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, ThermoFisher Scientific assumes no liability for any errors or
omissions, nor for any damages resulting from the application or use of
thisinformation. This instruction manual supersedes all previous editions.
Products are for Research Use Only. Not for use in
diagnosticprocedures.
The Product will operate substantially in conformance with Thermo Fisher
Scientific’s published specifications.
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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 StorageSolution. . . . . . . . . . . . . . . . . . 16
Preparation of the DNase I WorkingSolution .................16
Starting Material Handling and Storage .....................16
Chapter 5 Protocols and PipettingInstructions ....................17
Handling of KingFisher Magnetic Beads .....................17
Avoiding Ribonuclease (RNase) Contamination ................17
Instructions for KingFisher Flex with 96Deep 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 thePurity 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
PipettingInstructions”. 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 1.5-4µg 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
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