TITLE: Preliminary Evaluation of RNA Extraction Methods
AUTHORS: 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

[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 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.
Two types of biological samples were used:For preliminary evaluation of the RNA extraction methods, use saliva samples obtained from two asymptomatic volunteers. 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)
Saliva is routinely collected for the initial assessment of viral infection.
?. [RNA extraction]
Centrifuge a  at .
[aliquot]
Centrifuge: 12000 34, 30 s
?. [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]
Add  to  for a final reaction volume of 20 μl.
[RNA and primer mix]
[reverse transcription mix]
?. [RT-qPCR analysis]
Perform reverse transcription using ImProm-II Reverse Transcriptase (Promega) according to the instructions of the manufacturor.
?. [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]
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]
Use RT-qPCR against the human RNAseP gene with primers and a Taqman probe as previously described.
?. [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
?. [RT-qPCR analysis]
In a final reaction volume of 20 μl, perform RT-qPCR in a Step-One thermal cycler (Applied Biosystems).
?. [RNA extraction]
Directly heat  at  for .
[sample]
98 °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]
?. [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
?. [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]
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
?. [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]
Add .
[RNase-Free water]
?. [RT-qPCR analysis]
Add .
[2X TaqMan Fast Universal PCR Master Mix ]
?. [RT-qPCR analysis]
Add .
[EACH RNase P primer]
?. [RT-qPCR analysis]
Add .
[TaqMan RNase P probe]
?. [RT-qPCR analysis]
Add .
[RNA from saliva sample]
?. [RNA extraction]
Centrifuge saliva samples before taking an aliquot of supernatant for processing.
?. [RNA Extraction]
Centrifuge saliva samples before taking an aliquot of supernatant for processing using the acid pH-based method.
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).
?. [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]
Add .
[RNase-Free water]
?. [RT-qPCR Analysis]
Add .
[2X TaqMan Fast Universal PCR Master Mix ]
?. [RT-qPCR Analysis]
Add .
[EACH RNase P primer]
?. [RT-qPCR Analysis]
Add .
[TaqMan RNase P probe]
?. [RT-qPCR Analysis]
Add .
[RNA from saliva sample]
?. [RT-qPCR Analysis]
In a final reaction volume of 20 μl, perform RT-qPCR in a Step-One thermal cycler (Applied Biosystems).