VetMAX BRSV PI3 Kit Purification Kit€¦ · • Automated nucleic acid purification is performed using one of the following instruments: KingFisher™ Flex, MagMAX™ Express-96,

VetMAX™ BRSV PI3 KitNucleic acid purification protocols optimized for use with the kit (Cat. No. TRSVPI350)Pub. No. MAN0019168 Rev. A.0

Species Sample matrices Test type

Bovine

• Nasopharyngeal and tracheal swabs

• Organs (lungs)

• Tracheal and bronchoalveolar fluids

Individual or pooled(according to the type of sample)

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.

WARNING! BIOHAZARD. Read the biological hazard safety information at this product’s page at thermofisher.com. Follow allapplicable local, state/provincial, and/or national regulations for working with biological samples.

■ Purpose of this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

■ Sample selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

■ Sample storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

■ Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

■ Recommended RNA purification protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

■ Purify RNA using the MagMAX™ CORE Nucleic Acid Purification Kit (automated method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

■ Purify RNA using the MagVet™ Universal Isolation Kit (automated method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

■ Prepare samples for manual purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

■ Purify RNA using the RNeasy™ Mini Kit (manual method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

■ Purify RNA using the NucleoSpin™ RNA kit (manual method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

■ Purify RNA using the QIAamp™ Viral RNA Mini Kit (manual method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■ Purify RNA using the NucleoSpin™ RNA Virus kit (manual method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■ Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Appendix A (Alternative protocol) Parallel purification of RNA/DNA using the MagMAX™ CORE Nucleic Acid Purification Kit

■ Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

■ Purification procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Appendix B Purification with the KingFisher™ Duo Prime or KingFisher™ mL instrument

■ Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

■ Purification procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Appendix C Documentation and support

■ Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

■ Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

SUPPLEMENTAL INSTRUCTIONS

For Laboratory Use.

http://thermofisher.com/support

http://www.thermofisher.com

Purpose of this guideThis guide describes the bRSV and PI3 RNA purification protocols that have been validated and optimized for downstream use with theApplied Biosystems™ VetMAX™ BRSV PI3 Kit (Cat. No. TRSVPI350).

• Automated nucleic acid purification is performed using one of the following instruments: KingFisher™ Flex, MagMAX™ Express-96,KingFisher™ mL, or KingFisher™ Duo Prime.

• Manual nucleic acid purification uses silica-based spin columns.

Sample selection

Sample type Type ofanalysis Quantity required and sampling equipment

Nasopharyngeal and tracheal swabs Individual orpooled Swab in a dry tube

Organs (lungs) Individual 20–30 mg of tissue

Tracheal and bronchoalveolar fluids Individual Minimum volume: 5 mL of transtracheal aspirate (TTA) or bronchoalveolar lavage fluid

Sample storage

Sample type Storage

Nasopharyngeal andtracheal swabs

After collection, maintain the samples at 2°C to 8°C until use (up to 48 hours).

After elution of the swab, use the eluate directly to perform PCR. After use or after 48 hours, store samples below −16°C forup to 1 year, or below −70°C for long‑term storage.

Organs (lungs) After sampling, store as indicated:• Store samples at 2°C to 8°C if the analysis is to be performed within 24 hours of sampling.

• Store samples below −16°C if the analysis is to be performed more than 24 hours after sampling.

After use or after 24 hours, store samples below −16°C for up to 1 year, or below −70°C for long‑term storage.

Tracheal andbronchoalveolar fluids

After collection, maintain the samples at 2°C to 8°C until use (up to 48 hours).

After use or after 48 hours, store samples below −16°C for up to 1 year.

Required materials not suppliedUnless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that the material is available from fisherscientific.com or another major laboratory supplier.

Materials required for sample collection, preparation, and nucleic acid purification

Table 1 Materials required for all sample preparation methods

Item Source

Equipment

Type II Biological Safety Cabinet (BSCII) MLS

Benchtop microcentrifuge MLS

Laboratory mixer, vortex or equivalent MLS

Adjustable precision micropipettors (range of 1 μL to 1,000 μL) MLS

Consumables

Aerosol-resistant, nuclease-free pipette tips MLS

1.5‑mL and 2.0‑mL DNase/RNase-free microtubes MLS

Reagents

Nuclease-free water AM12450

PBS (1X), pH 7.4 MLS

2 VetMAX™ BRSV PI3 Kit Supplemental Instructions


http://fisherscientific.com

Table 2 Additional materials required for purification from organ samples

Item Source

Equipment

Tissue homogenizer for bead-beating, one of the following, or equivalent:• Fisher Scientific™ Bead Mill 24 Homogenizer

• Precellys™ 24 Homogenizer (Bertin)

• FastPrep‑24™ Instrument (MP Biomedical 116004500)

• Mixer Mill 400 (Verder 207450001)

• Fisher Scientific 15-340-163

• Bertin EQ03119.200.RD000.0

• Fisher Scientific MP116004500

• Fisher Scientific 08 418 241

Precision scale MLS

PYREX™ Solid Glass Beads for Distillation Columns (3 mm), or equivalent 3‑mm glass beads Fisher Scientific™ 11-312-10A

Scalpels and metallic forceps (sterile) MLS

Consumables

Petri dish (sterile) MLS

Additional materials required for automated nucleic acid purification

Table 3 Materials required for the MagMAX™ CORE Nucleic Acid Purification Kit

Item Source

Instrument, one of the following:

KingFisher™ Flex Purification SystemContact your local sales office.

