Applied Biosystems MagMAX FFPE DNA RNA Ultra Kit 产

MagMAX

™

FFPE DNA/RNA Ultra Kit

Automated or manual isolation of total nucleic acid (TNA) from FFPE samples using AutoLys tubes

Catalog Number A31881

Pub. No. MAN0017538 Rev. A.0

WARNING!

handling instructions. Wear appropriate protective eyewear,

Read the Safety Data Sheets (SDSs) and follow the

clothing, and gloves. Safety Data Sheets (SDSs) are available

from /support.

The Applied Biosystems

Product description

™

designed to isolate both DNA and RNA from the same section of

MagMAX

™

FFPE DNA/RNA Ultra Kit is

formaldehyde- or

tissues. The kit also allows for

paraformaldehyde-fixed, paraffin-embedded (FFPE)

only or total nucleic acid (TNA). The kit uses MagMAX

flexibility to isolate DNA only, RNA

™

bead technology, ensuring reproducible recovery of high-quality

magnetic-

nucleic acid through manual or automated processing. The isolated

nucleic acid is appropriate for use with a broad range of downstream

assays, such as quantitative real-time RT-PCR and next-generation

sequencing.

In addition to the use of traditional solvents, the kit is compatible with

Autolys M tubes that enable a faster and more convenient means of

deparaffinizing

solvents such as xylene or CitriSolv and ethanol. Samples are put into

FFPE samples by eliminating the need for organic

the tubes for protease digestion, tubes are lifted with the Auto-pliers

or Auto-Lifter and then samples are spun down. The wax and debris

are contained in the upper chamber while the lysate is passed through.

Afterwards, the

MagMAX

™

FFPE DNA/RNA Ultra Kit.

clarified lysate can be directly purified with the

For guides without using AutoLys M tubes for sequential DNA and

RNA isolation, or DNA isolation, or RNA isolation only, see

MagMAX

™

isolation)

FFPE DNA/RNA Ultra Kit User Guide (sequential DNA/RNA

Ultra Kit User Guide (DNA isolation only)

(Pub. No. MAN0015877), or MagMAX

™

MagMAX

™

(Pub. No. MAN0015905), or

FFPE DNA/RNA

(Pub. No. MAN0015906), respectively.

FFPE DNA/RNA Ultra Kit User Guide (RNA isolation only)

This guide describes isolation of TNA from FFPE tissue blocks or FFPE

slides using AutoLys M tubes. Three optimized methods for sections

or curls both up to 40 µm using AutoLys M tubes are included:

•

•

Manual sample processing.

KingFisher

™

Flex Magnetic Particle Processor with 96 Deep-Well

•

Head (DW96; 96-well deep well

KingFisher

setting).

™

well setting).

Duo Prime Magnetic Particle Processor (12-well deep

For sequential DNA and RNA isolation, or DNA isolation, or RNA

isolation only, see

(sequential DNA/RNA isolation)

MagMAX

™

FFPE DNA/RNA Ultra Kit User Guide

(Pub. No. MAN0017541), MagMAX

™

FFPE DNA/RNA Ultra Kit User Guide (DNA isolation only)

(Pub. No. MAN0017539), or

Guide (RNA isolation only) (Pub. No. MAN0017540), respectively.

MagMAX

™

FFPE DNA/RNA Ultra Kit User

For Research Use Only. Not for use in diagnostic procedures.

Contents and storage

Reagents provided in the kit are

sections up to 40 µm with the AutoLys

sufficient

workflow.

for 48 TNA isolations from

Table 1 MagMAX

™

FFPE DNA/RNA Ultra Kit (Cat. No. A31881)

ContentsAmountStorage

Protease960 µL–25°C to –15°C

Protease Digestion Buffer

[1]

10 mL

Binding Solution

[1]

38.5 mL

15°C to 30°C

Nucleic Acid Binding Beads

[2]

1.95 mL2°C to 8°C

DNA Wash Buffer

[3]

38.5 mL

Wash Solution 2 Concentrate210 mL

[4]

Elution Solution5 mL

15°C to 30°C

RNA Wash Buffer Concentrate115 mL

[4]

DNase

[3]

1.95 mL

DNase buffer

[3]

960 µL

–25°C to –15°C

[1]

Additional reagents are available separately; Protease Digestion Buffer, Binding

[2]

Solution, and DNA Wash Buffer are also available as Cat. No. A32796.

