Dec 08, 2021
Version 3
MAVRICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples V.3
- Mo Li1,
- Gerardo Ramos-Mandujano2
- 1King Abdullah University of Science and Technology;
- 2Laboratory of Stem Cell and Regeneration, Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology (KAUST)
Protocol Citation: Mo Li, Gerardo Ramos-Mandujano 2021. MAVRICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.b2p8qdrwVersion created by Mo Li
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 08, 2021
Last Modified: December 08, 2021
Protocol Integer ID: 55776
Keywords: SARS-CoV-2, MAVRICS, COVID-19, RNA extraction, Magnetic nanoparticle synthesis, Silica magnetic nanoparticles (SiMNP),
Funders Acknowledgements:
KAUST
Grant ID: BAS/1/1080-01
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.
Materials
MATERIALS
Bis-TrisGold BiotechnologyCatalog #B-020
TRIzol™ ReagentThermo FisherCatalog #15596018
SuperScript™ IV Reverse TranscriptaseThermo FisherCatalog #18090010
TaqMan™ Fast Advanced Master MixThermo FisherCatalog #4444556
Hydrochloric AcidFisher ScientificCatalog #A144S
Sodium hydroxideSigma-aldrichCatalog #306576
Ethanol absolute ≥99.8% AnalaR NORMAPUR® ACS Reag. Ph. Eur. analytical reagentCatalog #20821.330DP
Tetraethyl orthosilicate ≥99.0% (GC)Sigma-aldrichCatalog #86578
Iron (III) chloride anhydrous Extra PureFisher ScientificCatalog #10224390
Guanidine HydrochlorideFisher ScientificCatalog #BP178-500
RNaseOUT™ Recombinant Ribonuclease InhibitorInvitrogen - Thermo FisherCatalog #10777019
RNase HNew England BiolabsCatalog #M0297
2019-nCoV RUO KitCatalog #10006605
Iron(II) chloride tetrahydrate ≥98%
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.
Overnight
COVID-19 patient samples.
Oropharyngeal or nasopharyngeal swabs are steeped in 1 mL acid guanidinium thiocyanate-phenol-chloroform (AGPC, e.g., TRIzol Reagent or TRI reagent).
Safety information
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.
00:01:00
Making Bis-Tris Buffer 50 mL
Dissolve 14.33 g guanidinium hydrochloride and 104.6 mg Bis-Tris in 50 mL of 90% ethanol.00:10:00
Note
If Bis-Tris is not available, it may be substituted by Tris Base (10 mM final concentration)
Note
Add 90% ethanol to the chemicals to a final volume of 45-48 ml and wait for guanidinium hydrochloride to completely dissolve by mixing.
Adjust pH (<6.5) with HCl, and adjust the volume with 90% of ethanol to 50 mL .
00:05:00
Magnetic-nanoparticle-Aided Viral RNA Isolation of Contagious Samples.
In an Eppendorf tube add 200 µL clinical sample, 200 µL Bis-Tris buffer, mix well by vortexing.00:01:00
Note
We recommend Lo-Bind Eppendorf tubes or similar low binding tubes.
Safety information
Samples contain phenol. Cap the tubes slowly. Make sure the tubes can be closed securely during vortexing.
Add 40 µL SiMNP, mix at 1300 rpm 00:05:00
Note
The SiMNP stock is further diluted with RNase-free water to be used for RNA extraction. Recommended dilution for swab samples mixed with TRIzol is 1 volume of fully resuspended SiMNP stock to 4 volumes of RNase-free water. The optimal ratio for other samples should be determined empirically; typical dilution ranges from 1:4 to 1:10.
Spin the tube for 2-3 seconds, settle the SiMNPs on a magnetic stand and remove the supernatant.00:02:00
Safety information
The supernatant contains phenol. Follow local safety guidelines when handling and disposing these reagents.
Mix 200 µL of AGPC (TRIzol or TRI reagent) and 200 µL Bis-Tris buffer, add to the SiMNPs, mix well by vortexing.00:01:00
Settle the SiMNPs on a magnetic stand and remove the supernatant.00:02:00
Safety information
The supernantant contains phenol. Follow local safety guidelines when handling and disposing these reagents.
Add 400 µL of 90% ethanol and mix well, spin for 2-3 seconds, settle the siMNPs on a magnetic stand and remove the supernatant.00:02:00
Note
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 washes00:06:00
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. 00:20:00
Note
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 40 µL nuclease-free water, and mix at 1300 rpm for 00:05:00 at room temperature.
Settle the SiMNPs on a magnetic stand and transfer the eluted RNA to a new RNase-free tube.00:02:00
Note
Pipet slowly, avoid taking up SiMNPs.
Analyze RNA concentration and purity using a Qubit fluorometer or Nanodrop.00:10:00
Store RNA at -80℃ or use immediately.
Reverse transcription (RT): use 4 µL of eluted RNA and follow the instructions for SuperScript™ IV Reverse Transcriptase adding the RNase H incubation step. 01:00:00
Note
User should optimize the input RNA amount.
Real-time PCR: For each 10 µL qPCR reaction mix 1.5 µL cDNA, 0.5 µL SARS-CoV-2 (2019-nCoV) CDC qPCR Probe Assay, 5 µL TaqMan Fast Advanced Master Mix, and 1.5 µL 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. 01:20:00