MagMAX™ Express‑96 Magnetic Particle Processor

KingFisher™ Duo Prime Purification System 5400110

KingFisher™ mL Purification System 5400050

Equipment

Reagent reservoir MLS

Consumables

Consumables for the KingFisher™ Flex and MagMAX™ Express-96 instruments:• KingFisher™ Deepwell 96 Plate

• KingFisher™ 96 KF microplates

• KingFisher™ 96 tip comb for DW magnets

• 95040450

• 97002540

• 97002534

Consumables for the KingFisher™ Duo Prime and KingFisher™ mL instruments See Table 10 on page 20.

Adhesive PCR Plate Foils, or equivalent AB0626

Kits and reagents

MagMAX™ CORE Nucleic Acid Purification Kit A32700 or A32702

PBS, pH 7.4 (10X), RNase‑free AM9624

Table 4 Materials required for the MagVet™ Universal Isolation Kit

Item Source

Instrument, one of the following:

KingFisher™ Flex Purification SystemContact your local sales office.

MagMAX™ Express‑96 Magnetic Particle Processor

KingFisher™ mL Purification System 5400050

Equipment

Reagent reservoir MLS

VetMAX™ BRSV PI3 Kit Supplemental Instructions 3

Item Source

Kits and reagents

MagVet™ Universal Isolation Kit MV384

Ethanol, 80% MLS

Additional materials required for manual nucleic acid purification

Item Source

Consumables

(Optional) Spin‑column homogenizer, one of the following, depending on the protocol used[1].• NucleoSpin™ Filter

• QIAshredder

• Macherey Nagel 740606

• Qiagen 79654

Kits and reagents

One of the following kits:• RNeasy™ Mini Kit

• NucleoSpin™ RNA kit

• QIAamp™ Viral RNA Mini Kit

• NucleoSpin™ RNA Virus kit

• Qiagen 74104


• Qiagen 52904


Ethanol, 96–100% MLS

Ethanol, 70%[2] MLS

14.3 M β‑mercaptoethanol (βME)[2] MLS

[1] Recommended for the lysis of organs and viscous lysates using the NucleoSpin™ RNA kit or RNeasy™ Mini Kit.[2] Required only for purification using the NucleoSpin™ RNA kit or RNeasy™ Mini Kit.

Recommended RNA purification protocols

Sample type Automated purification Manual purification


• Organs (lungs) • MagMAX™ CORE Nucleic Acid PurificationKit (page 5)

• MagVet™ Universal Isolation Kit (page 9)

• RNeasy™ Mini Kit (page 11)

• NucleoSpin™ RNA kit (page 12)

Tracheal and bronchoalveolar fluids• QIAamp™ Viral RNA Mini Kit (page 14)

• NucleoSpin™ RNA Virus kit (page 15)


Purify RNA using the MagMAX™ CORE Nucleic Acid Purification Kit (automated method)Follow this procedure if you are using these instruments:

• KingFisher™ Flex

• MagMAX™ Express-96

Follow Appendix B, “Purification with the KingFisher™ Duo Prime or KingFisher™ mL instrument” if you are using these instruments:

• KingFisher™ Duo Prime

• KingFisher™ mL

OverviewThis procedure is designed for rapid purification of viral RNA from the following sample types:


• Organs (lungs)

• TTA

• Bronchoalveolar lavage fluid

If you are processing samples for use in other downstream applications, an alternative protocol is available that allows parallel purificationof RNA and DNA. See Appendix A, “(Alternative protocol) Parallel purification of RNA/DNA using the MagMAX™ CORE Nucleic AcidPurification Kit”.

Workflow

MagMAX™ CORE Nucleic Acid Purification Kit workflow

Set up the processing plates

Prepare the Bead/PK Mix

Prepare the Lysis/Binding Solution

Prepare the sample

Combine samples with the Bead/PK Mix and Lysis/Binding Solution

Process samples on the instrument

Procedural guidelines• Before use, invert bottles of solutions and buffers to ensure thorough mixing.

• Mix samples with reagents using a plate shaker or by pipetting up and down.

Note: Do not use a plate shaker with the tube strips required by the KingFisher™ mL instrument.

• To prevent cross-contamination:– Cover the plate or tube strip during the incubation and shaking steps, to prevent spill-over.

– Carefully pipet reagents and samples, to avoid splashing.

• To prevent nuclease contamination:– Wear laboratory gloves during the procedures. Gloves protect you from the reagents, and they protect the nucleic acid from

nucleases that are present on skin.

– Use nucleic acid-free pipette tips to handle the reagents, and avoid putting used tips into the reagent containers.

