RNA extraction from the SARS-CoV-2 virus

Oxford Nanopore Technologies
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Protocol

RNA extraction from the SARS-CoV-2 virus V RES_9100_v1_revA_01May2020

For Research Use Only

FOR RESEARCH USE ONLY

概要

For Research Use Only

Document version: RES_9100_v1_revA_01May2020

1. Overview of the protocol

Introduction

The MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit can efficiently purify viral DNA and RNA from:

  • throat swabs
  • saliva
  • serum
  • plasma
  • bronchoalveolar lavage (BAL) fluid
  • virus culture medium

The kit is suitable for use in downstream molecular detection. It is suitable for automated extraction on the MGISP-960 and MGISP-100 automation systems; other systems can also be used with further optimisation.

RNA extraction workflow

To extract viral RNA using the MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit, you will need to:

  • Prepare a master mix using the kit components
  • Add 200 µl of the clinical sample. The RNA will bind to the magnetic beads in the master mix
  • Perform three buffer exchanges using two buffers in the kit, and 100% ethanol
  • Elute the purified RNA from the beads in RNase-free water

RNA extraction

Safety information

  • Read this protocol carefully before starting the experiment.
  • All samples and reagents should not come into direct contact with skin and eyes. Do not swallow. If contact or ingestion occurs, rinse immediately with plenty of water and seek medical attention.
  • All samples and waste should be disposed of according to local regulations.

2. Equipment and consumables - individual samples

材料
  • MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit
消耗品
  • Ethanol, 100% (e.g. Fisher, 16606002)
  • Nuclease-free pipette filter tips
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 15 or 50 ml Falcon tubes
  • RNaseZap® RNase Decontamination Solution (ThermoFisher, cat # AM9780)
装置
  • Microfuge
  • ボルテックスミキサー
  • Thermomixer
  • マグネットラック
  • P1000 pipette
  • P200 pipette
  • P20 pipette
オプション装置
  • Agilent Bioanalyzer (or equivalent)
  • Qubit蛍光光度計（またはQCチェックのための同等品）
  • Nanodrop spectrophotometer

Kit contents

Each kit provides sufficient reagents for 1728 reactions.


 Contents Volume Quantity
Box 1 Buffer MLB
Buffer MW1
Buffer MW2
RNase-free water
Proteinase K
Magnetic Beads M		 380 ml
240 ml
200 ml
120 ml
26 ml
26 ml		 1 bottle
1 bottle
1 bottle
1 bottle
1 bottle
1 bottle
Box 2 Enhancer buffer 2 ml 1 tube

Buffer MLB and Buffer MW1 may arrive with some precipitation, which does not affect their function. If precipitation occurs, heat the reagent bottle in a 37°C water bath for ~10 min until the precipitation disappears, then mix thoroughly for use.

Storage and shelf life

Different reagents in the kit have different storage conditions. Store the reagents according to the following table:

Reagent Storage
Enhancer Buffer -25°C to -15°C
Proteinase K 2-8°C
Magnetic Beads M 2-8°C
All other reagents 0-30°C
重要

Reagents contained within a kit are intended to be used together. Do not mix reagents from different kit lots.

3. Equipment and consumables - 96-well plate

材料
  • MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit
消耗品
  • Ethanol, 100% (e.g. Fisher, 16606002)
  • Nuclease-free pipette filter tips
  • 96-well deepwell plates
  • 96-well plate lids or seals
  • 50 ml Falcon tubes
  • 1.5 ml Eppendorf DNA LoBind tubes
  • RNaseZap® RNase Decontamination Solution (ThermoFisher, cat # AM9780)
装置
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Vortex mixer with plate adapter
  • Plate thermomixer
  • Magnetic rack suitable for 96-well plates
  • Multichannel pipette and tips
  • P1000 pipette
  • P200 pipette
  • P20 pipette
オプション装置
  • Agilent Bioanalyzer (or equivalent)
  • Qubit蛍光光度計（またはQCチェックのための同等品）
  • Nanodrop spectrophotometer

Kit contents

Each kit provides sufficient reagents for 1728 reactions.


 Contents Volume Quantity
Box 1 Buffer MLB
Buffer MW1
Buffer MW2
RNase-free water
Proteinase K
Magnetic Beads M		 380 ml
240 ml
200 ml
120 ml
26 ml
26 ml		 1 bottle
1 bottle
1 bottle
1 bottle
1 bottle
1 bottle
Box 2 Enhancer buffer 2 ml 1 tube

Buffer MLB and Buffer MW1 may arrive with some precipitation, which does not affect their function. If precipitation occurs, heat the reagent bottle in a 37°C water bath for ~10 min until the precipitation disappears, then mix thoroughly for use.

