TITLE: Validation of Selected RNA Extraction Method
AUTHORS: Ariel Cerda, 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, Aniela Wozniak

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

[STEPS]
?. [Biological Samples]
Obtain nasopharyngeal swabs in Universal Transport Medium (UTM) from 50 patients that attend outpatient services at Red Salud UC-CHRISTUS (Santiago, Chile) because of suspected coronavirus infection.
Two types of biological samples were used:For preliminary evaluation of the RNA extraction methods, use saliva samples obtained from two asymptomatic volunteers. (This protocol can be found here: Preliminary Evaluation of RNA Extraction Methods)For validation of the RNA extraction method selected, use nasopharyngeal swabs in Universal Transport Medium (UTM).
Saliva is routinely collected for the initial assessment of viral infection.
?. [RNA extraction]
Extract RNA from the 50 nasopharyngeal swabs using High Pure viral RNA extraction kit (Roche) according to instructions provided by the manufacturer.
This RNA extraction method is considered as the gold standard for comparison purposes, and it is based in capture of RNA using columns with silica filters.
?. [RT-qPCR analysis]
Perform RT-qPCR using Taqman probes and primers recommended by the CDC, and using the following steps:
?. [RT-qPCR analysis]
Perform a one-step RT-qPCR reaction in a Step-One thermal cycler (Applied Biosystems).
?. [RNA extraction]
Centrifuge a  at .
[aliquot]
Centrifuge: 12000 34, 30 s
?. [RNA extraction]
Add  to  (1:20 ratio).
[supernatant]
[1 mg/mL BSA solution]
?. [RNA extraction]
Vortex for  and keep  or store at  until further use.
on ice
-80 °C
?. [RT-qPCR analysis]
Use RT-qPCR against the human RNAseP gene with primers and a Taqman probe as previously described.
?. [RT-qPCR analysis]
Treat all RNAs with DNaseI to remove any cellular DNA from samples: Mix  with  and incubate for  at .
[RNA]
[DNaseI]
0 Room temperature
?. [RT-qPCR analysis]
For cDNA synthesis and heat inactivation of DNaseI: Mix  with  (20 pmol) and incubate for  at .
[DNA-free RNA]
[RNAse P reverse primer]
70 °C
Keep samples   until the reverse transcription mix is added.
?. [RT-qPCR analysis]
Perform reverse transcription using ImProm-II Reverse Transcriptase (Promega) according to the instructions of the manufacturor.
?. [RT-qPCR analysis]
Add  to  for a final reaction volume of 20 μl.
[RNA and primer mix]
[reverse transcription mix]
?. [RT-qPCR analysis]
Use  for RT-qPCR in a final reaction volume of 20 μl, performing RT-qPCR in a Step-One thermal cycler (Applied Biosystems).
[RNA and RNAse free water]
?. [RNA extraction]
Add  to  and mix by pipetting 3 times.
[Lysis Buffer, pH 5]
[uncentrifuged sample]
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
?. [RNA extraction]
Add  and mix by inversion 10 times.
[Precipitation Buffer]
Precipitation Buffer contains:     5 g/L sodium citrate dihydrate    6.4 g/L anhydrous citric acid    234 g/L anhydrous NaCl
?. [RNA extraction]
Incubate samples  for .
on ice
?. [RNA extraction]
Centrifuge samples at .
Centrifuge: 15000 34, 3 min
?. [RNA extraction]
Transfer  to a clean tube containing  and incubate for   at .
[supernatant]
[isopropanol]
0 Room temperature
?. [RNA extraction]
Centrifuge samples at .
Centrifuge: 15000 34, 5 min
?. [RNA extraction]
Discard the supernatant.
?. [RNA extraction]
Wash the pellet with .
[cold 70% ethanol]
?. [RNA extraction]
Centrifuge at .
Centrifuge: 15000 34, 3 min
?. [RNA extraction]
Discard the supernatant and invert tubes in paper towel.
?. [RNA extraction]
Dry the pellet by leaving tubes open for .
?. [RNA extraction]
Resuspend pellet in .
[nuclease-free water (pre-warmed at 70°C)]
?. [RT-qPCR analysis]
Use RT-qPCR against the human RNAseP gene with primers and a Taqman probe as previously described.
