Thermo Scientific KingFisher Pure RNA Blood Kit Instruction Manual

Thermo Scientific
KingFisher Pure RNA
Blood Kit

Instruction Manual

Revision 1.0

N14035 March 2013

Copyright

© 2013 Thermo Fisher Scientific Inc. All rights reserved. PAXgene is a trademark
of PreAnalytiX GmbH (a QIAGEN / BD Company). Tempus is a trademark of
Life Technologies and its subsidiaries. Tween is a trademark of ICI Americas
Inc. Virkon is a trademark of E.I. du Pont de Nemours and Company or its
affiliates. All (other) trademarks are the property of Thermo Fisher ScientificInc.
and its subsidiaries. Reproduction of the accompanying user documentation in
whole or in part is prohibited.

Disclaimer

Thermo Fisher Scientific reserves the right to change its products and services
at any time to incorporate technological developments. This manual is subject
to change without prior notice as part of continuous product development.
Although this manual has been prepared with every precaution to ensure
accuracy, ThermoFisher Scientific assumes no liability for any errors or
omissions, nor for any damages resulting from the application or use of
thisinformation. This instruction manual supersedes all previous editions.

Products are for Research Use Only. Not for use in
diagnosticprocedures.

The Product will operate substantially in conformance with Thermo Fisher
Scientific’s published specifications.
THERMO FISHER SCIENTIFIC DISCLAIMS ALL OTHER WARRANTIES, WHETHER
EXPRESSED OR IMPLIED, ORAL OR WRITTEN, WITH RESPECT TO THE
PRODUCTS, INCLUDING WITHOUT LIMITATION ALL IMPLIED WARRANTIES
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OBTAINED THROUGH THE USE OF THE PRODUCTS, INCLUDING WITHOUT
LIMITATION ANY CLAIM OF INACCURATE, INVALID OR INCOMPLETE RESULTS.

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Thermo Fisher Scientific and its affiliates shall have no liability to an End User
arising out of the use or inability to use the product, including, without limitation,
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caused and regardless of whether an action in contract, tort, strict product
liability or otherwise.

Table of Contents

Chapter 1 Kit Content ..........................................5

Storage Conditions .....................................6

Additional Reagents Required .............................6

Chapter 2 Product Description ..................................9

Introduction ..........................................9

Intended Use .........................................9

Principle and Procedure ................................10

Kit Specifications .....................................10

KingFisher Magnetic Particle Processors ....................11

Chapter 3 Safety Information ..................................13

Chapter 4 Storage Conditions and Preparation of the Reagents ......15

Storage Conditions ....................................15

Preparation of the Lysis Buffer ...........................15

Preparation of the Wash Buffers and Rebinding Buffer ..........15

Preparation of the DNase I StorageSolution. . . . . . . . . . . . . . . . . . 16

Preparation of the DNase I WorkingSolution .................16

Starting Material Handling and Storage .....................16

Chapter 5 Protocols and PipettingInstructions ....................17

Handling of KingFisher Magnetic Beads .....................17

Avoiding Ribonuclease (RNase) Contamination ................17

Instructions for KingFisher Flex with 96Deep Well Plates ........18

Instructions for KingFisher Duo with 12-pin Magnet Head ............20

Instructions for Total RNA Purification from PAXgene Stabilized

Blood Samples .......................................23

Instructions for Total RNA Purification from Tempus Stabilized

Blood Samples .......................................24

Quantification and Determination of thePurity of RNA ..........24

Chapter 6 General Information .................................27

Reagent Specificity and Volumes ..........................27

Handling of Magnetic Beads .............................27

Binding, Wash, and Elution Steps .........................27

Decontamination and Disinfection of Sample Material ..........28

Appendix A Troubleshooting .....................................29

Appendix B Typical RNA Yields ..................................31

Appendix C Ordering Information ................................33

NOTE: For more details on storing the kit reagents, refer to “Storage Conditions” on

page 6.

Thermo Scientific KingFisher Pure RNA Blood Kit | 3Thermo Fisher Scientific

1

Kit Content

Table 1-1. Thermo Scientific™ KingFisher™ Pure RNA Blood Kit

Item KingFisher Pure RNA Blood Kit

Cat. No. 98020196

Package size 96 samples

Lysis Buffer 27 ml

Proteinase K 2 x 1.0 ml

KingFisher Magnetic Beads 1 x 1.6 ml

Rebinding Buffer (conc.)* 25 ml

Wash Buffer 1 (conc.)* 100 ml

Wash Buffer 2 (conc.)* 30 ml

DNase I (lyophilized) 2 vials

DNase I Reconstitution Buffer 2 x 1.0 ml

Manganese Chloride Solution 1 ml

2 x DNase I Buffer 3 x 1.5 ml

Nuclease-free water 30 ml

* Addition of ethanol or isopropanol required.

The KingFisher Pure RNA Blood Kit (Cat. No. 98020196) is intended for the
purification of blood samples, using the Thermo Scientific™ KingFisher™
Flex with a 96 deep well head or the Thermo Scientific™ KingFisher™ Duo
with a 12-pin head and a sample volume of 200 µl.