[3]

Shipped at room temperature.

[4]

Not used in TNA workflow

Final volume; see “Isolate TNA“ on page 4.

Required materials not supplied

Unless otherwise indicated, all materials are available through

other major laboratory supplier.

. MLS: Fisher Scientific () or

Table 2 Materials required for nucleic acid isolation (all methods)

ItemSource

Equipment

Incubators or ovens at 60°C and 90°CMLS

Centrifuge with plate adaptorsMLS

Adjustable micropipettorsMLS

Multi-channel micropipettorsMLS

Laboratory mixer (Vortex mixer or equivalent)MLS

Locking lid for Autolys M TubesA37954

AutoLys M TubeLifterA37956

AutoLys M Tube PliersA38261

AutoLys M Tube RackA37955

Tubes, plates, and other consumables

AutoLys M Tubes and CapsA38738

Nonstick, RNase-free Microfuge Tubes (1.5 mL)AM12450

Nonstick, RNase-free Microfuge Tubes (2 mL)AM12475

Aerosol-resistant pipette tipsMLS

Reagent reservoirsMLS

Reagents

Ethanol, 200 proof (absolute)MLS

Isopropanol, 100%MLS

Nuclease-Free WaterAM9938

Table 3 Additional materials required for manual isolation

ItemSource

Equipment

Fisher Scientific

™

Analog Vortex Mixer

Fisher

02-215-365

Scientific

Vortex Adapter-60AM10014

Accessories and tubes

DynaMag

™

-2 Magnet12321D

Table 4 Additional materials required for automated isolation

ItemSource

Magnetic particle processor, one of the following:

KingFisher

™

with 96 Deep-Well Head

Flex Magnetic Particle Processor

5400630

KingFisher

™

Processor

Duo Prime Magnetic Particle

5400110

Plates and combs

96 Deep-well plates, one of the following:

MagMAX

™

Express-96 Deep Well Plates4388476

96 Deep-Well Plates for KingFisher

™

Magnetic Particle Processor

Flex

95040450

96-well standard plates, one of the following:

MagMAX

™

Express-96 Standard Plates4388475

96 Standard-Well Plates for KingFisher

™

Magnetic Particle Processor

Flex

97002540

Tip comb, compatible with the magnetic particle processor used:

KingFisher

™

96 Tip Comb for DW Magnets97002534

MagMAX

™

Express-96 Deep Well Tip Combs4388487

KingFisher

™

Deepwell plate

Duo 12-Tip Comb, for Microtiter 96

97003500

Consumables

MicroAmp

™

Clear Adhesive Film4306311

If needed, download the KingFisher

™

program

Duo Prime or Flex

The programs required for this protocol are not pre-installed on the

KingFisher

™

KingFisher

™

Duo Prime Magnetic Particle Processor or on the

Flex Magnetic Particle Processor 96DW.

1.

On the MagMAX

™

scroll down to the

FFPE DNA/RNA Ultra Kit product web page,

Product Literature section.

2.

Right-click on the appropriate program

size to download the program to your computer:

file(s) for your sample

InstrumentSections ≤40 µm

KingFisher

™

Magnetic Particle Processor

Duo Prime

A31881_DUO_large_vol_TNA

KingFisher

™

Particle Processor 96DW

Flex Magnetic

A31881_FLEX_large_vol_TNA

3.

Select Save as Target to download to your computer.

4.

Refer to the manufacturer's documentation for instructions for

installing the program on the instrument.

Procedural guidelines

•Perform all steps at room temperature (20–25°C) unless otherwise

•

noted.

When mixing samples by pipetting up and down, avoid creating

•

bubbles.

When working with RNA:

–

–

Wear clean gloves and a clean lab coat.

Change gloves whenever you suspect that they are

–

contaminated.

Open and close all sample tubes carefully. Avoid splashing or

–

spraying samples.