?. [RT-qPCR analysis]
Treat all RNAs with DNaseI to remove any cellular DNA from samples: Mix  with  and incubate for  at .
[RNA]
[DNaseI]
0 Room temperature
?. [RT-qPCR analysis]
For cDNA synthesis and heat inactivation of DNaseI: Mix  with  (20 pmol) and incubate for  at .
[DNA-free RNA]
[RNAse P reverse primer]
70 °C
Keep samples   until the reverse transcription mix is added.
?. [RT-qPCR analysis]
Perform reverse transcription using ImProm-II Reverse Transcriptase (Promega) according to the instructions of the manufacturor.
?. [RT-qPCR analysis]
Add  to  for a final reaction volume of 20 μl.
[RNA and primer mix]
[reverse transcription mix]
?. [RT-qPCR analysis]
Use  for RT-qPCR in a final reaction volume of 20 μl, performing RT-qPCR in a Step-One thermal cycler (Applied Biosystems).
[RNA and RNAse free water]
?. [RNA extraction]
Directly heat  at  for .
[sample]
98 °C
?. [RNA extraction]
Cool sample at .
4 °C
?. [RNA extraction]
Mix  with  and keep  for immediate use or store at  for later use.
[sample]
[BSA (20 mg/mL)]
on ice
-80 °C
?. [RT-qPCR analysis]
Use RT-qPCR against the human RNAseP gene with primers and a Taqman probe as previously described.
?. [RT-qPCR analysis]
Treat all RNAs with DNaseI to remove any cellular DNA from samples: Mix  with  and incubate for  at .
[RNA]
[DNaseI]
0 Room temperature
?. [RT-qPCR analysis]
For cDNA synthesis and heat inactivation of DNaseI: Mix  with  (20 pmol) and incubate for  at .
[DNA-free RNA]
[RNAse P reverse primer]
70 °C
Keep samples   until the reverse transcription mix is added.
?. [RT-qPCR analysis]
Perform reverse transcription using ImProm-II Reverse Transcriptase (Promega) according to the instructions of the manufacturor.
?. [RT-qPCR analysis]
Add  to  for a final reaction volume of 20 μl.
[RNA and primer mix]
[reverse transcription mix]
?. [RT-qPCR analysis]
Use  for RT-qPCR in a final reaction volume of 20 μl, performing RT-qPCR in a Step-One thermal cycler (Applied Biosystems).
[RNA and RNAse free water]
?. [RNA extraction]
Use an aliquot directly taken from the original RNA sample to perform RT-qPCR analysis.
?. [RT-qPCR analysis]
Use RT-qPCR against the human RNAseP gene with primers and a Taqman probe as previously described.
?. [RT-qPCR analysis]
Treat all RNAs with DNaseI to remove any cellular DNA from samples: Mix  with  and incubate for  at .
[RNA]
[DNaseI]
0 Room temperature
?. [RT-qPCR analysis]
For cDNA synthesis and heat inactivation of DNaseI: Mix  with  (20 pmol) and incubate for  at .
[DNA-free RNA]
[RNAse P reverse primer]
70 °C
Keep samples   until the reverse transcription mix is added.
?. [RT-qPCR analysis]
Perform reverse transcription using ImProm-II Reverse Transcriptase (Promega) according to the instructions of the manufacturor.
?. [RT-qPCR analysis]
Add  to  for a final reaction volume of 20 μl.
[RNA and primer mix]
[reverse transcription mix]
?. [RT-qPCR analysis]
Use  for RT-qPCR in a final reaction volume of 20 μl, performing RT-qPCR in a Step-One thermal cycler (Applied Biosystems).
[RNA and RNAse free water]
?. [RT-qPCR analysis]
Amplify two viral targets: the nucleocapsid viral proteins N1 and N2.
?. [RT-qPCR analysis]
Additionally, amplify the RNAse P target as a quality control measure for the extraction method and to corroborate the absence of PCR-inhibitors in the sample.
?. [RT-qPCR analysis]
Required cutoff points for Ct values (Cycle Threshold) to decide whether a result is COVID-19 positive or negative are specified by the CDC as follows: To report a positive result, both viral targets N1 and N2 must be CTTo report a negative result, both viral targets must be CT≥40.If one of the viral targets is CTundetermined. The RNAse P target must be CT≤35.