI, Pan CY, Li KH, Huang YC, Lu CW, Tang CY, Su YW, Tseng LW, Tseng KC, Lin CY, Chen CD, Lin SS, Chen YT - Sci Rep (2015)

Detection of the endogenous miRNA by SiNW-FET.(A) A flow diagram of a reusable DNAprobe/SiNW-FET device. The MPTMS-modified SiNW-FET (SH/SiNW-FET) provides reversible disulfide bonding sites for the DNAprobe tagged with a thiol group at the 3′ end (DNAprobe/SiNW-FET). After the targeted miRNAs bind to the DNAprobe/SiNW-FET, the bound DNAprobe-miRNA complex can be eluted by flushing dithiothreitol (DTT) to break the disulfide bond, returning the device surface to SH/SiNW-FET. (B) The electrical conductance changes (ΔGs) of SH/SiNW-FET during repeated cycles of DTT-buffer washing, miR159probe modification, and RNA binding (0.3 μg/μL total RNA extracted from Arabidopsis). (C) miRNA159 in the Input and Eluted mixtures. After using miR159probe/SiNW-FET, the relative amounts of miR159 (upper panel) and miR168 (lower panel) in the bound fraction (Eluted) to the total RNA (Input) extracted from Arabidopsis were analyzed by RT-qPCR. (D,E) Comparison of the detection limits between RT-qPCR and SiNW-FET. We determined the amounts of miR21 expressed in different concentrations of total RNA extracted from cancer cell lines, MCF-7 and M10, by (D) RT-qPCR with specific primers or (E) miR21probe/SiNW-FET.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4663627&req=5

f1: Detection of the endogenous miRNA by SiNW-FET.(A) A flow diagram of a reusable DNAprobe/SiNW-FET device. The MPTMS-modified SiNW-FET (SH/SiNW-FET) provides reversible disulfide bonding sites for the DNAprobe tagged with a thiol group at the 3′ end (DNAprobe/SiNW-FET). After the targeted miRNAs bind to the DNAprobe/SiNW-FET, the bound DNAprobe-miRNA complex can be eluted by flushing dithiothreitol (DTT) to break the disulfide bond, returning the device surface to SH/SiNW-FET. (B) The electrical conductance changes (ΔGs) of SH/SiNW-FET during repeated cycles of DTT-buffer washing, miR159probe modification, and RNA binding (0.3 μg/μL total RNA extracted from Arabidopsis). (C) miRNA159 in the Input and Eluted mixtures. After using miR159probe/SiNW-FET, the relative amounts of miR159 (upper panel) and miR168 (lower panel) in the bound fraction (Eluted) to the total RNA (Input) extracted from Arabidopsis were analyzed by RT-qPCR. (D,E) Comparison of the detection limits between RT-qPCR and SiNW-FET. We determined the amounts of miR21 expressed in different concentrations of total RNA extracted from cancer cell lines, MCF-7 and M10, by (D) RT-qPCR with specific primers or (E) miR21probe/SiNW-FET.
Mentions: The expressions of RNAs are under strict regulation and usually in a small quantity except those house-keeping genes. RT-qPCR is the most sensitive technique to identify the expression of a specific RNA from a mixture owing to its precise amplification procedure. To verify the detection sensitivity and target selectivity of a SiNW-FET in probing the miRNAs of interest from total extracted RNA, we anchored the single-strand DNAs (ss-DNAs) onto a 3-mercaptopropyl-trimethoxysilane (MPTMS)-modified SiNW-FET (referred to as SH/SiNW-FET) via disulfide bonding (referred to as DNAprobe/SiNW-FET) (Fig. 1A) and perfused the DNAprobe/SiNW-FET with extracted RNA for the selective binding of the complementary miRNA to the DNAprobe. We then eluted the bound DNAprobe-miRNA complexes using dithiothreitol (DTT) to reduce the disulfide bonds and returned the SiNW-FET surface to its original state for device reusability.

Bottom Line: In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET.Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET.After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, National Taiwan University, Taipei 106, Taiwan.

ABSTRACT
Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3'-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.

No MeSH data available.


Related in: MedlinePlus