The user will need the KingFisher Flex or KingFisher Duo magnetic particle
processor for conducting purification (Table 1-2). In addition, several
common laboratory instruments and consumables are necessary to conduct

Thermo Scientific KingFisher Pure RNA Blood Kit | 5Thermo Fisher Scientific

Chapter 1 | Kit Content

an efficient purification. For more details, refer to Chapter 5: “Protocols and

PipettingInstructions”. Suitable consumables for the KingFisher Duo and

KingFisher Flex are listed in Table 1-3 and Table 1-4.

Storage Conditions

Upon arrival of the kit, store the DNase I, DNase I Reconstitution Buffer,
and 2 x DNase Buffer at -20°C. Also after dissolving of the DNase I,
continue to keep it at -20°C. The Proteinase K solution is stable at room
temperature until the seal of the vial is broken. After being opened, the vial
should be stored at -20°C. Store the Thermo Scientific™ KingFisher™
Magnetic Beads at +4°C. Other kit components can be stored at room
temperature (15−25°C). The reagents are stable for up to two years from
the manufacturing date.

Additional Reagents Required

• 96−100% ethanol (EtOH), molecular biology grade

• 100% isopropanol, molecular biology grade

• 2 M DTT (dithiothreitol) solution (store at -20°C)

• PBS (Phosphate buffered saline for Tempus™ protocol)

Table 1-2. Thermo Scientific™ KingFisher™ magnetic particle processors

Cat. No. Product

5400100 KingFisher Duo magnetic particle processor

5400630 KingFisher Flex magnetic particle processor with 96 deep well head

Table 1-3. Thermo Scientific™ KingFisher™ Flex consumables

Cat. No. Product Package size

97002534 KingFisher Flex 96 tip comb for deep well magnet 100 pcs

97002540 KingFisher Flex 96 KF plate (200 µl) 48 pcs

95040450 Microtiter deep well 96 plate 50 pcs

95040460 Microtiter deep well 96 plate, sterile 50 pcs

6 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

Kit Content | Chapter 1

Table 1-4. Thermo Scientific™ KingFisher™ Duo consumables

Cat. No. Product Package size

97003500 KingFisher Duo 12-tip comb for Microtiter deep well

96 plate

97003520 KingFisher Duo elution strip 40 pcs

95040450 Microtiter deep well 96 plate 50 pcs

95040460 Microtiter deep well 96 plate, sterile 50 pcs

97003530 KingFisher Duo Combi pack for Microtiter deep well

96 plate

(tip combs, plates, and elution strips for 96 samples)

50 pcs

1 box

Thermo Scientific KingFisher Pure RNA Blood Kit | 7Thermo Fisher Scientific

2

Product Description

Introduction

The KingFisher Pure RNA Blood Kit is designed for rapid automated RNA
purification from up to 200 µl of mammalian blood samples using Thermo
Scientific™ KingFisher™ instruments. See the detailed list below.

1) Fresh or frozen whole human blood samples anticoagulated by EDTA
(ethylenediaminetetraacetic acid) or citrate, and samples stabilized by
commercial stabilizers PAXgene or Tempus

2) Fresh, frozen, or stabilized (PAXgene and Tempus) blood of different
species: mouse, rabbit, canine, horse, cow, sheep, and goat

3) Human buffy coat, blood leucocytes, erythrocytes, and bone marrow samples

The RNA purified using the KingFisher Pure RNA Blood Kit is of high quality
and free of proteins, nucleases, and other contaminants or inhibitors. It is,
therefore, suitable for direct use in many different downstream applications,
such as RT-qPCR (reverse transcription quantitative PCR), RT-PCR, and
several other enzymatic reactions.

Intended Use

The KingFisher Pure RNA Blood Kit is developed for the purification of RNA from
blood samples using paramagnetic particles. The reagents and specific plastic
consumables are designed for use with the KingFisher Flex and KingFisher Duo
magnetic particle processors as part of an integrated system. The KingFisher
Pure RNA Blood Kit enables extraction of RNA from fresh or stored blood. The
KingFisher Pure RNA Blood Kit is only intended for research use, not for clinical
or diagnostic use. The user is responsible for validating the performance of the

Thermo Scientific KingFisher Pure RNA Blood Kit | 9Thermo Fisher Scientific

Chapter 2 | Product Description

Thermo Scientific™ KingFisher™ instrument and the KingFisher Pure RNA
Blood Kit for any particular use, as the performance of the kits has not been
validated for any specific organism or downstream application.

Principle and Procedure

The KingFisher Pure RNA Blood Kit uses magnetic-particle technology for
RNA purification. The Thermo Scientific™ KingFisher™ technology combines
the speed and efficiency of RNA purification with easy handling of magnetic
particles. The purification process requires no phenol/chloroform extraction
and needs very little hands-on time.

The first step of the protocol lyses the cells, after which the NA (nucleic acid)
can bind to the surface of the KingFisher Magnetic Beads in the presence of a
chaotropic salt. Copurified DNA is removed during a DNase I treatment step.

The following effective wash steps dispose of proteins, cell debris, and any
residual contaminants, while the RNA bound to the KingFisher Magnetic
Beads is transferred through the wash steps.

Two different Wash Buffers are used, followed by an air drying step. High­quality RNA is eluted into nuclease-free water, and is ready for subsequent
downstream processes.

Kit Specifications

The KingFisher Pure RNA Blood Kit is designed for rapid automated
preparation of highly pure total RNA from up to 200 µl of blood samples
using KingFisher magnetic particle processors.