– Decontaminate lab benches and pipettes before you begin.


Before first use of the kit

(Optional) Determine the optimal bead mill homogenizer settings

We recommend using the Fisher Scientific™ Bead Mill 24 Homogenizer for maximum nucleic acid yield. If an alternative instrument isused, follow the manufacturer's guidelines to determine the speed and time settings necessary to achieve sufficient cell lysis.

Determine the maximum plate shaker setting

If a plate shaker is used, determine the maximum setting.

1. Verify that the plate fits securely on your shaker.

2. Add 1 mL of water to each well of the plate, then cover with sealing foil.

3. Determine the maximum setting that you can use on your shaker without any of the water splashing onto the sealing foil.

Download and install the script

The appropriate script for the MagMAX™ CORE Nucleic Acid Purification Kit must be installed on the instrument before first use.

1. On the MagMAX™ CORE Nucleic Acid Purification Kit product web page (at thermofisher.com, search by catalogue number), scrollto the Product Literature section.

2. Right‑click the appropriate file to download the latest version of the MagMAX_CORE script for your instrument.

Table 5 Recommended scripts

Instrument Script name

KingFisher™ Flex MagMAX_CORE_Flex.bdzKingFisher™ 96

MagMAX™ Express-96

MagMAX_CORE_KF-96.bdz

KingFisher™ Duo Prime MagMAX_CORE_DUO.bdzKingFisher™ mL MagMAX_CORE_mL_no_heat.bdz

If required by your laboratory, use one of the following scripts, which do not heat the samples during the elution step.

Table 6 Alternate scripts without heated elution step

Instrument Script name

KingFisher™ Flex MagMAX_CORE_Flex_no_heat.bdzKingFisher™ 96

MagMAX™ Express-96

MagMAX_CORE_KF-96_no_heat.bdz

KingFisher™ Duo Prime MagMAX_CORE_DUO_no_heat.bdzKingFisher™ mL MagMAX_CORE_mL_no_heat.bdz

3. See your instrument user guide or contact Technical Support for instructions for installing the script.



Perform the purification procedure

a. Set up the processing plates.

Table 7 Plate setup: KingFisher™ Flex or MagMAX™ Express-96 instrument

Plate ID Plateposition[1] Plate type Reagent Volume per well

Wash Plate 1 2 Deep Well MagMAX™ CORE Wash Solution 1 500 µL


Elution 4 Standard MagMAX™ CORE Elution Buffer 90 µL

Tip Comb 5 Standard Place a tip comb in the plate.

[1] Position on the instrument.

b. (Optional) To prevent evaporation and contamination, cover the prepared processing plates withsealing foil until they are loaded into the instrument.

1 Set up the processingplates

Prepare new Bead/PK Mix for each processing run.

a. Vortex the MagMAX™ CORE Magnetic Beads thoroughly to ensure that the beads are fullyresuspended.

b. Combine the following components for the required number of samples plus 10% overage.

Component Volume per sample

MagMAX™ CORE Magnetic Beads 20 µL

MagMAX™ CORE Proteinase K 10 µL

Total Bead/PK Mix 30 µL

(Optional) Store the Bead/PK Mix at 4°C for up to 1 week.

2 Prepare the Bead/PK Mix

a. Combine the following components for the required number of samples plus 10% overage.


MagMAX™ CORE Lysis Solution 350 µL

MagMAX™ CORE Binding Solution 350 µL

Total Lysis/Binding Solution 700 µL

b. Invert the tube or bottle at least 10 times to mix.

(Optional) Store the Lysis/Binding Solution at room temperature for up to 24 hours.

3 Prepare the Lysis/BindingSolution


Prepare samples as described.

Sample type Action

Nasopharyngeal or trachealswab

Follow the manufacturer's recommended protocol, or follow this procedure:1. Break off the tip of the swab and add to a 2‑mL tube.

2. Add 1 mL of PBS (1X), pH 7.4 to each sample.

3. Vortex for 3 minutes.

4. Proceed with 200 µL of supernatant.

Organ (lung) 1. Add the following components to a 2-mL tube.• Tissue—20 to 30 mg

• PBS (1X), pH 7.4—1 mL

• PYREX™ Solid Glass Beads for Distillation Columns (3 mm)—2 beads

2. Disrupt (bead-beat) the samples.• Fisher Scientific™ Bead Mill 24 Homogenizer—6 m/s for 45 seconds

• Mixer Mill 400—30 Hz for 5 minutes

3. Centrifuge at 1,000 × g for 1 minute.

4. Proceed with 100 μL of supernatant.

TTA or bronchoalveolarlavage fluid

Proceed with 200 µL of fluid.

4 Prepare the sample

a. Invert the tube of Bead/PK Mix several times to resuspend the beads, then add 30 µL of theBead/PK Mix to the required wells in the plate or tube strip.

b. Transfer the appropriate volume of each prepared sample to a well with Bead/PK Mix.

For... Use...