Use a positive-displacement

tips.

pipettor and RNase-free pipette

2

–Clean lab benches and equipment periodically with an RNase

decontamination solution, such as RNase

•Incubation at 60°C can be extended overnight to increase DNA

No. AM9780).

Zap

™

Solution (Cat.

•

yields, followed by incubation at 90°C for 1 hour.

Volumes for reagent mixes are given per sample. We recommend

that you prepare master mixes for larger sample numbers. To

calculate volumes for master mixes, refer to the per-well volume

and add 5–10% overage.

Before you begin

Before

first use of the kit

•Prepare the Wash Solutions from the concentrates:

–Add 46 mL of isopropanol to RNA Wash Buffer Concentrate ,

–

mix, and store at room temperature.

Add 168 mL of ethanol to Wash Solution 2 Concentrate , mix,

and store at room temperature.

Before each use of the kit

•

•

Equilibrate the Nucleic Acid Binding Beads to room temperature.

•

Pre-heat the incubators or ovens to 60°C and 90°C.

Prepare the following solutions according to the following tables.

Table 5 Protease solution

ReagentsVolume

Protease10 µL

Protease Digestion Buffer225 µL

Total Protease Solution235 µL

Table 6 TNA Binding Buffer

ReagentsVolume

Binding Solution400 µL

Isopropanol500 µL

Total TNA Binding Buffer900 µL

MagMAX

™

FFPE DNA/RNA Ultra Kit User Guide (TNA isolation only)

Prepare the FFPE samples

•For curls from FFPE tissue blocks: proceed to “Prepare the curls from FFPE tissue blocks“ on page 3.

•For FFPE slide-mounted sections: proceed to “Prepare samples from FFPE slides“ on page 3.

Prepare the curls from FFPE tissue blocks

1

2

Section FFPE tissue blocks

a.

Cut sections from FFPE tissue blocks using a microtome.

Note: For miRNA extraction, we recommend using sections of 10 µm or thicker.

b.

Collect each section in an AutoLys M tube.

Digest with Protease

a.

Add 235 µL of the Protease Solution (see Table 5).

Note: If working with curls, they might stick straight up so make sure to submerge samples in the

Protease Solution with a tip or a 1 mL syringe plunger or do a quick spin down at 3000 rpm for 1 minute

prior to the addition of buffer to collapse the curl. Time may be extended.

b.

Incubate at 60°C for 1 hour or longer.

Note: Use the AutoLys racks and place in an incubator or oven.

c.

Incubate at 90°C for 1 hour.

Note: For automated isolation, set up the processing plates during the incubation.

·

For isolation using KingFisher

™

Duo Prime Magnetic Particle Processor, proceed to “Set up the

processing plate“ on page 4.

·

For isolation using KingFisher

™

Flex Magnetic Particle Processor 96DW, proceed to “Set up the TNA

processing plates“ on page 5.

3

Lift the tubes

a.

b.

c.

d.

e.

f.

g.

Allow samples to cool down for 3–5 minutes before proceeding to lift the tubes.

Use the Auto-plier for individual tube lifting or the Auto-lifter for multiple tube lifting of up to 24 tubes.

Lock the tubes in position by hand or use the locking lid.

Centrifuge at 2000 × g for 10 minutes in a benchtop centrifuge with plate adapters.

Unlock the tubes by hand or remove the locking lid.

Use the Auto-plier or Auto-lifer to lift the inner tube for sample access.

Proceed to purification. See “Isolate TNA“ on page 4

Prepare samples from FFPE slides

4

Scrape the samples and

digest with Protease

a.

Pipet 2–4 µL of Protease Digestion Buffer depending on the tissue size evenly across the FFPE tissue

section on the slide to pre-wet the section.

Note: You can adjust the volume of Protease Digestion Buffer if the tissue is smaller or larger.

b.

Scrape the tissue sections in a single direction with a clean razor blade or scalpel, then collect the tissue

on the slide into a cohesive mass.

c.

Transfer the tissue mass into an AutoLys M tube with the scalpel or a pipette tip.

d.