If a dispense step requiring the addition of Rebinding Buffer is excluded, the
approximate processing time is 45 min for the purification of 96 samples
on the KingFisher Flex and 12 samples on the KingFisher Duo. The obtained
RNA can be used directly in various downstream applications.

Fresh or stored blood samples can be used. Suitable sample storage is
essential for obtaining a high yield and good quality total RNA.

Typically 1.5-4µg of total RNA can be purified from a 200 µl volume of
blood sample with an A
purified RNA depend on the sample type, and the method of sample
collection, and storage. Refer to Appendix B for typical total RNA yields from
various sources.

10 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

ratio of ≥ 1.8–2.1. The yields of acquired

260/A280

Product Description | Chapter 2

KingFisher Magnetic Particle Processors

The KingFisher magnetic particle processors are designed for the automated
transfer and processing of magnetic particles in microplate format. The
patented technology of the Thermo Scientific™ KingFisher™ systems is based
on the use of magnetic rods covered with a disposable, specially designed
tip comb and plates or tubes. Use only Thermo Scientific™ KingFisher™
plastic consumables, as use of products from other manufacturers may
cause unsuitable mixing or even instability in the KingFisher instrument. The
instrument functions without any dispensing or aspiration parts or devices.
Samples and reagents, including magnetic particles, are dispensed onto the
plates according to the corresponding instructions. Dispensing can be carried
out manually or partially automatically using automatic dispensers, for example,
the Thermo Scientific™ Multidrop™ Combi and/or the Thermo Scientific™
Versette™. Thermo Scientific™ BindIt™ Software 3.2 can be used for running
ready-made and optimized protocols for the Thermo Scientific™ KingFisher™
Pure Kits. It is also possible to transfer the developed protocol onto the onboard
software and run it directly from the instrument. The KingFisher instruments
provide a rapid and automated solution for complicated and time-consuming
purification processes, resulting in high-purity RNA without risk of carryover
or cross-contamination.

The KingFisher instrument family comprises four systems covering working
volumes from 20 to 5000 µl. Each system consists of an instrument, specially
designed plastic consumables, and the easy-to-use BindIt Software 3.2.
The KingFisher Pure RNA Blood Kit is optimized and ready for use with the
KingFisher Flex or KingFisher Duo.

KingFisher magnetic particle processors are intended for professional
research use by trained personnel. Detailed information and user instructions
for the KingFisher instruments can be found in their respective user manuals.

The BindIt Software 3.2 protocols optimized for the KingFisher Pure RNA
Blood Kit are available for the KingFisher Flex 96 and 24 and KingFisher Duo.
For more information, go to www.thermoscientific.com/kingfisherinfo or
contact your local authorized distributor.

Thermo Scientific KingFisher Pure RNA Blood Kit | 11Thermo Fisher Scientific

Chapter 2 | Product Description

Table 2-1. Overview of KingFisher Flex and KingFisher Duo magnetic
particleprocessors

KingFisher Flex KingFisher Duo

96 deep well
format

Processing
volume

Capacity Up to 96

Magnetic head 96inter-

Plates KingFisher Flex

Tip combs KingFisher Flex

Heating
temperature

50–1000 µl* 200–5000 µl 50–1000 µl* 200–5000 µl

samples per
run (sample
volume approx.
200 µl)

changeable
formats for
Microtiter deep
well 96 plate,
PCR plate and
KingFisher Flex
96 KF plate

96 KF plate
(20–200 µl),
96 well PCR
plate, skirted
(20–100 µl),
Microtiter deep
well 96 plate
(50–1000 µl)

96 tip comb for
PCR magnets,
KingFisher Flex
tip comb for KF
magnets,
KingFisher Flex
96 tip comb for
deep well
magnets

Heating block temperature from
+5°C above ambient room
temperature to +115°C

24 format 12 format 6 format

Up to 24
samples per
run (sample
volume approx.
1 ml)

24 format for
KingFisher Flex
24 deep well
plate

KingFisher Flex
24 deep well
plate
(200–5000 µl)

KingFisher Flex
24 tip comb for
deep well
magnets

* See the details above on the Plates row.

Up to 12
samples per
run (sample
volume approx.
200 µl)

12-pin magnet
head for
Microtiter deep
well 96 plate

Microtiter deep
well 96 plate
(50–1000 µl),
KingFisher Duo
elution strip
(30–130 µl)

KingFisher Duo
12-tip comb

Heating block temperature from
+10°C to +75°C, elution strip
+4°C to +75°C at room
temperature

Up to 6
samples per
run (sample
volume approx.
1 ml)

6-pin magnet
head for
KingFisher Flex
24 deep well
plate

KingFisher Flex
24 deep well
plate
(200–5000 µl)

KingFisher Duo
6-tip comb

12 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

3

Safety Information

The following components of the KingFisher Pure RNA Blood Kit contain
hazardous contents (Table 3-1).

Always wear a laboratory coat, disposable gloves and goggles, and follow the
safety instructions provided in the kit instruction manual. It is recommended
that Good Laboratory Practice (GLP) is followed to guarantee reliable
analyses.

Table 3-1. Safety precautions

Reagent Hazardous contents Safety instructions

Lysis Buffer Guanidium thiocyanate Harmful by inhalation. Liberates

Continued

Thermo Scientific KingFisher Pure RNA Blood Kit | 13Thermo Fisher Scientific

very toxic gas in contact with
acids. Harmful to aquatic
organisms and may cause
long-term adverse effects in the
aquatic environment.