Nasopharyngeal and tracheal swab 200 µL of supernatant

Organ (lung) 100 µL of supernatant

TTA and bronchoalveolar lavage fluid 200 µL of fluid

Mock‑purified sample —

c. Mix the sample with the Bead/PK Mix for 2 minutes at room temperature according to yourmixing method.

• Using a plate shaker—Shake vigorously for 2 minutes (see “Determine the maximum plateshaker setting” on page 6).

• By pipetting—Pipet up and down several times, then incubate for 2 minutes at roomtemperature. (For downstream processing on the KingFisher™ mL Magnetic ParticleProcessor, you must mix by pipetting.)

d. Add 700 µL of the Lysis/Binding Solution to each sample-containing well or tube.

e. Immediately proceed to process samples on the instrument (next section).

5 Combine samples with theBead/PK Mix andLysis/Binding Solution

a. Select the appropriate script on the instrument (see “Download and install the script” on page 6).

b. Start the run, then load the prepared plates in the appropriate positions when prompted by theinstrument.

Store purified nucleic acid on ice for immediate use, at −20°C for up to 1 month, or at −80°C forlong‑term storage.

6 Process samples on theinstrument


Purify RNA using the MagVet™ Universal Isolation Kit (automated method)The following protocol can be used with the KingFisher™ Flex, KingFisher™ mL, and MagMAX™ Express-96 instruments.


• Prepare the NM1 Buffer—Transfer 100 mL of N1 Buffer to the bottle of M1 Buffer (25 mL), then vortex to mix thoroughly.

Store the NM1 Buffer at room temperature for up to 1 year.

Before each use of the kitPrepare NM2+Beads Mix—Combine the following components for the required number of samples plus 5–10% overage, then vortex tomix thoroughly.


NM2 Binding Solution 600 µL

NM_LSI_Beads 20 µL

Discard the NM2+Beads Mix after use.



Sample type Action


1. Add 1 mL of PBS (1X), pH 7.4 to the swab in the transport tube, thenswirl the swab in the PBS.For pooled samples, add 1 mL of PBS (1X), pH 7.4 to 3–5 swabs fromthe pool.

2. Press the swab against the wall of the tube to squeeze out the liquid, thendiscard the swab.For pooled samples, repeat this procedure for the remaining swabs.

3. Centrifuge at 1,500 × g for 10 minutes.

4. Carefully remove the supernatant, leaving approximately 100 μL ofsupernatant in the tube to avoid aspiration of the cell pellet.

5. Gently pipet up and down to resuspend the cell pellet.

6. Proceed with 100 μL of the cell suspension.

Organ (lung) 1. Finely mince the organ piece in a sterile petri dish, using sterile forcepsand a scalpel.

2. Add the following components to a 2‑mL tube:• Tissue—20 mg

• PBS (1X), pH 7.4—1 mL

• Glass beads (3‑mm diameter)—2 beads

3. Thoroughly vortex to mix.

4. Disrupt (bead‑beat) the samples.• Precellys™ 24 Homogenizer—6,000 rpm for 40 seconds

• FastPrep‑24™ Instrument—6 m/s for 45 seconds





1. Transfer a minimum volume of 5 mL per sample to a conical tube, thencentrifuge at 1,500 × g for 10 minutes.

2. Carefully remove the supernatant, leaving approximately 100 μL ofsupernatant in the tube to avoid aspiration of the cell pellet.

3. Gently pipet up and down to resuspend the cell pellet.

4. Proceed with 100 μL of the cell suspension.



Add the following components to the appropriate wells in position 1 of the strip or plate 1, dependingon the instrument used.

Component Volume per test sample Volume per mock-purified sample

Prepared sample• 100 µL of cell suspension

• 100 µL of organ supernatant—

NM1 Buffer 250 µL 250 µL

2 Lyse the samples

Set up the processing plates or strips outside the instrument as described in the following table.

Position[1] Plate type[2] Reagent Volume per well

2 Deep Well NM3 Buffer 600 µL

3 Deep Well NM4 Buffer 600 µL

4 Deep Well 80% ethanol 600 µL

5 Standard NM6 Buffer 80 µL

6 Deep Well Place a tip comb in the plate or strip.

[1] Position on the instrument.[2] Does not apply if using tube strips.

3 Set up the processingplates or strips

a. Vortex the NM2+Beads Mix thoroughly to ensure that the beads are fully resuspended.

b. Add 620 µL of NM2+Beads Mix to each sample and control.

c. Select the appropriate script on the instrument.

• KingFisher™ mL: NM_LSI_15prep

• KingFisher™ Flex/MagMAX™ Express-96: NM_LSI_RRC96

d. Start the run, then load the prepared processing plates or strips in their positions whenprompted by the instrument.

e. Load the plate or strip containing the samples and controls at position 1 when prompted by theinstrument.

f. At the end of the run, when prompted by the instrument, remove the plate or tubes containingthe purified nucleic acid.

Instrument Procedure

KingFisher™ mL Remove the tube strip at position 5, then transfer the purified nucleic acidto new microcentrifuge tubes.