Add 235 µL of the Protease Solution (see Table 5).

Note: Be sure to submerge samples in the Protease Solution with a tip or a 1 mL syringe plunger

e.

Incubate at 60°C for 1 hour or longer.

Note: Use the AutoLys racks and place in an incubator or oven.

f.

Incubate at 90°C for 1 hour.

Note: For automated isolation, set up the processing plates during the incubation.

·

For isolation using KingFisher

™

Duo Prime Magnetic Particle Processor, proceed to “Set up the

processing plate“ on page 4.

·

For isolation using KingFisher

™

Flex Magnetic Particle Processor 96DW, proceed to “Set up the TNA

processing plates“ on page 5.

5

Lift the tubes

a.

b.

c.

d.

e.

f.

g.

Allow samples to cool down for 3–5 minutes before proceeding to lift the tubes.

Use the Auto-plier for individual tube lifting or the Auto-lifter for multiple tube lifting of up to 24 tubes.

Lock the tubes in position by hand or use the locking lid.

Centrifuge at 2000 × g for 10 minutes in a benchtop centrifuge with plate adapters.

Unlock the tubes by hand or remove the locking lid.

Use the Auto-plier or Auto-lifer to lift the inner tube for sample access.

Proceed to purification. See “Isolate TNA“ on page 4

MagMAX

™

FFPE DNA/RNA Ultra Kit User Guide (TNA isolation only)

3

Isolate TNA

•To isolate TNA manually, proceed to “Isolate TNA manually“ on page 4.

™

•To isolate TNA using the KingFisher Duo Prime Magnetic Particle Processor, proceed to “Isolate TNA using KingFisher

™

Duo Prime Magnetic

Particle Processor“ on page 4.

™

•To isolate TNA using the KingFisher Flex Magnetic Particle Processor 96DW, proceed to “Isolate TNA using KingFisher

™

Flex Magnetic

Particle Processor 96DW“ on page 5.

Isolate TNA manually

Use microcentrifuge tubes to perform manual TNA isolations.

1

Bind the TNA to beads

a.

b.

c.

d.

After the Protease digestion is complete, add 20 µL of Nucleic Acid Binding Beads to the samples.

Add 900 µL of TNA Binding Buffer (see Table 6) to the sample.

Shake for 5 minutes at speed 10 or 1150 rpm.

Place the sample on the magnetic stand for 2 minutes or until the solution clears and the beads are

pelleted against the magnet.

e.

Carefully discard the supernatant with a pipette.

2

Wash TNA on the beads

a.

Wash the beads with 500 µL of RNA Wash Buffer.

b.

Shake for 1 minute at speed 10 or 1150 rpm until the mixture is thoroughly chocolate brown in color.

c.

Place the sample on the magnetic stand for 2 minutes or until the solution clears and the beads are

pelleted against the magnet.

d.

Carefully discard the supernatant with a pipette.

e.

Repeat steps a-d.

f.

Wash the beads with 500 µL of Wash Solution 2.

g.

Shake for 1 minute at speed 10 or 1150 rpm until the mixture is thoroughly chocolate brown in color.

h.

Place the sample on the magnetic stand for 2 minutes or until the solution clears and the beads are

pelleted against the magnet.

i.

Carefully discard the supernatant with a pipette.

j.

Repeat steps f-i.

k.

Shake for 1–2 minutes at speed 10 or 1150 rpm to dry the beads.

Do not over-dry the beads. Over-dried beads results in low TNA recovery yields.

a.

Add 50 µL of Elution Solution to the beads.

b.

Shake for 5 minutes at speed 10 or 1150 rpm and at 55°C until the mixture is thoroughly chocolate

brown in color.

c.

Place the sample on the magnetic stand for 2 minutes or until the solution clears and the beads are

pelleted against the magnet.

The supernatant contains the purified TNA

The purified TNA is ready for immediate use. Store at –20°C or –80°C for long-term storage.

3

Elute the TNA

Isolate TNA using KingFisher

™

Duo Prime Magnetic Particle Processor

4

Set up the processing plate

During the protease incubation, add processing reagents to the wells of a MagMAX

™

Express-96 Deep Well

Plate as indicated in the following table.