Keep the container in a
well-ventilated place. Do not
breathe gas/fumes/vapor/spray.
Wear suitable protective clothing
and gloves. This material and its
container must be disposed of as
hazardous waste. Avoid release to
the environment. Refer to special
instructions/safety data sheets.

Chapter 3 | Safety Information

Cont.

Reagent Hazardous contents Safety instructions

Proteinase K Proteinase, Tritirachium album

Rebinding Buffer
(conc.)

Wash Buffer 1
(conc.)

serine

Sodium perchlorate Harmful if swallowed. Explosive

Guanidium chloride Harmful if swallowed. Irritating to

May cause sensitization by
inhalation.

Do not breathe gas/fumes/vapor/
spray. Wear suitable protective
clothing. In case of accident or if
you feel unwell, seek medical
advice immediately. This material
and its container must be
disposed of as hazardous waste.

when mixed with combustible
material. Keep away from
combustible material. Do not
breathe gas/fumes/vapor/spray.
Take off immediately all
contaminated clothing. Wear
suitable protective clothing. This
material and its container must be
disposed of as hazardous waste.

eyes and skin.

Do not breathe gas/fumes/vapor/
spray. In case of contact with
eyes, rinse immediately with
plenty of water and seek medical
advice. Wear suitable protective
clothing and gloves. This material
and its container must be
disposed of as hazardous waste.

14 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

4

Storage Conditions and
Preparation of the Reagents

Storage Conditions

Upon arrival of the kit, store the DNase I, DNase I Reconstitution Buffer,
and 2 x DNase Buffer at -20°C. The Proteinase K solution is stable at room
temperature until the seal of the vial is broken. After being opened, the vial
should be stored at -20°C. Store the KingFisher Magnetic Beads at +4°C.
Other kit components can be stored at room temperature (15−25°C). The
reagents are stable for up to two years from the manufacturing date.

Preparation of the Lysis Buffer

Before each RNA purification experiment, a fresh aliquot of Lysis Buffer
should be supplemented with 2 M DTT solution (not provided). It is required
to use 200µl of Lysis Buffer for the purification of one sample. Add 20 µl of
2MDTT to 1 ml of Lysis Buffer.

Preparation of the Wash Buffers and Rebinding Buffer

Add 100% isopropanol and 96−100% ethanol to the Wash Buffer 1, Wash
Buffer 2, and Rebinding Buffer, as indicated below in Table 4-1 prior to the
first use.

Thermo Scientific KingFisher Pure RNA Blood Kit | 15Thermo Fisher Scientific

Chapter 4 | Storage Conditions and Preparation of the Reagents

Table 4-1. Instructions for the preparation of Wash Buffer 1, Wash Buffer2,
and Rebinding Buffer. Add the indicated volume of 100% isopropanol or
96−100% ethanol to each bottle.

Wash Buffer 1 Wash Buffer 2 Rebinding Buffer

Concentrated buffer 100 ml 30 ml 25 ml

Ethanol (96−100%)

Isopropanol (100%) 90 ml − 33.4 ml

Total volume 190 ml 150 ml 58.4 ml

After preparing each solution, mark the bottle to indicate that the step has
been completed. The buffers can be stored at room temperature.

Preparation of the DNase I
StorageSolution

To prepare the DNase I storage solution, add 1100 µl of DNase I Reconstitution
Buffer to each vial of lyophilized DNaseI. Incubate at room temperature for
5min. Occasional gentle rotation of the vial helps to dissolve the DNaseI, but
avoid forceful mixing. Store the DNaseI storage solution at -20°C. Repeated
freezing and thawing should be avoided.

− 120 ml −

Preparation of the DNase I
WorkingSolution

Calculate the amount of DNase I working solution needed. For the purification
of one sample, mix 35 µl of 2 x DNase I Buffer, 1.5 µl Manganese Chloride
Solution, 10 µl of DNase I storage solution, and 23.5 µl of nuclease-free water.
The DNase I working solution should be used immediately after preparation.

Starting Material Handling and Storage

RNA purification from blood is recommended to be carried out on the
day of collection. Fresh blood samples collected in anticoagulants
(ethylenediaminetetraacetic acid (EDTA) or citrate) containing vials are not
recommended to be stored at +4°C for longer than six hours. Aliquots
of fresh blood samples, if frozen immediately, can be stored at -80°C for
several months.

Blood samples collected in PAXgene or Tempus tubes should be stored
according to the manufacturer’s instructions.

16 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

5

Protocols and
PipettingInstructions

Before beginning the RNA purification protocol, carefully read through the

Thermo Scientific™ KingFisher™ Flex User Manual
Thermo Scientific™ KingFisher™ Duo User Manual

the

Thermo Scientific™ KingFisher™ mL User Manual

and the

Thermo Scientific™ BindIt™ Software for KingFisher Instruments

version 3.2 User Manual

BindIt Software protocols for the KingFisher Pure RNA Blood Kit can be found
in BindIt Software 3.2 and at www.thermoscientific.com/kingfisher.

Handling of KingFisher Magnetic Beads

A homogeneous distribution of the KingFisher Magnetic Beads in the container
is essential before the beads are transferred to the wells or tubes in order
to ensure a high consistency between the wells or tubes. To gain complete
resuspension of the beads, shake the container vigorously or vortex briefly.