• MagMAX™ Express-96Remove the plate at position 5, then cover with an adhesive film.

Store the purified nucleic acid at 2–8°C for immediate use or below –16°C for long-term storage.



Prepare samples for manual purificationPrepare samples as described.

Sample type Action

Nasopharyngeal or tracheal swab Follow the procedure described in the kit protocol.

Organ (lung) 1. Finely mince the organ piece in a sterile petri dish, using sterile forceps and a scalpel.

2. Proceed with 20 mg of minced organ.

TTA and bronchoalveolar lavage fluid Proceed with 70 μL of fluid.

Purify RNA using the RNeasy™ Mini Kit (manual method)The RNeasy™ Mini Kit can be used to purify RNA from the following sample types:


• Organs (lungs)


• Prepare the RLT+βME Buffer—Follow the recommendations of the supplier.

• Reconstitute the RPE Buffer—Add the required volume of 96–100% ethanol according to the recommendations of the supplier.



Sample type Action


1. Add a swab to a 2‑mL microtube.For pooled samples, add 3–5 swabs from the pool.

2. Add 800 μL of RLT+βME Buffer, then swirl the swab in the buffer.


4. Proceed with a minimum of 600 μL of swab eluate.

Organ (lung) See “Prepare samples for manual purification” on page 11.


a. Combine the following components in the order indicated, then immediately proceed to the nextstep.

Component Volume per swabsample

Volume per organsample

Volume per mock-purified sample

Prepared sample 600 µL of swab eluate 20 mg of minced organ —

Nuclease‑free water — — 100 µL

RLT+βME Buffer — 600 µL 600 µL

b. Vortex for 1 minute.

c. (Optional) For organs and viscous lysates, homogenize the lysate in a spin column.

1. Place a QIAshredder column on a collection tube, then transfer the lysate to the column.


3. Discard the column and proceed with the sample in the collection tube.

d. Add 600 μL of 70% ethanol to each sample, then pipet up and down 4–5 times to mix. Do notcentrifuge.

2 Lyse, then homogenizethe samples


a. Insert an RNeasy™ Mini Kit column into a new collection tube, then transfer 700 μL of the sampleto the column.

b. Cap the column, then centrifuge the assembly at 10,000 × g for 30 seconds.

c. Discard the collection tube, then place the column on a new collection tube.

d. Transfer the remaining sample volume to the column, cap the column, then centrifuge at10,000 × g for 30 seconds.

e. Discard the collection tube, then place the column on a new collection tube.

3 Bind the RNA to thecolumn

a. Add 700 μL of RW1 Buffer to each column, cap the column, then centrifuge at 10,000 × g for30 seconds.

b. Discard the collection tube, then place the column on a new collection tube.

c. Add 700 μL of RPE Buffer to each column, cap the column, then centrifuge at 10,000 × g for30 seconds.

d. Discard the collection tube, then place the column on a new collection tube.

e. Add 500 μL of RPE Buffer to each column, cap the column, then centrifuge at 10,000 × g for30 seconds.

f. Discard the collection tube, then place the column on a new collection tube.

g. Centrifuge at 13,000 × g for 1 minute to dry the membrane.

h. Discard the collection tube.

i. Place the column on a new 1.5‑mL microtube, then add 50 μL of nuclease‑free water.

j. Cap the column, then incubate at room temperature for 1 minute.

k. Centrifuge at 8,000 × g for 1 minute, then discard the column.The purified RNA is in the microtube.

Store the purified RNA at 2–8°C for immediate use or below –16°C for long-term storage.

4 Wash, then elute the RNA

Purify RNA using the NucleoSpin™ RNA kit (manual method)The NucleoSpin™ RNA kit can be used to purify RNA from the following sample types:


• Organs (lungs)


• (Optional) Prepare RA1+βME Buffer—In a laminar flow hood, combine the following components for the required number of samplesplus 10% overage.


RA1 Buffer 350 μL

βME 3.5 μL

If necessary, store the RA1+βME Buffer at room temperature for up to 1 month.

• Reconstitute the RA3 Buffer—Add the required volume of 96–100% ethanol according to the recommendations of the supplier.

• Reconstitute the rDNase Buffer—Follow the recommendations of the supplier.




Sample type Action


1. Add a swab to a 2‑mL microtube.For pooled samples, add 3–5 swabs from the pool.

2. Add 800 μL of PBS (1X), pH 7.4, then swirl the swab in the PBS.


4. Proceed with 100 μL of swab eluate.

Organ (lung) See “Prepare samples for manual purification” on page 11.



Component Volume per swabsample

Volume per organsample

Volume per mock-purified sample

Prepared sample 100 µL of swab eluate 20 mg of minced organ —

PBS (1X), pH 7.4 — — 100 µL

RA1+βME Buffer[1] 350 µL 350 µL 350 µL

[1] Alternatively, add 350 µL of RA1 Buffer + 3.5 µL βME to each sample.

b. Vortex for 1 minute.

c. (Optional) For organs and viscous lysates, homogenize the lysate in a spin column.