Table 7 TNA plate setup

Row ID

Elution

Wash Solution 2 - 1

Wash Solution 2 - 2

RNA Wash Buffer 1

RNA Wash Buffer 2

Empty

Sample

Tip Comb

[1]

Plate row

[1]

A

B

C

D

E

F

G

H

ReagentVolume per well

Elution Solution50 µL

Wash Solution 21 mL

Wash Solution 21 mL

RNA Wash Buffer500 µL

RNA Wash Buffer500 µL

Empty

TNA Binding Buffer (see Table 6)900 µL

™

Place a KingFisher Duo 12-Tip Comb.

Row on the MagMAX

™

Express-96 Deep Well Plate.

5

Bind, wash, rebind, and

elute the TNA

a.

Ensure that the instrument is set up for processing with the deep well 96–well plates and select the

appropriate program A31881_DUO_large_vol_TNA on the instrument.

b.

At the end of the protease incubation, add 200 µL of sample to each well in Row G of the TNA plate.

c.

Add 20 µL of Nucleic Acid Binding Beads to each sample well in Row G.

d.

Start the run and load the prepared processing plates when prompted by the instrument.

4

MagMAX

™

FFPE DNA/RNA Ultra Kit User Guide (TNA isolation only)

5

Bind, wash, rebind,

and elute the TNA

(continued)

e.

At the end of the run, remove the Elution Plate from the instrument and transfer the eluted TNA

(Row A of TNA plate) to a new plate and seal immediately with a new MicroAmp

™

Clear Adhesive

Film.

IMPORTANT! Do not allow the purified samples to sit uncovered at room temperature for more than 10

minutes, to prevent evaporation and contamination.

The purified TNA is ready for immediate use. Store at –20°C or –80°C for long-term storage.

Isolate TNA using KingFisher

™

Flex Magnetic Particle Processor 96DW

6

Set up the TNA processing

During the protease incubation, add processing reagents to the wells of MagMAX

™

Express-96 Plates as

indicated in the following table.

plates

Table 8 TNA plates setup

Plate ID

Sample

RNA Wash Buffer Plate 1

RNA Wash Buffer Plate 2

Wash Solution 2 Plate 1

Wash Solution 2 Plate 2

Elution Plate

Tip Comb

[1]

Plate

position

[1]

1

2

3

4

5

6

7

Plate typeReagentVolume per well

Deep WellTNA Binding Buffer (see Table 6)900 µL

Deep WellRNA Wash Buffer500 µL

Deep WellRNA Wash Buffer500 µL

Deep WellWash Solution 21 mL

Deep WellWash Solution 21 mL

StandardElution Solution50 µL

Place a MagMAX

™

Express-96 Deep Well Tip Comb in a plate.

Position on the instrument

7

Bind, wash, rebind, and

elute the TNA

a.

Ensure that the instrument is set up for processing with the deep well magnetic head and select the

A31881_FLEX_large_vol_TNA program on the instrument.

b.

At the end of the protease incubation, add 200 µL of sample to each well in Plate 1.

c.

Add 20 µL of Nucleic Acid Binding Beads to each sample well in Plate 1.

d.

Start the run and load the prepared processing plates in their positions when prompted by the

instrument (see “Set up the TNA processing plates“ on page 5).

e.

At the end of the run, remove the Elution Plate from the instrument and seal immediately with a new

MicroAmp

™

Clear Adhesive Film.

IMPORTANT! Do not allow the purified samples to sit uncovered at room temperature for more than 10

minutes, to prevent evaporation and contamination.

The purified TNA is ready for immediate use. Store at –20°C or –80°C for long-term storage. .

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of

Sale found on Life Technologies' website at /us/en/home/global/. If you have any questions,

please contact Life Technologies at /support.

Limited product warranty

Manufacturer: Life Technologies Corporation | 2130 Woodward Street | Austin, TX 78744

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

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES 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. MAN0017538

Revision

A.0

Date

26 February 2018

Description

New document

Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all

applicable Limited Use Label Licenses.

©2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

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26 February 2018