Viral RNA Purification Protocol Using GenElute™ Mammalian Total RNA Purification Kits

For Research Use Only; not for use in diagnostic procedures. This protocol is relevant for catalog numbers RTN70 and RTN350.

The GenElute^™ Mammalian Total RNA Purification Kit provides a rapid method for the isolation and purification of total RNA from viruses such as the 2019 Novel Coronavirus (SARS-CoV-2), which causes COVID-19 disease. Purification is based on spin column chromatography using a silica resin as the separation matrix. The following protocol is recommended for purification of viral RNA from cell-free samples including sera, plasma, and nasal and throat swabs.

Reagent Preparation

Required materials:

• GenElute^™ Mammalian Total RNA Purification Kit (RTN70 or RTN350)
• 96-100% ethanol (non-denatured)
• RNase-free water
• Poly-A carrier RNA
• Microcentrifuge
• Sterile, RNAse/DNase-free pipette tips and tubes

Before you begin:

1. Clean the workspace
2. Prepare Poly-A carrier RNA
   • Resuspend lyophilized carrier RNA with lysis buffer to a concentration of 1 µg/µL and divide into 20 µL aliquots. Store aliquots frozen at -20 °C. Avoid freeze-thaw.
3. Prepare lysis buffer
   • Determine how much lysis buffer you will need. Use 500 µL lysis buffer for each 120 µL plasma sample. Samples may need to be concentrated.
   • Add 10 µL/mL β-mercaptoethanol (BME) to lysis buffer
   • Add 10 µL/mL carrier RNA to lysis buffer
     • It may be necessary to adjust the amount of carrier RNA for your application
   • Vortex lysis buffer to mix

RNA Extraction Protocol for Serum and Plasma Samples

1. Pipette 500 µL of prepared lysis buffer containing β-mercaptoethanol and carrier RNA into a 1.5 mL RNAse-free microcentrifuge tube (not provided).
   • If sample volume is larger than 120 µL, increase the amount of lysis buffer proportionately.
2. Add 120 µL plasma or cell-free fluid. Mix by pulse-vortexing for 10 seconds.
3. Spin tubes briefly at low speed.
4. Incubate at room temperature for 5 minutes.
5. Add 500 µL of 100% ethanol. Mix by pulse-vortexing for 10 seconds.
   • Do not use denatured alcohol
   • If the sample volume is greater than 120 µL, increase ethanol volume proportionately.
6. Proceed to viral RNA purification step.

RNA Extraction Protocol for Nasal or Throat Swabs

1. Add 600 µL of prepared lysis buffer containing β-mercaptoethanol and carrier RNA into an RNase-free microcentrifuge tube.
2. Gently brush a sterile, single-use cotton swab inside the nose or mouth of the subject.
3. Using sterile techniques cut the cotton tip where the nasal or throat cells were collected and place into the microcentrifuge tube containing the lysis buffer.
4. Close the tube.
5. Vortex gently and incubate for 5 minutes at room temperature.
6. Using a pipette, transfer the lysate into another RNase-free microcentrifuge tube.
7. Note the volume of the lysate.
8. Add an equal volume of 70% ethanol to the lysate volume collected (100 µL of ethanol is added to every 100 µL of lysate).
9. Vortex to mix.
10. Proceed to viral RNA purification step.

Viral RNA Purification

1. Pipette up to 700 µL of sample/ethanol mixture into binding column and spin at 14,000 RPM for 15 seconds.
2. Carefully discard flow through and repeat above step as needed for entire sample.
3. Add 500 µL of Wash Buffer 1 to column and spin at 14,000 RPM for 15 seconds.
4. Carefully move column to a new collection tube (provided) and label.
5. Add 500 µL of Wash Buffer 2 to column and spin at 14,000 RPM for 15 seconds.
6. Carefully discard flow through and repeat above step.
7. Carefully discard flow through and spin at maximum speed for 2 minutes to dry column. Trace amounts of ethanol will have a negative effect on downstream processes.
8. Move column to a new collection tube and label appropriately.
9. Add 50 µL of elution buffer and incubate at room temperature for 5 minutes.
10. Spin at 14,000 RPM for 1 minute.
11. Use eluted RNA immediately or store at -80 °C.

Figure 1. Yield of viral RNA purified with GenElute™ Mammalian Total RNA Purification Kit. An Armored RNA SARS-CoV-2 panel was serially diluted in Universal Transport Media and purified according to the above protocol. Viral RNA fragments were detected with TaqMan RT-qPCR kit (Norgen). Amplification curves from the N1 primer/ probe set (A) and the RNase P primer/ probe set (B) were quantified using standard curves to determine RNA yield (C and D).