TITLE: MARVICS: A Robust and Safe Magnetic Nanoparticle based  RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples
AUTHORS: Mo Li, Gerardo Ramos-Mandujano

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[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  acid guanidinium thiocyanate-phenol-chloroform (AGPC, e.g., TRIzol Reagent or TRI reagent).
1 mL
Follow CDC or institutional safety guidlines when handling potential infectious samples. AGPC, TRIzol and TRI reagent contain phenol. Follow local safety guidelines when handling and disposing these reagents.
?. Making Bis-Tris Buffer
50 mL
?. Dissolve  guanidinium hydrochloride and  Bis-Tris in   of 100% ethanol.
14.33 g
104.6 mg
45 mL
If Bis-Tris is not available, it may be substituted by Tris Base (10 mM final concentration)
Add 40 ml of 100% ethanol to the other chemicals, and wait for guanidinium hydrochloride to completely dissolve before adding the remaining volume of 100% Ethanol.
?. Adjust pH (2O to .
50 mL
?. Magnetic-nanoparticle-Aided Viral RNA Isolation of Contagious Samples.
?. In an Eppendorf tube add    clinical sample,  Bis-Tris buffer, mix well by vortexing.
200 µl
200 µl
We recommend Lo-Bind Eppendorf tubes or similar low binding tubes.
Samples contain phenol. Cap the tubes slowly. Make sure the tubes can be closed securely during vortexing.
?. Add   SiMNP, mix at
40 µl
1300
The SiMNP stock is composed of 1 volume of SiMNPs and 1 volume of RNase-free water. The stock is further diluted to be used for RNA extraction. Typical dilution ranges from 1:4 to 1:10. The optimal ratio should determined empirically.
?. Spin the tube for 2-3 seconds, settle the SiMNPs on a magnetic stand and remove the supernatant.
The supernatant contains phenol. Follow local safety guidelines when handling and disposing these reagents.
?. Mix    of AGPC (TRIzol or TRI reagent) and  Bis-Tris buffer, add to the SiMNPs, mix well by vortexing.
200 µl
200 µl
?. Settle the SiMNPs on a magnetic stand and remove the supernatant.
The supernantant contains phenol. Follow local safety guidelines when handling and disposing these reagents.
?. Add   90% ethanol,  spin for 2-3 seconds, settle the siMNPs on a magnetic stand and remove the supernatant.
400 µl
It is highly recommended to prepare fresh 90% ethanol before use. Make sure the 90% ethanol container is closed tightly to prevent evaporation.
?. Repeat Setp 4.6 three more times for a total of 4 ethanol washes
?. 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.
Drying may take less than 20 min. Monitor the appearance of the SiMNPs during drying. The SiMNPs appear in a rusty brownish color when dried.
?. To elute the RNA, add  nuclease-free water, and mix at for  at room temperature.
40 µl
1300
?. Settle the SiMNPs on a magnetic stand and transfer the eluted RNA to a new RNase-free tube.
Pipet slowly, avoid taking up SiMNPs.
?. Analyze RNA concentration and purity using a Qubit fluorometer or Nanodrop.
?. Reverse transcription (RT): use  of eluted RNA and follow the instructions for SuperScript™ IV Reverse Transcriptase adding the RNase H incubation step.
4 µl
User should optimize the input RNA amount.
?. Real-time PCR: For each  qPCR reaction mix   cDNA,  SARS-CoV-2 (2019-nCoV) CDC qPCR Probe Assay,  TaqMan Fast Advanced Master Mix, and  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.
10 µl
1.5 µl
0.5 µl
5 µl
1.5 µl
?. Store RNA at -80℃ or use immediately.