Storage and shelf life

Different reagents in the kit have different storage conditions. Store the reagents according to the following table:

Reagent Storage
Enhancer Buffer -25°C to -15°C
Proteinase K 2-8°C
Magnetic Beads M 2-8°C
All other reagents 0-30°C
重要

Reagents contained within a kit are intended to be used together. Do not mix reagents from different kit lots.

4. Viral RNA extraction - individual samples

材料
  • MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit
消耗品
  • Ethanol, 100% (e.g. Fisher, 16606002)
  • 15 or 50 ml Falcon tubes
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Nuclease-free pipette filter tips
  • RNaseZap® RNase Decontamination Solution (ThermoFisher, cat # AM9780)
装置
  • ボルテックスミキサー
  • Microfuge
  • マグネットラック
  • Thermomixer
  • P1000 pipette
  • P200 pipette
  • P20 pipette
オプション装置
  • Agilent Bioanalyzer (or equivalent)
  • Qubit蛍光光度計（またはQCチェックのための同等品）
  • Nanodrop spectrophotometer
重要

RNA handling best practice

It is important to follow the below best practice recommendations to avoid RNA degradation:

  • Perform the sample handling and extraction in a PCR hood
  • Use dedicated pipettes that are reserved only for RNA work
  • Use filter pipette tips for every step of the extraction protocol
  • Decontaminate all equipment and surfaces using RNaseZap™ RNase Decontamination Solution before starting the extraction

Bring all kit reagents to room temperature. Mix the bottle contents briefly by vortexing.

オプショナルステップ

If Buffer MLB or Buffer MW1 has a precipitate, incubate the bottle in a 37°C water bath until the precipitate dissolves. Shake and mix well before use.

Add pure (100%) ethanol into Buffer MW1 and Buffer MW2 according to the amount indicated on the bottle label.

Prepare the patient samples according to the manufacturer's instructions.

Mix Magnetic Beads M thoroughly before the next step.

In a Falcon tube, prepare a master mix using the reagents in the table below:

N represents the number of samples to be tested.

Reagent Volume
Buffer MLB 200x N µl
Pure ethanol 250x N µl
Proteinase K 15x N µl
Magnetic Beads M 15x N µl
Enhancer Buffer 1x N µl
Total 481x N µl

Mix gently by flicking the tube, and spin down.

重要

The prepared master mix needs to be used within 30 mins of preparation. If preparing the master mix in advance, omit the Proteinase K and add it to the master mix shortly before dispensing to avoid proteinase inactivation.

Dispense 460 μl of the master mix for each sample into 1.5 ml Eppendorf DNA LoBind tubes.

Add 200 μl of the patient samples to each tube containing master mix.

Mix the contents of each tube by vortexing, and spin down.

Incubate for 10 minutes at room temperature.

Spin down the samples and pellet on a magnet for 1 min. When the liquid clears, carefully remove the supernatant without disturbing the pellet.

Remove the tubes from the magnetic rack, and add 500 μl Buffer MW1 (ensure that absolute ethanol has been added) into each tube. Mix well for 1 min, and spin down.

Return the tubes to the magnetic rack, and pellet the beads for 1 min. When the liquid clears, carefully remove the supernatant without disturbing the pellet.

Remove the tubes from the magnetic rack, and add 500 μl Buffer MW2 (ensure that absolute ethanol has been added) into each tube. Mix well for 1 min, and spin down.

Return the tubes to the magnetic rack, and pellet the beads for 1 min. When the liquid clears, carefully remove the supernatant without disturbing the pellet.

Remove the tubes from the magnetic rack, and add 600 μl pure ethanol into each tube. Mix well for 1 min, and spin down.

Return the tubes to the magnetic rack, and pellet the beads for 1 min. When the liquid clears, carefully remove the supernatant without disturbing the pellet.

Spin down the tubes, and place them back in the magnetic rack. Remove all residual ethanol, and leave the tubes on the bench with the lids open for 30 seconds.

Remove the tubes from the magnetic rack and add 50 μl of RNase-free Water to each tube. Mix well by vortexing.

Place the tubes on a thermomixer and incubate at 56°C, shaking at 1,000 rpm for 5 min. Briefly spin down.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain ~45 µl of each eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

最終ステップ

The extracted RNA can be stored at -80°C until ready to be analysed.