1. Place a NucleoSpin™ Filter column on a collection tube, then transfer the lysate to thecolumn.


3. Discard the column and proceed with the sample in the collection tube.

d. Add 350 μL of 70% ethanol to each sample, then pipet up and down 4–5 times to mix. Do notcentrifuge.


a. Insert a NucleoSpin™ RNA kit column into a new collection tube, then transfer 700 μL of thesample to the column.





f. (Optional) Desalt the silica membrane, then digest DNA.

1. Add 350 μL of MDB Buffer to each column, cap the column, then centrifuge at 11,000 × gfor 1 minute.

2. Discard the collection tube, then place the column on a new collection tube.

3. Add 95 μL of rDNase Buffer to each column, then incubate at room temperature for15 minutes.

Keep the column‑tube assembly and proceed to the next section.



a. Add 200 μL of RAW2 Buffer to each column, cap the column, then centrifuge at 11,000 × g for30 seconds.


c. Add 600 μL of RA3 Buffer to each column, cap the column, then centrifuge at 11,000 × g for30 seconds.


e. Add 250 μL of RA3 Buffer to each column, cap the column, then centrifuge at 11,000 × g for30 seconds.

f. Discard the collection tube, then place the column on a new collection tube.

g. Centrifuge at 11,000 × g for 2 minutes to dry the membrane.

h. Discard the collection tube.

i. Place the column on a new 1.5‑mL microtube, then add 60 μL of nuclease‑free water.

j. Cap the column, then incubate at room temperature for 2 minutes.

k. Centrifuge at 11,000 × g for 1 minute, then discard the column.The purified RNA is in the microtube.



Purify RNA using the QIAamp™ Viral RNA Mini Kit (manual method)The QIAamp™ Viral RNA Mini Kit can be used to purify RNA from TTA and bronchoalveolar lavage fluid.


• Reconstitute the AVL+Carrier Buffer—Follow the recommendations of the supplier.

• Reconstitute the AW1 and AW2 Buffers—Add the required volume of 96–100% ethanol according to the recommendations of thesupplier.




Prepared sample 70 µL of fluid —

Nuclease‑free water 70 µL 140 µL

AVL+Carrier Buffer 560 µL 560 µL

b. Vortex for 15 seconds.

c. Incubate at room temperature for 10 minutes.

d. Add 560 μL of 96–100% ethanol to each sample, vortex for 15 seconds, then briefly centrifugeto collect the contents.



a. Insert a QIAamp™ Viral RNA Mini Kit column into a collection tube, then transfer 630 μL of thesample to the column.






a. Add 500 μL of AW1 Buffer to each column, cap the column, then centrifuge at 10,000 × g for30 seconds.


c. Add 700 μL of AW2 Buffer to each column, cap the column, then centrifuge at 10,000 × g for30 seconds.


e. Centrifuge at 10,000 × g for 2 minutes to dry the membrane.

f. Discard the collection tube.

g. Place the column on a new 1.5‑mL microtube, then add 60 μL of AVE Buffer.

h. Cap the column, then incubate at room temperature for 1 minute.

i. Centrifuge at 8,000 × g for 1 minute, then discard the column.The purified RNA is in the microtube.



Purify RNA using the NucleoSpin™ RNA Virus kit (manual method)The NucleoSpin™ RNA Virus kit can be used to purify RNA from TTA and bronchoalveolar lavage fluid.


• Reconstitute the RAV1+Carrier Buffer—Follow the recommendations of the supplier.

• Reconstitute the RAV3 Buffer—Add the required volume of 96–100% ethanol according to the recommendations of the supplier.




Prepared sample 70 µL of fluid —

Nuclease‑free water 70 µL 140 µL

RAV1+Carrier Buffer 600 µL 600 µL

b. Vortex for 15 seconds.



1 Lyse, thenhomogenize thesamples (continued)

c. Incubate at room temperature for 10 minutes.

d. Add 600 μL of 96–100% ethanol to each sample, vortex for 15 seconds, then briefly centrifugeto collect the contents.

a. Insert a NucleoSpin™ RNA Virus kit column into a collection tube, then transfer 630 μL of thesample to the column.

b. Cap the column, then centrifuge the assembly at 10,000 × g for 1 minute.


d. Transfer the remaining sample volume to the column, cap the column, then centrifuge at10,000 × g for 1 minute.



a. Add 500 μL of RAW Buffer to each column, cap the column, then centrifuge at 10,000 × g for1 minute.


c. Add 600 μL of RAV3 Buffer to each column, cap the column, then centrifuge at 10,000 × g for1 minute.


e. Centrifuge at 11,000 × g for 3 minutes to dry the membrane.

f. Discard the collection tube.

g. Place the column on a new 1.5‑mL microtube, then add 60 μL of nuclease‑free water.

h. Cap the column, then incubate at room temperature for 1 minute.

i. Centrifuge at 8,000 × g for 1 minute, then discard the column.The purified RNA is in the microtube.




Good laboratory practices for PCR and RT-PCR• Wear clean gloves and a clean lab coat.