Avoiding Ribonuclease (RNase) Contamination

(Cat. No. N07974).

(Cat. No. N07669), the
(Cat. No. N12420) or

(Cat. No. 1508260),

RNA purity and integrity is essential for downstream applications. RNase is a
ubiquitously found enzyme, which degrades RNA. RNases are highly stable
contaminants found in any laboratory environment. Keep all kit components
tightly sealed when not in use.

Skin is a common source of RNases. Always wear gloves when handling
reagents and RNA samples. Use sterile, RNase-free pipette tips when working
with RNA. Remove RNase contamination from work surfaces and non-disposable
items (pipettes, centrifuges) with reagents designed to remove RNase.

Thermo Scientific KingFisher Pure RNA Blood Kit | 17Thermo Fisher Scientific

Chapter 5 | Protocols and PipettingInstructions

Instructions for KingFisher Flex with
96Deep Well Plates

These instructions are intended for RNA purification from 200 µl of whole
blood, using the KingFisher Pure RNA Blood Kit (Cat. No. 98020196) and the
KingFisher Flex with Thermo Scientific™ Microtiter™ deep well 96 plates.

When using the KingFisher Pure RNA Blood Kit for the first time, prepare the
DNase I storage solution, Rebinding Buffer, Wash Buffer 1, and Wash Buffer2.
For each run, prepare the DNase I working solution and Lysis Buffer with
2M DTT. For more instructions, refer to Chapter 4: “Storage Conditions and

Preparation of the Reagents”.

Check all the solutions in the kit for salt precipitation before each use.
Redissolve precipitates by warming the solution at 37°C and equilibrate to
room temperature (15−25°C).

Blood samples of certain species, e.g. mouse, contain a high amount of
RNA. With such species, it is recommended to use up to 50 µl of blood
sample to obtain RNA of equal yield and purity. When working with less than
a 200 µl volume of blood sample, it is recommended to equalize the volume
by adding Lysis Buffer, thus bringing the total volume to 400 µl.

1. Take six empty Microtiter deep well 96 plates and two empty Thermo

Scientific™ KingFisher™ Flex 96 KF plates.

2. Fill the plates as follows.

Plate
number

1

2 Wash 1_1 Wash Buffer 1 900 µl

3 DNase DNase I working

4 Wash 1_2 Wash Buffer 1 700 µl

5 Wash 2_1 Wash Buffer 2 700 µl

6 Wash 2_2 Wash Buffer 2 700 µl

7 KingFisher Flex

18 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

Plate type Plate name Content

Sample Lysis Buffer

Blood sample

Proteinase K

Microtiter deep
well 96 plate

Elution Nuclease-free

96 KF plate

solution

water

Reagent volume
per well

200 µl

200 µl

20 µl

70 µl

75 µl

Protocols and PipettingInstructions | Chapter 5

3. Place a Thermo Scientific™ KingFisher™ Flex 96 tip comb for deep

well magnets on a Tip Plate (i.e. an empty KingFisher Flex 96 KF plate).

4. Start the PURE_RNABlood_Flex96 protocol using the KingFisher Flex 96

and load the plates as instructed on the KingFisher Flex 96 instrument
display.

Switch on the KingFisher Flex making sure that you are using the Thermo
Scientific™ KingFisher™ Flex 96 deep well head and heating block.

Connect the PC with BindIt Software 3.2 to the KingFisher Flex. Start the
PURE_RNABlood_Flex96 protocol. Insert the Tip Plate and the filled plates
into the instrument as indicated on the KingFisher Flex display. After all the
plates have been loaded into the instrument, the protocol will start.

5. When the KingFisher Flex is to be run as a standalone instrument,

transfer the PURE_RNABlood_Flex96 protocol to the KingFisher Flex.
The instructions for transferring the protocol can be found in Chapter 4:
“Using the software” in the

version 3.2 User Manual

BindIt Software for KingFisher Instruments

.

6. Add the KingFisher Magnetic Beads and isopropanol to the Sample plate

during the first dispense step.

When the KingFisher Flex pauses at the dispense step after the lysis step at
approximately 5 minutes after starting the protocol run, remove the Sample
plate from the instrument, and add the KingFisher Magnetic Beads and
isopropanol to the Sample plate.

Plate name Add

Sample Isopropanol

KingFisher Magnetic Beads*

Added reagent volume
perwell

400 µl

15 µl

* Resuspend the KingFisher Magnetic Beads well by vortexing before use.

7. Place the Sample plate back into the instrument and press Start.

8. Add the Rebinding Buffer (supplemented with isopropanol) to the

Sample plate during the second dispense step.

Thermo Scientific KingFisher Pure RNA Blood Kit | 19Thermo Fisher Scientific

Chapter 5 | Protocols and PipettingInstructions

When the KingFisher Flex pauses at the second dispense step after the
DNase step at approximately 10 minutes after the first dispense step,
remove the DNase plate from the instrument, and add the Rebinding Buffer
(supplemented with ethanol) to the DNase plate to rebind the RNA.

Plate name Add

DNase Rebinding Buffer 490 µl

Added reagent volume
per well

9. Place the DNase plate back into the instrument and press Start. After

the pause, the protocol will continue to completion.

