Viral RNA Purification Protocol Using GenElute™ Spin Prep Kits

Research use only. Not for use in diagnostic procedures. This protocol is relevant for catalog number RNB100.

• Products
• Reagent Preparation
• Lysate Preparation
• RNA Purification

The GenElute™ 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 proprietary resin as the separation matrix. The RNA is preferentially purified from other cellular components, such as proteins, without the use of phenol or chloroform. The procedure involves:

• Lysing cells or sample with provided buffer
• Loading sample onto spin column
• Washing to remove impurities
• Elution of purified RNA

The protocol is designed to yield purified RNA of the highest integrity that can be used in various downstream applications including PCR, Northern blotting, and expression array assays.

Reagent Preparation

• Prepare a working concentration of “Wash Solution A” by adding 90 mL of 96-100% ethanol (provided by the user) to the supplied bottle(s) containing concentrated “Wash Solution A”. This will give a final volume of 128 mL. The label on the bottle has a box that may be checked to indicate that the ethanol has been added.
  
  
• The use of β-mercaptoethanol in lysis is highly recommended for most animal tissues, particularly those known to have a high RNAse content (example: pancreas), as well as for nasal and throat swabs. It is also recommended for users who wish to isolate RNA for sensitive downstream applications. Add 10 µL of β-mercaptoethanol (provided by the user) to each 1 mL of “Buffer RL” required. β-mercaptoethanol is toxic and should be dispensed in a fume hood. Alternatively, the “Buffer RL” can be used as provided. It is important to work quickly during this procedure.

Lysate Preparation

• Cell culture
• Tissue
• Blood
• Nasal or throat swab
• Free viral particles

Cell Culture Samples (Mammalian Cells)

• Adherent cells
• Suspension cells

Note: Ensure that all solutions are at room temperature prior to use. The maximum recommended number of cells is 3 x 10⁶. Cell pellets can be stored at –70 °C for later use or used directly in the procedure. Determine the number of cells present before freezing. Frozen pellets should be stored for no longer than 2 weeks to ensure that the integrity of the RNA is not compromised. Frozen cell pellets should not be thawed prior to beginning the protocol. Add the Buffer RL directly to the frozen cell pellet.

Adherent cell lysis protocol:

1. Aspirate culture medium and wash cell monolayer with an appropriate amount of PBS.
2. Aspirate PBS.
3. Add 350 µL of Buffer RL directly to culture plate.
4. Lyse cells by gently tapping culture dish and swirling buffer around plate surface for five minutes.
5. Transfer lysate to a microcentrifuge tube.
6. Add 200 µL of 96-100% ethanol (provided by the user) to the lysate. Mix by vortexing for 10 seconds.¹
7. Proceed to RNA Purification step.

¹ For starting amounts greater than 10⁶ cells, it is recommended that the lysate is passed through a 25 gauge needle attached to a syringe 5-10 times at this point, in order to shear the genomic DNA prior to loading onto the column.

Suspension cell lysis protocol:

1. Transfer cell suspension to an RNase-free tube (not provided) and centrifuge at no more than 200 x g (~2,000 rpm) for 10 minutes to pellet cells.
2. Carefully decant the supernatant.
3. Add 350 µL of Buffer RL to the pellet.
4. Lyse cells by vortexing for 15 seconds. Ensure that the entire pellet is completely dissolved before proceeding to the next step.
5. Add 200 µL of 96-100% ethanol to the lysate. Mix by vortexing for 10 seconds.²
6. Proceed to RNA Purification step.

² For starting amounts greater than 10⁶ cells, it is recommended that the lysate is passed through a 25 gauge needle attached to a syringe 5-10 times at this point, in order to shear the genomic DNA prior to loading onto the column.

Tissue Samples

Notes:

• The GenElute™ Total RNA Purification Kit is designed for isolating RNA from small amounts of tissue sample (up to 10 mg).
• RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized. It is therefore important that the procedure is carried out as quickly as possible, particularly the Cell Lysate Preparation step.
• Fresh or frozen tissues may be used for the procedure.
• Tissues should be flash-frozen in liquid nitrogen and transferred immediately to a –70 °C freezer for long-term storage. Tissues may be stored at –70 °C for several months.
• When isolating total RNA from frozen tissues, ensure that the tissue does not thaw during weighing or prior to grinding with the mortar and pestle.
• Tissues stored in RNA stabilization reagents are compatible with this isolation procedure. Prior to isolation, carefully remove the tissue from the storage reagent using forceps and dry excessive liquid.

Tissue lysis procedure:

1. Excise tissue sample.
2. Determine the amount of tissue by weighing and refer to Table 1 for maximum weight. 10 mg is optimal for tissues not mentioned in the table.

1. Transfer the tissue into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample. Grind the tissue thoroughly using a pestle. Allow the liquid nitrogen to evaporate, without allowing the tissue to thaw.
2. Add 600 µL of Buffer RL to the tissue sample and continue to grind until the sample has been homogenized.
3. Homogenize by passing the lysate 5-10 times through a 25 gauge needle attached to a syringe.
4. Using a pipette, transfer the lysate into an RNase-free microcentrifuge tube.
5. Spin the lysate for 2 minutes to pellet any cell debris.
6. Transfer the supernatant to another RNase-free microcentrifuge tube. Note the volume of the supernatant/lysate.
7. Add an equal volume of 70% ethanol to the lysate volume collected (100 µL of ethanol is added to every 100 µL of lysate).
8. Vortex to mix.
9. Proceed to RNA Purification step

Blood Samples

• Whole blood
• Plasma and serum

Whole Blood

Notes: This procedure is for the isolation of RNA from whole blood. It is recommended that no more than 100 µL of blood be used in order to prevent clogging of the column. We recommend the use of this kit to isolate RNA from non-coagulating fresh blood using EDTA as the anti-coagulant. Working quickly is important for this procedure.