After extraction, we recommend using the Real-Time Fluorescent RT-PCR kit for detecting 2019-nCoV (SARS-CoV-2).

The instructions for using this kit are found in the RT-PCR for detecting the SARS-CoV-2 virus protocol.

5. Viral RNA extraction - 96-well plate

材料
  • MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit
消耗品
  • Ethanol, 100% (e.g. Fisher, 16606002)
  • 50 ml Falcon tubes
  • 96-well deepwell plates
  • 96-well plate lids or seals
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Nuclease-free pipette filter tips
  • RNaseZap® RNase Decontamination Solution (ThermoFisher, cat # AM9780)
装置
  • Vortex mixer with plate adapter
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Magnetic rack suitable for 96-well plates
  • Plate thermomixer
  • Multichannel pipette and tips
  • P1000 pipette
  • P200 pipette
  • P20 pipette
オプション装置
  • Agilent Bioanalyzer (or equivalent)
  • Qubit蛍光光度計（またはQCチェックのための同等品）
  • Nanodrop spectrophotometer
重要

RNA handling best practice

It is important to follow the below best practice recommendations to avoid RNA degradation:

  • Perform the sample handling and extraction in a PCR hood
  • Use dedicated pipettes that are reserved only for RNA work
  • Use filter pipette tips for every step of the extraction protocol
  • Decontaminate all equipment and surfaces using RNaseZap™ RNase Decontamination Solution before starting the extraction

Bring all kit reagents to room temperature. Mix the bottle contents briefly by vortexing.

オプショナルステップ

If Buffer MLB or Buffer MW1 has a precipitate, incubate the bottle in a 37°C water bath until the precipitate dissolves. Shake and mix well before use.

Add pure (100%) ethanol into Buffer MW1 and Buffer MW2 according to the amount indicated on the bottle label.

Prepare the patient samples according to the manufacturer's instructions.

Mix Magnetic Beads M thoroughly before the next step.

In a Falcon tube, prepare a master mix using the reagents in the table below:

These volumes are sufficient for 96 reactions.

Reagent Volume
Buffer MLB 19,200 µl
Pure ethanol 24,000 µl
Proteinase K 1,440 µl
Magnetic Beads M 1,440 µl
Enhancer Buffer 96 µl
Total 44,176 µl

Mix the contents of the tube, and spin down.

重要

The prepared master mix needs to be used within 30 mins of preparation. If preparing the master mix in advance, omit the Proteinase K and add it to the master mix shortly before dispensing to avoid proteinase inactivation.

Dispense 460 μl of the master mix for each sample into the wells of the 96-well plate.

Add 200 μl of the patient samples to each well of the 96-well plate.

Mix the contents of the plate by vortexing, and spin down.

Incubate for 10 minutes at room temperature.

Spin down the plate and pellet on a magnet for 1 min. When the liquid clears, carefully remove the supernatant without disturbing the pellet.

Remove the plate from the magnetic rack, and add 500 μl Buffer MW1 (ensure that absolute ethanol has been added) into each well of the plate. Mix well for 1 min, and spin down.

Return the plate to the magnetic rack, and pellet the beads for 1 min. When the liquid clears, carefully remove the supernatant without disturbing the pellet.

Remove the plate from the magnetic rack, and add 500 μl Buffer MW2 (ensure that absolute ethanol has been added) into each well of the plate. Mix well for 1 min, and spin down.

Return the plate to the magnetic rack, and pellet the beads for 1 min. When the liquid clears, carefully remove the supernatant without disturbing the pellet.

Remove the plate from the magnetic rack, and add 600 μl pure ethanol into each well of the plate. Mix well for 1 min, and spin down.

Return the plate to the magnetic rack, and pellet the beads for 1 min. When the liquid clears, carefully remove the supernatant without disturbing the pellet.

Spin down the plate, and place it back in the magnetic rack. Remove all residual ethanol, and leave the plate on the bench with the lids off for 30 seconds.

Remove the plate from the magnetic rack and add 50 μl of RNase-free Water to each well of the plate. Mix well by vortexing.

Place the plate on a thermomixer and incubate at 56°C, shaking at 1,000 rpm for 5 min. Briefly spin down.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain ~45 µl of each eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

最終ステップ

The extracted RNA can be stored at -80°C until ready to be analysed.

After extraction, we recommend using the Real-Time Fluorescent RT-PCR kit for detecting 2019-nCoV (SARS-CoV-2).

The instructions for using this kit are found in the RT-PCR for detecting the SARS-CoV-2 virus protocol.

Last updated: 5/1/2020

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