10. When the protocol is completed, remove the plates according to the

instructions on the KingFisher Flex display and switch off the instrument.
The purified RNA is ready for use in downstream applications. When
working with RNA, keep the purified samples on ice. Store the purified
RNA at -20°C or -80°C.

NOTE: The final RNA concentration in the nuclease-free water may
increase if the purified RNA is eluted into a smaller than recommended
volume of water, but this can slightly reduce the overall RNA yield.

Instructions for KingFisher Duo with 12-pin Magnet Head

These instructions are intended for RNA purification from 200 µl of blood,
using the KingFisher Pure RNA Blood Kit (Cat. No. 98020196) and the
KingFisher Duo with 12-pin magnet head.

When using the KingFisher Pure RNA Blood Kit for the first time, prepare
the DNase I storage solution, Rebinding Buffer, Wash Buffer 1, and Wash
Buffer2. For each run, prepare the DNase I working solution and Lysis Buffer
with 2M DTT. For more instructions, refer to Chapter 4: “Storage Conditions

and Preparation of the Reagents”.

Check all the solutions in the kit for salt precipitation before each use.
Redissolve precipitates by warming the solution at 37°C and equilibrate to
room temperature (15−25°C).

1. Take one empty Microtiter deep well 96 plate and one Thermo Scientific™

KingFisher™ Duo elution strip.

20 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

Protocols and PipettingInstructions | Chapter 5

2. Prepare the Blood RNA plate (i.e. a Microtiter deep well 96 plate). Add

the following reagents to the rows. Note that row B is reserved for the tip
comb and should be left

empty

. Note that row D is also left

empty

.

Plate
name and
type

Blood RNA
plate

Microtiter
deep well
96 plate

Row Row name Content

A DNase DNase I working

solution

B Tip 12-tip comb Empty

C Empty Empty Empty

D Wash 2_2 Wash Buffer 2 700 µl

E Wash 2_1 Wash Buffer 2 700 µl

F Wash 1_2 Wash Buffer 1 700 µl

G Wash 1_1 Wash Buffer 1 900 µl

H Sample Lysis Buffer

Blood sample

Proteinase K

Sample/reagent
volume per well

70 µl

200 µl

200 µl

20 µl

3. Fill the KingFisher Duo elution strip as follows. Make sure that the elution

strip is placed in the correct direction into the elution block. Ensure that
the perforated end is facing towards the user and the nuclease-free
water is pipetted into the correct wells.

Elution strip Content

KingFisher Duo elution strip Nuclease-free water 75 µl

Reagent volume
perwell

4. Place a Thermo Scientific™ KingFisher™ Duo 12-tip comb into row B on

the Blood RNA plate.

5. Start the PURE_RNABlood_Duo protocol using the KingFisher Duo and

load the plate and elution strip.

Switch on the KingFisher Duo making sure that you are using the Thermo
Scientific™ KingFisher™ Duo 12-pin magnet head and heating block.

Connect the PC with BindIt Software 3.2 to the KingFisher Duo. Start the
PURE_RNABlood_Duo protocol. Insert the Blood RNA plate and elution strip
into the instrument as indicated on the KingFisher Duo display and pressOK.

Thermo Scientific KingFisher Pure RNA Blood Kit | 21Thermo Fisher Scientific

Chapter 5 | Protocols and PipettingInstructions

Make sure that the elution strip is placed in the correct direction into the
elution block. Ensure that the perforated end is facing towards the user.

When the KingFisher Duo is to be run as a standalone instrument, transfer
the PURE_RNABlood_Duo protocol to the KingFisher Duo. The instructions
for transferring the protocol can be found in Chapter 4: “Using the software”
in the

BindIt Software for KingFisher Instruments version 3.2 User Manual

6. Add the KingFisher Magnetic Beads and isopropanol to row C during the

dispense step.

When the KingFisher Duo pauses at the first dispense step after the lysis
step at approximately 5 minutes after starting the protocol run, remove the
plate from the instrument, and add the KingFisher Magnetic Beads and
isopropanol to row C on the Blood RNA plate.

.

Row Row name Add

C Sample Isopropanol

KingFisher Magnetic
Beads*

Added reagent
volume per well

400 µl

15 µl

* Resuspend the KingFisher Magnetic Beads well by vortexing before use.

7. Place the plate back into the instrument and press OK.

8. Add the Rebinding Buffer (supplemented with ethanol) to row A during

the dispense step.

When the KingFisher Duo pauses at the dispense step after the lysis step
at approximately 10 minutes after the first dispense step, remove the plate
from the instrument, and add the Rebinding Buffer into row A on the Blood

RNA plate to rebind the RNA.

Row Row name Add

A DNase Rebinding Buffer 490 µl

Added reagent
volume per well

9. Place the plate back into the instrument and press OK. After the pause,

the protocol will continue to completion.

10. After the run is completed, remove the plate and elution strip, and store

the purified RNA.

22 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

Protocols and PipettingInstructions | Chapter 5

When the protocol is completed, remove the plate and elution strip according
to the instructions on the KingFisher Duo display and switch off the
instrument. The purified RNA is ready for use in downstream applications.
When working with RNA, keep the purified samples on ice. Store the purified
RNA at -20°C or -80°C.

NOTE: The final RNA concentration in the nuclease-free water may
increase if the purified RNA is eluted into a smaller than recommended
volume of water, but this can slightly reduce the overall RNA yield.