Whole blood lysis procedure:

1. Transfer up to 100 µL of non-coagulating blood to an RNase-free microcentrifuge tube.
2. Add 350 µL of Buffer RL to the blood.
3. Lyse cells by vortexing for 15 seconds. Ensure that mixture becomes transparent before proceeding to the next step.
4. Add 200 µL of 96–100% ethanol to the lysate.
5. Mix by vortexing for 10 seconds.
6. Proceed to RNA Purification step.

Plasma and Serum Samples

Notes:

• Plasma or serum of all human and animal subjects is considered potentially infectious. All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with plasma or serum.
• We recommend the use of this kit to isolate RNA from plasma or serum prepared by standard protocol from non-coagulating fresh blood using EDTA or sodium citrate as the anti-coagulant. Plasma prepared from fresh blood using heparin as an anti-coagulant is not suitable for use with this protocol. For heparin-prepared samples follow the protocol for Whole Blood samples.
• It is recommended that no more than 200 µL of plasma or serum be used in order to prevent clogging of the column. Avoid multiple freeze-thaw cycle of the plasma or serum sample. Aliquot to the appropriate volume for usage prior to freezing. It is important to work quickly during this procedure.
• The yield of RNA from plasma and serum is highly variable. In general, the expected yield could vary from 1 to 100 ng per 100 µL of plasma or serum used. In addition, the expected A260:A280 ratio as well as the A260:A230 ratio will be lower (<1.80) than the normal acceptable range from other cells or tissues.

Plasma and serum lysis procedure:

1. Transfer up to 200 µL of plasma or serum to an RNase-free microcentrifuge tube.
2. Add 300 µL of Buffer RL to every 100 µL of plasma or serum.
3. Mix by vortexing for 10 seconds.
   
   Optional: Add 0.7 µL of 0.8 µg/µL MS2 RNA per sample. The use of MS2 RNA could increase the consistency of downstream applications such as RT-PCR. However, the use of MS2 RNA is not recommended for applications involving global gene expression analysis such as microarrays or sequencing.
   
   
4. Add 400 µL of 96–100% ethanol (provided by the user) to every 400 µL of the lysate (equivalent to every 100 µL of plasma or serum used).
5. Mix by vortexing for 10 seconds.
6. Proceed to RNA Purification step.

Nasal or Throat Swabs

Notes: Body fluid of all human and animal subjects is considered potentially infectious. It is important to work quickly during this procedure.

Nasal or throat swab lysis procedure:

1. Add 600 µL of Buffer RL to 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 Buffer RL.
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 RNA Purification step.

Free Viral Particles

Notes:

• It is recommended that no more than 100 µL of viral suspension be used in order to prevent clogging of the column.
• It is important to work quickly during this procedure.

Free viral particle lysis procedure:

1. Transfer up to 100 µL of viral suspension to an RNase-free microcentrifuge tube (not provided).
2. Add 350 µL of Buffer RL.
3. Lyse viral particles by vortexing for 15 seconds.
4. Add 200 µL of 96–100% ethanol (provided by the user) to the lysate.
5. Mix by vortexing for 10 seconds.
6. Proceed to RNA Purification step.

RNA Purification

Binding RNA to Column

1. Assemble a column with one of the provided collection tubes.
2. Apply up to 600 µL of the lysate with the ethanol onto the column
3. Centrifuge for 1 minute at ~3,500 x g (~6,000 rpm). Ensure the entire lysate volume has passed through into the collection tube by inspecting the column. If the entire lysate volume has not passed, spin for an additional minute at 14,000 x g (~14,000 rpm).
4. Discard the flowthrough.
5. Reassemble the spin column with its collection tube.
6. Depending on the lysate volume, repeat the above steps as necessary.

The GenElute™ Total RNA Purification Kit isolates total RNA with minimal amounts of genomic DNA contamination. As an optional step, the On-Column DNA Removal Protocol can be performed at this point for maximum removal of residual DNA that may affect sensitive downstream applications. Please refer to separate instructions provided in the user guide to perform the On-Column DNA Removal using Catalog Number DNASE10-1SET (not included in kit).

Column Wash

1. Apply 400 µL of Wash Solution A to the column and centrifuge for 1 minute. Ensure the entire ethanol solution has passed through into the collection tube by inspecting the column. If the entire wash volume has not passed, spin for an additional minute.
2. Discard the flowthrough and reassemble the spin column with its collection tube.
3. Repeat steps to wash column two additional times.
4. After the final wash, spin the column for 2 minutes in order to thoroughly dry the resin.
5. Discard the collection tube.

RNA Elution

1. Place the column into a fresh 1.7 mL Elution tube provided with the kit.
2. Add 50 µL of Elution Solution A to the column.
3. Centrifuge for 2 minutes at 200 x g (~2,000 rpm), followed by 1 minute at 14,000 x g (~14,000 rpm).
4. Note the volume eluted from the column. If the entire 50 µL has not been eluted, spin the column at 14,000 x g (~14,000 rpm) for 1 additional minute.
5. For maximum RNA recovery, it is recommended that a second elution be performed into a separate microcentrifuge tube (repeat the above steps).

The purified RNA sample may be stored at –20 °C for a few days. It is recommended that samples be placed at –70 °C for long term storage.