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.
Bibliographical noteFunding 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).
© 2018 American Chemical Society.
All Science Journal Classification (ASJC) codes
- Analytical Chemistry