Instructions for Total RNA Purification
from PAXgene Stabilized Blood Samples

1. Collect and store the blood samples in PAXgene tubes according to the

manufacturer’s instructions.

2. If the PAXgene samples were stored in the cold, equilibrate the samples

at room temperature before purification according to the manufacturer’s
instructions for approx. 2 h.

3. Centrifuge the PAXgene blood RNA tubes for 10 min at 3,000−5,000 x g.

4. Carefully discard the supernatant from each tube and wash the pellet.

Add1−3 ml of nuclease-free water, vortex for 20−30 s, and collect the
pellet again by centrifuging for 10 min at 3,000–5,000 x g.

• For 0.5−2 ml of PAXgene blood lysate, wash the obtained pellet with

1 ml of water.

• For a whole tube of PAXgene blood lysate, wash the obtained pellet with

3 ml of water.

5. Carefully discard the supernatant, add 100 µl of nuclease-free water,

and vortex for 10−20 s to suspend precipitates. Add 400 µl of Lysis
Buffer (supplemented with DTT) and 20 µl of Proteinase K. Mix the lysate
and continue the purification as in “Instructions for KingFisher Flex with

96Deep Well Plates” on page 18 or as in “Instructions for KingFisher
Duo with 12-pin Magnet Head” on page 20.

Thermo Scientific KingFisher Pure RNA Blood Kit | 23Thermo Fisher Scientific

Chapter 5 | Protocols and PipettingInstructions

Instructions for Total RNA Purification
from Tempus Stabilized Blood Samples

1. Collect and store the blood samples in Tempus tubes according to the

manufacturer’s instructions.

2. If the sample is frozen, thaw the sample in the Tempus tube at room

temperature according to the manufacturer’s instructions.

3. Transfer the entire contents of the Tempus tube into a 50 ml conical

tube. Bring the total volume to 12 ml by adding the required amount of
PBS. Typically a Tempus stabilized blood sample volume is 9 ml, which
requires the addition of 3 ml of PBS to bring the total volume to 12 ml.

4. Vortex efficiently for 30 s to ensure proper mixing of the contents.

5. Centrifuge the appropriate volume (1−2 ml) of Tempus blood lysate in

conical tubes at maximal speed (about 12,000−16,000 × g) for 10 min
at 4°C. If the Tempus blood volume is more than 2 ml, increase the time
of centrifugation up to 30 min.

6. Carefully discard the supernatant from each tube and add 450 µl of

Lysis Buffer (supplemented with DTT) and 20 µl of Proteinase K. Vortex
the samples and incubate at room temperature for 10 min. Continue
the purification as in “Instructions for KingFisher Flex with 96Deep Well

Plates” on page 18 or as in “Instructions for KingFisher Duo with
12-pin Magnet Head” on page 20.

Quantification and Determination of
thePurity of RNA

It is recommended to measure the absorbance at 320 nm, 280 nm, and
260 nm. The concentration of RNA can be defined with the absorbance at
260 nm (A
ratio between the A
should be ≥ 1.8–2.1.

24 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

). One unit at 260 nm corresponds to 40 μg of RNA per ml. The

260

indicates the purity of the RNA. The value for RNA

260/A280

Protocols and PipettingInstructions | Chapter 5

It is recommended that A
Subtract the A

from the A

320

correction is used for the absorbance values.

320

and A

260

ratios to remove the effects of

280

carryover of the magnetic particles.

• Concentration of RNA sample = 40 μg/ml x (A

– A

260

) x dilution factor

320

• Total amount of RNA isolated = concentration x volume of sample in ml

• Purity of RNA sample = (A

– A

)/( A

– A

260

320

)

280

320

Thermo Scientific KingFisher Pure RNA Blood Kit | 25Thermo Fisher Scientific

6

General Information

Reagent Specificity and Volumes

A reagent must not be used with any kit other than that for which it is
intended. It is strongly recommended that the volume of reagents in each
well or tube is kept within the limits specified in the

Manual

or

KingFisher Duo User Manual

efficiency of performance.

Handling of Magnetic Beads

The KingFisher Magnetic Beads should be mixed thoroughly before use to
avoid the risk of transferring variable amounts of the beads to the respective
wells or tubes. The amount of beads in the wells or tubes affects the yield of
the purified RNA.

Binding, Wash, and Elution Steps

The binding between the purified RNA and the KingFisher Magnetic Beads is
strong in the presence of a chaotropic salt.

to avoid spillover and to maximize

KingFisher Flex User

The volume of the Elution Buffer can be modified depending on user
requirements concerning the purified RNA concentration. The final RNA
concentration in the Elution Buffer may increase if the purified RNA is eluted
into a smaller than recommended volume of the buffer, but this can slightly
reduce the overall RNA yield. The modifications of the elution step must be
done in BindIt Software 3.2 and according to the volume ranges suitable for
the KingFisher instrument. The table below indicates the available elution
volumes of the KingFisher instruments.

