Jun 04, 2020

Public workspacePreliminary Evaluation of RNA Extraction Methods

This protocol is a draft, published without a DOI.
  • Aniela Wozniak1,
  • Catalina Ibarra-Henriquez2,
  • Valentina Sebastian3,
  • Grace Armijo2,
  • Liliana Lamig2,
  • Carolina Miranda3,
  • Marcela Lagos1,
  • Sandra Solari1,
  • Ana María Guzmán1,
  • Teresa Quiroga1,
  • Susan Hitschfeld2,
  • Eleodoro Riveras2,
  • Marcela Ferres1,
  • Rodrigo A. Gutiérrez2,
  • Patricia García1,
  • Ariel Cerda2
  • 1Departamento de Laboratorios Clínicos. Escuela de Medicina. Facultad de Medicina. Pontificia Universidad Católica de chile;
  • 2FONDAP Center for Genome Regulation. Millennium Institute for Integrative Biology (iBio), Departamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile, Santiago, 8331150, Chile;
  • 3Laboratorio de Microbiología. Servicio de laboratorios Clínicos. Red de Salud UC-CHRISTUS
  • Coronavirus Method Development Community
  • Reclone.org (The Reagent Collaboration Network)
Catalina Ibarra-Henriquez
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External link: https://doi.org/10.1101/2020.05.07.083048
Protocol Citation: Aniela Wozniak, Catalina Ibarra-Henriquez, Valentina Sebastian, Grace Armijo, Liliana Lamig, Carolina Miranda, Marcela Lagos, Sandra Solari, Ana María Guzmán, Teresa Quiroga, Susan Hitschfeld, Eleodoro Riveras, Marcela Ferres, Rodrigo A. Gutiérrez, Patricia García, Ariel Cerda 2020. Preliminary Evaluation of RNA Extraction Methods. protocols.io https://protocols.io/view/preliminary-evaluation-of-rna-extraction-methods-bghmjt46
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: May 17, 2020
Last Modified: June 04, 2020
Protocol Integer ID: 37133
Keywords: Coronavirus, SARS-CoV-2, RNA extraction,
Abstract
The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.
Materials
MATERIALS
ReagentSDSBio Basic Inc.Catalog #SB0485.SIZE.100g
ReagentTRIZOL reagentInvitrogen - Thermo FisherCatalog #15596-026
Reagent100% EtOHGold Shield DistributorsCatalog #DSP-CA-151
ReagentIsopropanolFisher ScientificCatalog #A464-4
ReagentBSAMerck MilliporeSigma (Sigma-Aldrich)Catalog ##A8806
ReagentEDTAFisher ScientificCatalog #16 004Y
ReagentChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #372978
ReagentNuclease-free Water (1.75 ml/tube)Thermo FisherCatalog #AM9914G
ReagentSodium citrate dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #W302600
ReagentCitric Acid AnhydrousMerck MilliporeSigma (Sigma-Aldrich)Catalog #PHR1071
ReagentSodium chloride (anhydrous free-flowing Redi-Dri ≥99%)Merck MilliporeSigma (Sigma-Aldrich)Catalog #793566
Biological samples
Obtain saliva samples from two asymptomatic volunteers.

RNA extraction methods evaluated
(1) TRIzol
TRIzol
chloroform
isopropanol
70% ethanol
RNAse-free water

(2) BSA-based method
1 mg/mL BSA solution

(3) Acid pH-based method
Lysis Buffer
20 g/L sodium dodecyl sulfate (SDS)
20 g/L sodium citrate dihydrate
25.36 g/L anhydrous citric acid
10 mM EDTA
Precipitation Buffer
5 g/L sodium citrate dihydrate
6.4 g/L anhydrous citric acid
234 g/L anhydrous NaCl
isopropanol
70% ethanol (cold)
Nuclease-free water (pre-warmed at 70°C)

(4) High temperature-based method
BSA (20 mg/mL)

