MAVRICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples
Mo Li, Gerardo Ramos-Mandujano
SARS-CoV-2
MAVRICS
COVID-19
RNA extraction
Magnetic nanoparticle synthesis
Silica magnetic nanoparticles (SiMNP)
Abstract
Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magneticnanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities.
Steps
Silica magnetic nanoparticles (SiMNP) synthesis .
SiMNP synthesis was done following the published protocols in BOMB.bio: BOMB magnetic core nanoparticles synthesis and BOMB coating ferrite MNPs with silica oxide.
COVID-19 patient samples .
Oropharyngeal or nasopharyngeal swabs are steeped in 1mL acid guanidinium thiocyanate-phenol-chloroform (AGPC, e.g., TRIzol Reagent or TRI reagent).
0h 1m 0s Making Bis-Tris Buffer 50mL
Dissolve 14.33g guanidinium hydrochloride and 104.6mg Bis-Tris in 50mL of 90% ethanol.0h 10m 0s
Adjust pH (<6.5) with HCl, and adjust the volume with 90% of ethanol to 50mL.
0h 5m 0s
Magnetic-nanoparticle-Aided Viral RNA Isolation of Contagious Samples .
In an Eppendorf tube add 200µL clinical sample, 200µL Bis-Tris buffer, mix well by vortexing.0h 1m 0s
Settle the SiMNPs on a magnetic stand and transfer the eluted RNA to a new RNase-free tube.0h 2m 0s
Analyze RNA concentration and purity using a Qubit fluorometer or Nanodrop.0h 10m 0s
Store RNA at -80℃ or use immediately.
Add 40µL SiMNP, mix at 1300rpm 0h 5m 0s
Spin the tube for 2-3 seconds, settle the SiMNPs on a magnetic stand and remove the supernatant.0h 2m 0s
Mix 200µL of AGPC (TRIzol or TRI reagent) and 200µL Bis-Tris buffer, add to the SiMNPs, mix well by vortexing.0h 1m 0s
Settle the SiMNPs on a magnetic stand and remove the supernatant.0h 2m 0s
Add 400µL of 90% ethanol and mix well, spin for 2-3 seconds, settle the siMNPs on a magnetic stand and remove the supernatant.0h 2m 0s
Repeat Setp 4.6 three more times for a total of 4 ethanol washes0h 6m 0s
After removing the supernatant from the last ethanol wash, dry the SiMNPs on a heat block at 50°C. Keep the lid open, no shaking. Do not elute before the SiMNPs are dried. 0h 20m 0s
To elute the RNA, add 40µL nuclease-free water, and mix at 1300rpmfor 0h 5m 0s at room temperature.
Reverse transcription (RT) : use 4µL of eluted RNA and follow the instructions for SuperScript™ IV Reverse Transcriptase adding the RNase H incubation step. 1h 0m 0s
Real-time PCR : For each 10µL qPCR reaction mix 1.5µL cDNA, 0.5µL SARS-CoV-2 (2019-nCoV) CDC qPCR Probe Assay, 5µL TaqMan Fast Advanced Master Mix, and 1.5µL nuclease-free water. Run qPCR on a Biorad CFX384 Touch Real-Time PCR Detection System (or similar instrument) using the following program: 50°C for 2 min, 95°C for 2 min followed by 45 cycles of 95°C for 5 sec and 59°C for 30 sec. 1h 20m 0s
