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[Analytical Methods]Dynamic Solid-Phase RNA Extraction from a Biological Sample in a Polyester-Toner


Abstract:

The standard molecular diagnostic techniques for dengue virus infection involve sample preparation (RNA extraction) followed by reverse transcription-nucleic acid amplification and separation/detection of DNA fragments. Efficient and reliable RNA extraction is crucial for all downstream steps. Solid-phase extraction (SPE) is one of the most used methodologies for RNA purification, and it has been largely adapted for microfluidic applications. Here, we describe a methodology for RNA purification of dengue virus in polyester-toner (PeT) microfluidic devices using a magnetic silica-based dynamic solid phase extraction (dSPE). PeT microdevices were produced by a print, cut, and laminate (PCL) methodology previously described by Duarte et al.1The dSPE methodology involved performing adsorption, washing, and elution steps. The PeT microchip has already proven to be biocompatible and capable of performing various steps of genetic analysis involving DNA. In this work, we show for the first time that PeT microdevices can also be used for RNA analysis. High-quality pure RNA (amplifiable via RT-PCR and/or RT-LAMP) was obtained in dSPE experiments carried out in PeT microchips, demonstrating that RNA is compatible with reagents used in dSPE and with substrates used in PeT microchip fabrication. Dengue virus RNA was purified from human serum samples using the proposed dSPE methodology in PeT microchips, making this a viable technique for isolating RNA towards integration with downstream sample analysis, such as RT-PCR or RT-LAMP, to produce a fully integrated device for genetic analysis involving RNA.

Thiago Dadalto Gimenez, Alexandre Melo Bailão, Célia Maria de Almeida Soares, Fabíola Souza Fiaccadori, Menira Borges de Lima Dias Souza and Gabriela Rodrigues Mendes Duarte

Anal. Methods, 2017, Accepted Manuscript

DOI: 10.1039/C6AY03481K

Link: http://pubs.rsc.org/en/content/articlelanding/2017/ay/c6ay03481k#!divAbstract

#031317 #RNA #magnetic #solidphaseextraction #LAMP #DigitalPCR

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