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PCR-Free, Multiplexed Expression Profiling of microRNAs Using Silicon Photonic Microring Resonators.

ABSTRACT: We describe an approach for multiplexed microRNA analysis using silicon photonic microring resonators to detect cDNA reverse transcription products via a subsequent enzymatic signal enhancement strategy. Key to this method is a modified stem loop primer that facilitates downstream signal amplification via enzymatic turnover and improves the sensor signal 20-fold when compared to traditional stem loop primers. This approach facilitates targeted microRNA quantification in only 2.5 h and without requiring target amplification via the polymerase chain reaction (PCR). Primers for 7 miRNA targets were orthogonally designed to avoid cross-hybridization between capture probes. This approach was applied to the detection of total RNA from human tissues and found to display differential expression profiles consistent with literature precedent. This development holds promise as an alternative to single-plex RT-qPCR methods and more expensive RNA-seq by offering a cost-effective method to analyze targeted miRNA panels in emerging diagnostic applications.

SUBMITTER: Graybill RM

PROVIDER: S-EPMC5812356 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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PCR-Free, Multiplexed Expression Profiling of microRNAs Using Silicon Photonic Microring Resonators.

Graybill Richard M RM Para Christopher S CS Bailey Ryan C RC

Analytical chemistry 20161019 21

We describe an approach for multiplexed microRNA analysis using silicon photonic microring resonators to detect cDNA reverse transcription products via a subsequent enzymatic signal enhancement strategy. Key to this method is a modified stem loop primer that facilitates downstream signal amplification via enzymatic turnover and improves the sensor signal 20-fold when compared to traditional stem loop primers. This approach facilitates targeted microRNA quantification in only 2.5 h and without re ...[more]

PMID: 27726377

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Project description:The ability to perform multiple simultaneous protein biomarker measurements in complex media with picomolar sensitivity presents a large challenge to disease diagnostics and fundamental biological studies. Silicon photonic microring resonators represent a promising platform for real-time detection of biomolecules on account of their spectral sensitivity toward surface binding events between a target and antibody-modified microrings. For all refractive index-based sensing schemes, the mass of bound analytes, in combination with other factors such as antibody affinity and surface density, contributes to the observed signal and measurement sensitivity. Therefore, proteins that are simultaneously low in abundance and have a lower molecular weight are often challenging to detect. By employing a more massive secondary antibody to amplify the signal arising from the initial binding event, it is possible to improve both the sensitivity and the specificity of protein assays, allowing for quantitative sensing in complex sample matrices. Herein, a sandwich assay is used to detect the 15.5 kDa human cytokine interleukin-2 (IL-2) at concentrations down to 100 pg/mL (6.5 pM) and to quantitate unknown solution concentrations over a dynamic range spanning 2.5 orders of magnitude. This same sandwich assay is then used to monitor the temporal secretion profile of IL-2 from Jurkat T lymphocytes in serum-containing cell culture media in the presence of the entire Jurkat secretome. The same temporal secretion analysis is performed in parallel using a commercial ELISA, revealing similar IL-2 concentration profiles but superior precision for the microring resonator sensing platform. Furthermore, we demonstrate the generality of the sandwich assay methodology on the microring resonator platform for the analysis of any biomolecular target for which two high-affinity antibodies exist by detecting the approximately 8 kDa cytokine interleukin-8 (IL-8) with a limit of detection and dynamic range similar to that of IL-2. This work demonstrates the first application of silicon photonic microring resonators for detecting cellular secretion of cytokines and represents an important advance for the detection of protein biomarkers on an emerging analytical platform.

Dataset Information

PCR-Free, Multiplexed Expression Profiling of microRNAs Using Silicon Photonic Microring Resonators.

Publications

PCR-Free, Multiplexed Expression Profiling of microRNAs Using Silicon Photonic Microring Resonators.

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