RNA – COVID-19
Viral RNA Extraction
Viral RNA Isolation Protocol
The protocol is developed for processing 50-300 μL samples.
- Pipet 20 µL Proteinase K, 2 µL L Solution, and 300 µL Buffer LYE to a 1.5 mL tube. Calculate the number of samples to be processed and make master mix of proteinase K, L Solution and Buffer LYE.
- Pipet 300 µL nasopharyngeal or oropharyngeal aspirates or washes, nasopharyngeal or oropharyngeal swabs, broncheoalveolar lavage, tracheal aspirates, or plasma, serum, into the 1.5 mL tube from Step 1. Mix well by pulse-vortexting for 10 seconds. Incubate at room temperature for 10 min to lyse the cells and virus.
- Add 600 µL isopropanol and mix well by pulse-vortexing for 5 seconds. Spin briefly to collect the drop from the lid.
Note: Maintain the ratio of (Sample+ Buffer LYE): Isopropanol= 1:1
- Transfer 600 µL of the sample from step 3 into a RNA column and centrifuge at 10,000 rpm for 30 seconds. Discard the flow-through carefully to a waste container by pipetting and put the column back to the collection Transfer the remaining sample to the column and centrifuge at 10,000 rpm for 30 seconds. Discard the flow through and put the column back to the collection tube. Note: When transferring the column/collection tubes, always hold the upper rim of the collection tube, not the column, to avoid possible drop off of the collection tube.
- Add 500 µL Buffer RB to the column and centrifuge at 10,000 rm for 30 seconds. Discard the flow-through. Put the column back to the collection tube.
- Add 500 µL RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 seconds. Discard the flow-through. Put the column back to the collection tube.
- Centrifuge the empty column at 12,000 rpm for 2 min. It is critical to remove residual ethanol for optimal elution.
- Transfer the RNA column to a RNase-free 5 mL tube, add 35-50 µL DEPC-treated water to the column and centrifuge at 10,000 rpm for 30 seconds. The viral RNA is in the flow-through liquid.
- Optional: Add the eluent back to the column for a second elution.
Note: The first elution normally yields 70% of the RNA while the second elution yields another 20-30% of the RNA bound to the column.
Note: The purified RNA should be put on ice for downstream application or store at -20°C.