(5) Direct use of the samples

Machines required
Step-One thermal cycler (Applied Biosystems)

Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards. Obtain all necessary approvals from relevant Ethics Committees.
Biological Samples
Biological Samples
Obtain two saliva samples from each asymptomatic volunteer and perform at least three independent RNA extractions from each sample, obtaining a minimum of six RNA preparations to test each experimental procedure.
Note
Two types of biological samples were used:
  1. For preliminary evaluation of the RNA extraction methods, use saliva samples obtained from two asymptomatic volunteers.
  2. For validation of the RNA extraction method selected, use nasopharyngeal swabs in Universal Transport Medium (UTM). (This protocol can be found here: Validation of Selected RNA Extraction Method)

Note
Saliva is routinely collected for the initial assessment of viral infection.

RNA Extraction
RNA Extraction
Centrifuge saliva samples before taking an aliquot of supernatant for processing using the acid pH-based method.
Note
Under acidic pH, RNA can be separated from DNA and other molecules due to the differential polarity given by its hydroxyl groups which maintains it in solution.

Based on the methods described in the following protocols:

Heath, E. Low pH RNA isolation reagents, method, and kit. USA patent (1999).

Sambrook, J. & Russell, D. Molecular cloning: a laboratory manual. 3rd edn, (Cold Spring Harbor Laboratory, 2001).

Chomczynski, P. & Sacchi, N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nature protocols 1, 581-585, doi:10.1038/nprot.2006.83 (2006).

Add Amount300 µL Lysis Buffer, pH 5 to Amount200 µL uncentrifuged sample and mix by pipetting 3 times.
Note
Lysis Buffer contains:
20 g/L sodium dodecyl sulfate (SDS)
20 g/L sodium citrate dihydrate
25.36 g/L anhydrous citric acid
10 mM EDTA

Add Amount150 µL Precipitation Buffer and mix by inversion 10 times.
Note
Precipitation Buffer contains:
5 g/L sodium citrate dihydrate
6.4 g/L anhydrous citric acid
234 g/L anhydrous NaCl


Incubate samples TemperatureOn ice for Duration00:05:00 .

Centrifuge samples at Centrifigation15000 x g, Room temperature, 00:03:00 .

Transfer Amount600 µL supernatant to a clean tube containing Amount600 µL isopropanol and incubate for Duration00:10:00 at TemperatureRoom temperature .

Centrifuge samples at Centrifigation15000 x g, Room temperature, 00:05:00 .

Discard the supernatant.
Wash the pellet with Amount300 µL cold 70% ethanol .

Centrifuge at Centrifigation15000 x g, Room temperature, 00:03:00 .

Discard the supernatant and invert tubes in paper towel.
Dry the pellet by leaving tubes open for Duration00:10:00 .

Resuspend pellet in Amount50 µL nuclease-free water (pre-warmed at 70°C) .

RT-qPCR Analysis
RT-qPCR Analysis
Use RT-qPCR against the human RNAseP gene with primers and a Taqman probe as previously described.
CITATION
Waggoner, J. J. et al. (2013). Development of an internally controlled real-time reverse transcriptase PCR assay for pan-dengue virus detection and comparison of four molecular dengue virus detection assays.. Journal of clinical microbiology.
LINK
doi:10.1128/jcm.00548-13

Add Amount2 µL RNase-Free water .

Add Amount10 µL 2X TaqMan Fast Universal PCR Master Mix .
Add Amount1 µL EACH RNase P primer .

Add Amount1 µL TaqMan RNase P probe .

Add Amount5 µL RNA from saliva sample .

In a final reaction volume of 20 μl, perform RT-qPCR in a Step-One thermal cycler (Applied Biosystems).

Citations
Step 15
Waggoner, J. J. et al.. Development of an internally controlled real-time reverse transcriptase PCR assay for pan-dengue virus detection and comparison of four molecular dengue virus detection assays.
doi:10.1128/jcm.00548-13