Improved Reversible Cross-Linking-Based Solid-Phase RNA Extraction for Pathogen Diagnostics

Fei Zhao, Eun Yeong Lee, Yong Shin

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

In this study, we developed an amine-functionalized, diatomaceous earth-based, dimethyl suberimidate assisted (ADD) system as a novel binding strategy to improve the solid-phase extraction method for rapid and simple purification of RNA from biological samples including human cells and pathogenic bacteria. This ADD system is based on reversible cross-linking reactions between RNA and the silica matrix. The formation of robust covalent bonds protects RNA from both the sufferance of washing steps and isolation with ribonuclease (RNase)-rich samples, leading to the extraction of higher quality RNA. This improved RNA extraction system integrated with quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is evaluated for pathogen diagnostics. Compared to standard solid-phase extraction based commercial kits, this improved method shows highly enhanced sensitivity with 1000-fold higher sensitivity for human cells and 100-fold higher sensitivity for Brucella bacteria, according to the cycle threshold value of RT-qPCR. We envision that the ADD system can be tailored for commercial applications for RNA expression analysis in forensics studies, as well as for disease diagnostics in clinical applications.

Original languageEnglish
Pages (from-to)1725-1733
Number of pages9
JournalAnalytical Chemistry
Volume90
Issue number3
DOIs
Publication statusPublished - 2018 Feb 6

Bibliographical note

Funding Information:
This study was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (HI15C-2774-020015) and also by the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation of Korea (NRF) (2017R1A2B4005288).

Publisher Copyright:
© 2018 American Chemical Society.

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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