– Do not wear the same gloves and lab coat that you have previously used when handling amplified products or preparingsamples.

• Change gloves if you suspect that they are contaminated.

• Maintain separate areas and dedicated equipment and supplies for:– Sample preparation and reaction setup.

– Amplification and analysis of products.

• Do not bring amplified products into the reaction setup area.

• Open and close all sample tubes carefully. Avoid splashing or spraying samples.

• Keep reactions and components capped as much as possible.

• Use a positive-displacement pipettor or aerosol‑resistant barrier pipette tips.

• Clean lab benches and equipment periodically with 10% bleach solution or DNA decontamination solution.

Appendix A (Alternative protocol) Parallel purification of RNA/DNA using the MagMAX™ CORENucleic Acid Purification KitFollow this procedure if you are using these instruments:


• MagMAX™ Express-96

Follow Appendix B, “Purification with the KingFisher™ Duo Prime or KingFisher™ mL instrument” if you are using these instruments:

• KingFisher™ Duo Prime

• KingFisher™ mL

Required materials not supplied

A heat block at 55℃ is required for parallel purification of RNA and DNA using the MagMAX™ CORE Nucleic Acid Purification Kit.

Purification procedure

a. Set up the processing plates.

Table 8 Plate setup: KingFisher™ Flex or MagMAX™ Express-96 instrument

Plate ID Plateposition[1] Plate type Reagent Volume per well



Elution 4 Standard MagMAX™ CORE Elution Buffer 90 µL

Tip Comb 5 Standard Place a tip comb in the plate.


b. (Optional) To prevent evaporation and contamination, cover the prepared processing plates withsealing foil until they are loaded into the instrument.

1 Set up the processingplates



Sample type Action

Nasopharyngeal ortracheal swab

1. Break off the tip of the swab and add to a 2‑mL tube.

2. Add 1 mL of PBS (1X), pH 7.4 to each sample.

3. Vortex for 3 minutes.



Proceed with 200 μL of fluid.

Organ (lung)[1] Bead‑beating method: Prepare organ samples using a tissue homogenizer.1. Add the following components to a 2‑mL tube:

• Tissue—20 to 30 mg

• PBS (1X), pH 7.4—1 mL


2. Disrupt (bead‑beat) the samples.• Fisher Scientific™ Bead Mill 24 Homogenizer—6 m/s for 45 seconds




Non‑bead‑beating method: Prepare organ samples using a vortex mixer.1. Finely mince the organ piece in a sterile petri dish, using sterile forceps and a

scalpel.

2. Add the following components to a 2‑mL tube:• Tissue (finely minced)—20 to 30 mg

• PBS (1X), pH 7.4—500 μL


3. Vortex vigorously.



[1] Select the preparation method that is appropriate for your laboratory.


Calculate the number of samples. Scale the components proportionally based on the volume persample, then add 10% overage.

a. For each sample, combine the following components as indicated.

IMPORTANT! Add the components in the order indicated at the time of use; do not mix inadvance.

1. Combine the MagMAX™ CORE Lysis Solution with PBS (1X), pH 7.4.


MagMAX™ CORE Lysis Solution 200 μL

PBS (1X), pH 7.4 200 μL

Total diluted Lysis Solution 400 μL

2. Invert the tube several times to mix, then centrifuge briefly to collect contents at the bottomof the tube.

3 Prepare the Lysis/PKSolution


3 Prepare the Lysis/PKSolution (continued)

3. Add MagMAX™ CORE Proteinase K to the diluted Lysis Solution.

Note: PK Buffer is not required for this protocol.


Diluted Lysis Solution 400 μL

MagMAX™ CORE Proteinase K 10 μL

Total Lysis/PK Solution 410 μL

b. Invert the tube several times to mix, then centrifuge briefly to collect contents at the bottom ofthe tube.

Perform this procedure in single tubes – do not use plates – to avoid contamination.

a. Combine the following components in the order indicated.

Component Volume per sample Volume per mock‑purifiedsample

Prepared sample 200 μL —

PBS (1X), pH 7.4 — 200 μL

Lysis/PK Solution 410 μL 410 μL

b. Vortex briefly to mix the sample with the Lysis/PK Solution.

c. Incubate the sample according to the sample type.

For... Do this...

• Nasal or tracheal swab supernatant

• TTA or bronchoalveolar fluidIncubate for 30 minutes at 55°C.

Organ supernatant Incubate for 30 minutes to 2 hours at 55°C.

d. Centrifuge briefly to collect contents at the bottom of the tube.

4 Treat samples with theLysis/PK Solution

a. Vortex the MagMAX™ CORE Magnetic Beads thoroughly to ensure that the beads are fullyresuspended.

b. Combine the following reagents in the order indicated.

Table 9 Final Sample Plate volumes: KingFisher™ Flex or MagMAX™ Express-96 instrument

Plate ID Plateposition[1] Plate type Reagent Volume per

well

Sample Plate 1 Deep Well

Sample after lysis Up to 610 μL

MagMAX™ CORE BindingSolution 400 μL

MagMAX™ CORE MagneticBeads 20 μL


c. Immediately proceed to process samples on the instrument (next section).

