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Combining asymmetric PCR-based enzymatic amplification with silicon photonic microring resonators for the detection of lncRNAs from low input human RNA samples.

ABSTRACT: A method for quantifying biologically relevant long-non-coding RNAs by combining nucleic acid amplification via asymmetric polymerase chain reaction (PCR) with label-free PCR product detection using silicon photonic microring resonator arrays is described. This approach eliminates the need for fluorophores, which presents a limit for spectral multiplexing in conventional qPCR methods, and rather offers potential for much higher levels of plexity by spatially arraying capture probes. Here, we demonstrate the potential of this technique to detect two differentially expressed lncRNA transcripts and an internal control mRNA transcript in different commercial human tissue specimens, as well as in a glioblastoma cell line using only nanogram input amounts of total RNA. The obtained results were validated using single-plex RT-qPCR and found to be in good agreement, demonstrating the potential of this technique for lncRNA quantification applications.

SUBMITTER: Cardenosa-Rubio MC

PROVIDER: S-EPMC5826891 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Combining asymmetric PCR-based enzymatic amplification with silicon photonic microring resonators for the detection of lncRNAs from low input human RNA samples.

Cardenosa-Rubio Maria C MC Graybill Richard M RM Bailey Ryan C RC

The Analyst 20180201 5

A method for quantifying biologically relevant long-non-coding RNAs by combining nucleic acid amplification via asymmetric polymerase chain reaction (PCR) with label-free PCR product detection using silicon photonic microring resonator arrays is described. This approach eliminates the need for fluorophores, which presents a limit for spectral multiplexing in conventional qPCR methods, and rather offers potential for much higher levels of plexity by spatially arraying capture probes. Here, we dem ...[more]

PMID: 29431756

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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

Combining asymmetric PCR-based enzymatic amplification with silicon photonic microring resonators for the detection of lncRNAs from low input human RNA samples.

Publications

Combining asymmetric PCR-based enzymatic amplification with silicon photonic microring resonators for the detection of lncRNAs from low input human RNA samples.

Similar Datasets

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