Thermo Scientific KingFisher Pure RNA Blood Kit | 27Thermo Fisher Scientific

Chapter 6 | General Information

Table 6-1. Available elution volumes of the KingFisher Flex and KingFisher Duo

KingFisher instrument

KingFisher Flex with 96 deep well head, elution in a KingFisher Flex 96
KF plate

KingFisher Flex with 96 deep well head, elution in a Microtiter deep well
96 plate

KingFisher Flex with 24 deep well head 200–5000 µl

KingFisher Duo with 12-pin magnet head, elution in an elution strip 30−130 µl

KingFisher Duo with 12-pin magnet head, elution in a Microtiter deep
well 96 plate

Elution
volumes

50–150 µl

50–1000 µl

50–1000 µl

To maximize the yield of purified RNA, avoid the lowest permitted volumes of
Elution Buffer in the KingFisher instruments. The Elution Buffer should cover
the KingFisher Magnetic Beads completely, and any possible sedimented
magnetic-bead pellet(s) should be completely resuspended. In addition, the
volume of the Elution Buffer should be adequate for efficient mixing of the
beads in order to obtain a maximal release of the purified RNA from the beads.

Decontamination and Disinfection of Sample Material

You should decontaminate the sample material and the reagents and plastics
that have been in contact with the sample material in order to minimize the
risk of contamination. Use a decontaminant, such as Virkon™, paying due
attention to the manufacturer’s instructions. You should also take care of the
appropriate treatment and/or disposal of waste.

28 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

Troubleshooting

Problem Possible cause and actions

A

Low RNA yield There should be an adequate volume of the Elution Buffer to cover

Low purity Prolonged storage of the sample material may reduce the quality

Continued

the KingFisher Magnetic Beads completely during the elution step.

Do not let the KingFisher Magnetic Beads dry as this may result in
lower elution efficiency.

Prolonged storage of the sample material may reduce the total
RNA yield.

Use only Thermo Scientific plates, strips, and tip combs with the
KingFisher instruments. Use of products from other manufacturers
may cause unsuitable mixing and affect the yield of purified RNA.

and quantity of the total RNA.

Insufficient washing causes impurities in the eluted RNA.

Thermo Scientific KingFisher Pure RNA Blood Kit | 29Thermo Fisher Scientific

Appendix A | Troubleshooting

Cont.

Problem Possible cause and actions

Magnetic particles
remaining in the
sample or elution well

Starting material that is too viscose prevents efficient collection of
the KingFisher Magnetic Beads from the lysed sample.
Themagnetic rods will not be able to collect all the particles
unless the viscose samples are diluted before the beginning of
thepurification process. Improper lysis may also cause problems
collecting the KingFisher Magnetic Beads.

If the KingFisher Magnetic Beads are inefficiently collected from
the Elution Buffer, the addition of a small amount of detergent
(e.g.Tween™ 20) may improve the results.

KingFisher Magnetic Beads that occasionally remain attached to
the tip combs at the end of the process do not affect the total RNA
yield, as the RNA has already been released from the KingFisher
Magnetic Beads into the Elution Buffer.

If the KingFisher magnetic particle processor does not work
properly, refer to the relevant user manual of the KingFisher
instrument in use.

30 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

B

Typical RNA Yields

Table B-1. Typical total RNA yields from whole mammalian blood of different
species using KingFisher magnetic particle processors

Source

1. Fresh human blood 11−20

2. Frozen human blood 7−17

3. Stabilized (PAXgene, Tempus) human blood 9−12

4. Human bone marrow 15−80

5. Fresh mouse blood 120−160

6. Fresh rabbit blood 25−30

7. Fresh canine blood 11−15

8. Fresh animal (horse, cow, sheep, goat) blood 2−5

Yield of RNA
(µg/ml blood)

Thermo Scientific KingFisher Pure RNA Blood Kit | 31Thermo Fisher Scientific

C

Ordering Information

Table C-1. KingFisher Pure RNA Blood Kits

Cat. No. Product Package size

98020196 KingFisher Pure RNA Blood Kit 96

Table C-2. KingFisher Flex consumables

Cat. No. Product Package size

97002514 KingFisher Flex 96 tip comb for PCR magnet 80 pcs

97002524 KingFisher Flex 96 tip comb for KF magnet 100 pcs

97002534 KingFisher Flex 96 tip comb for deep well magnet 100 pcs

97002610 KingFisher Flex 24 deep well tip comb and plate 50 pcs

97002540 KingFisher Flex 96 KF plate (200 µl) 48 pcs

95040450 Microtiter deep well 96 plate 50 pcs

95040460 Microtiter deep well 96 plate, sterile 50 pcs

95040470 KingFisher Flex 24 deep well plate 50 pcs

95040480 KingFisher Flex 24 deep well plate, sterile 50 pcs

Thermo Scientific KingFisher Pure RNA Blood Kit | 33Thermo Fisher Scientific

Appendix C | Ordering Information

Table C-3. KingFisher Duo consumables

Cat. No. Product Package size

97003500 KingFisher Duo 12-tip comb for Microtiter deep well

96plate

97003510 KingFisher Duo 6-tip comb for KingFisher Flex 24 deep

well plate

97003520 KingFisher Duo elution strip 40 pcs

95040450 Microtiter deep well 96 plate 50 pcs

95040460 Microtiter deep well 96 plate, sterile 50 pcs

95040470 KingFisher Flex 24 deep well plate 50 pcs

95040480 KingFisher Flex 24 deep well plate, sterile 50 pcs

97003530 KingFisher Duo Combi pack for Microtiter deep well

96plate

(tips combs, plates, and elution strips for 96 samples)

50 pcs

48 pcs

1 box

34 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific

General Information | Chapter C

Thermo Scientific KingFisher Pure RNA Blood Kit | 35Thermo Fisher Scientific

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