5 Combine samples with thebinding solution andbeads

a. Select the appropriate script on the instrument (see “Download and install the script” on page 6).

b. Start the run, then load the prepared plates in the appropriate positions when prompted by theinstrument.



6 Process samples onthe instrument(continued)

Store purified nucleic acid on ice for immediate use, at −20°C for up to 1 month, or at −80°C forlong‑term storage.

Appendix B Purification with the KingFisher™ Duo Prime or KingFisher™ mL instrumentFollow this procedure for purification with the MagMAX™ CORE Nucleic Acid Purification Kit, using the KingFisher™ Duo Prime orKingFisher™ mL instrument.

Required materials not suppliedTable 10 Materials required for processing on the KingFisher™ Duo Prime and KingFisher™ mL instruments

Item Source[1]

Consumables for the KingFisher™ Duo Prime instrument

KingFisher™ Duo Combi pack for Microtiter 96 Deepwell plate (tip combs, plates, and elution stripsfor 96 samples) 97003530

KingFisher™ Duo Elution Strip (40 pieces)[2] 97003520

KingFisher™ Duo 12-tip comb for Microtiter 96 Deepwell plate (50 pieces)[2] 97003500

KingFisher™ Flex Microtiter Deepwell 96 plates[2] 95040460

Consumables for the KingFisher™ mL instrument

KingFisher™ mL Tubes and tip combs (for 240 samples) 97002141

KingFisher™ mL Tip comb (800 pieces) 97002111

KingFisher™ mL Tube (20 x 45 pieces) 97002121

[1] Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that the material is available from fisherscientific.com or another major laboratory supplier.

[2] Included in the KingFisher™ Duo Combi pack (Cat. No. 97003530).



http://fisherscientific.com

Purification procedure

Note: When performing this procedure for processing on the KingFisher™ mL instrument, mix samples by pipetting up and down. Do not

use a plate shaker with the large tube strips required by this instrument.

1. Follow the protocol, starting with sample lysate preparation through combining the samples with beads and lysis solution.

Note: Do not set up processing plates or tubes before preparing samples.

2. Add MagMAX™ CORE Wash Solutions and MagMAX™ CORE Elution Buffer to the indicated positions, according to your instrument.

Load the Tip Comb and all of the plates or tube strips at the same time. The instrument does not prompt you to load itemsindividually.

Table 11 Plate setup: KingFisher™ Duo Prime instrument

Row ID Row in the plate Plate type Reagent Volume per well

Sample A Deep Well Sample lysate/bead mix Varies by sample

Wash 1 B MagMAX™ CORE Wash Solution 1 500 µL

Wash 2 C MagMAX™ CORE Wash Solution 2 500 µL

Elution[1] Separate tube strip[2] Elution strip MagMAX™ CORE Elution Buffer 90 µL

Tip Comb H Deep Well Place a tip comb in the plate.

[1] Ensure that the elution strip is placed in the correct direction in the elution block. [2] Placed on the heating element.

Table 12 Tube strip setup: KingFisher™ mL instrument

Position ID Tube strip position Tube Reagent Volume per well

Sample 1 Standard Sample lysate/bead mix Varies by sample

Wash 1 2 MagMAX™ CORE Wash Solution 1 500 µL

Wash 2 3 MagMAX™ CORE Wash Solution 2 500 µL

Elution 4 MagMAX™ CORE Elution Buffer 90 µL

Tip Comb N/A N/A Slide the tip comb into the tip comb holder.

3. Select the appropriate script on the instrument (see “Download and install the script” on page 6).

4. Start the run, then load the prepared plates or tube strips in the appropriate positions when prompted by the instrument.

Store purified nucleic acid on ice for immediate use, at −20°C for up to 1 month, or at −80°C for long‑term storage.

Appendix C Documentation and support

Customer and technical supportVisit thermofisher.com/support for the latest service and support information.

• Worldwide contact telephone numbers

• Product support information– Product FAQs

– Software, patches, and updates

– Training for many applications and instruments

• Order and web support

• Product documentation– User guides, manuals, and protocols

– Certificates of Analysis

– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.



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Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0019168

Revision Date Description

A.0 13 May 2020

New document translated from the French document (MAN0008896 Rev. B.0) with the following updates:• Added MagMAX™ CORE Nucleic Acid Purification Kit protocols.

• Made minor wording and formatting updates for consistency with related documents.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of allapplicable Limited Use Label Licenses.

©2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. FastPrep‑24 is atrademark of MP Biomedicals, LLC. Mixer/Mill is a trademark of SPEX SamplePrep, LLC. NucleoSpin is a trademark of MACHEREY‑NAGEL GMBH & Co. Precellys is a trademark ofBertin Technologies. Precess 24 is a trademark of Bio‑Rad Laboratories, SA. QIAamp, RNeasy, and QIAGEN are trademarks of QIAGEN GmbH.

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