TissueLyser LT Handbook
TissueLyser LT Handbook
TissueLyser LT Handbook
TissueLyser LT Handbook
For disruption of up to 12 biological samples
Contents
Product Contents
Storage
Technical Assistance
Safety Information
Introduction
Principle
Applications
11
12
Important Notes
13
13
13
16
Protocols
Purification of RNA or Multiple Analytes from Animal and
Human Tissues
17
19
21
22
23
25
27
29
30
Ordering Information
32
Product Contents
TissueLyser LT
Catalog no.
85600
Allen Key
User Manual
Handbook
Storage
The TissueLyser LT should be stored upright in a dry environment at 540C.
meet your expectations, simply call your local Technical Service Department or
distributor. We will credit your account or exchange the product as you wish.
Separate conditions apply to QIAGEN scientific instruments, service products,
and to products shipped on dry ice. Please inquire for more information.
A copy of QIAGEN terms and conditions can be obtained on request, and is
also provided on the back of our invoices. If you have questions about product
specifications or performance, please call QIAGEN Technical Services or your
local distributor (see back cover or visit www.qiagen.com).
Technical Assistance
At QIAGEN, we pride ourselves on the quality and availability of our technical
support. Our Technical Service Departments are staffed by experienced
scientists with extensive practical and theoretical expertise in sample and assay
technologies and the use of QIAGEN products. If you have any questions or
experience any difficulties regarding the TissueLyser LT or QIAGEN products in
general, please do not hesitate to contact us.
QIAGEN customers are a major source of information regarding advanced or
specialized uses of our products. This information is helpful to other scientists as
well as to the researchers at QIAGEN. We therefore encourage you to contact
us if you have any suggestions about product performance or new applications
and techniques.
For technical assistance and more information, please see our Technical
Support Center at www.qiagen.com/Support or call one of the QIAGEN
Technical Service Departments or local distributors (see back cover or visit
www.qiagen.com).
Safety Information
When working with chemicals, always wear a suitable lab coat, disposable
gloves, and protective goggles. For more information, please consult the
appropriate material safety data sheets (MSDSs). These are available online in
convenient and compact PDF format at www.qiagen.com/Support/MSDS.aspx
where you can find, view, and print the MSDS for each QIAGEN kit and kit
component.
24-hour emergency information
Emergency medical information in English, French, and German can be
obtained 24 hours a day from:
Poison Information Center Mainz, Germany
Tel: +49-6131-19240
Introduction
Principle
The TissueLyser LT provides rapid and efficient disruption of up to 12 biological
samples, including animal and human tissues, plant tissues, bacteria, and
yeast. Disruption and homogenization are achieved through the beating and
grinding effect of beads on the sample material as they are shaken together in
2 ml sample tubes.
Disruption is critically important in order to release the nucleic acids from the
sample material. Homogenization of the material acts to shear carbohydrates
that may otherwise reduce binding of DNA and RNA to silica membranes or
magnetic particles. Sample disruption using, for example, a mortar and pestle
does not result in efficient homogenization. The TissueLyser LT both disrupts and
homogenizes sample material in one simple and reliable step.
The TissueLyser LT is easily programmed to provide variable speeds from 15 to
50 Hz (9003000 oscillations/minute) and run times from 1 second to 1 hour
59 minutes.
Applications
The ability to process up to 12 samples per run makes the TissueLyser LT the
ideal front-end solution to access biological information for genomics,
transcriptomics, and proteomics applications.
The TissueLyser LT enables fast and uniform disruption of animal and human
tissues, plant tissues, bacteria, and yeast in the TissueLyser LT Adapter, which
holds up to twelve 2 ml sample tubes. QIAGEN offers 2 ml microcentrifuge
tubes as well as stainless steel beads for use with the TissueLyser LT Adapter.
For more details about these and other accessories for the TissueLyser LT, see
Appendix A (page 29) and the ordering information (page 32).
The TissueLyser LT provides efficient disruption of biological material in each
sample tube for reproducible, high-quality results in downstream applications
such as the purification of total DNA, RNA, or protein from a variety of human,
animal, and plant tissues. A wide range of QIAGEN sample purification kits are
compatible with the TissueLyser LT (see Tables 16, pages 710). Sample
purification can be performed manually or can be automated using the
QIAcube , QIAsymphony SP, QIAxtractor, EZ1 Advanced, EZ1 Advanced
XL, or BioRobot or BioSprint workstations. For more information about
automated solutions from QIAGEN, see Appendix B (page 30) and visit
www.qiagen.com/automation.
This handbook provides guidelines on disrupting and homogenizing various
sample materials for subsequent purification of DNA, RNA, or protein. Specific
details on disruption and homogenization and nucleic acid or protein
6
purification, such as the amount of starting material and lysis buffer to use, can
be found in the handbook supplied with each QIAGEN sample purification kit.
Table 1. Kits for RNA purification from animal or human tissues using
spin columns
Sample type Kit
Kit format
Page
Up to 5 mg tissue; automatable
on QIAcube
17
Up to 30 mg tissue; automatable 17
on QIAcube
Up to 20 mg RNAlater stabilized 17
tissue; automatable on QIAcube
Up to 5 mg tissue; includes
gDNA Eliminator spin columns
17
17
RNeasy Fibrous
Tissue Mini Kit
Up to 30 mg tissue; automatable 17
on QIAcube
RNeasy Fibrous
Tissue Midi Kit
Up to 250 mg tissue
17
Up to 100 mg tissue;
automatable on QIAcube
17
Up to 100 mg tissue;
automatable on QIAcube
17
Any type of
RNeasy Lipid Tissue
tissue,
Mini Kit
including
miRNeasy Mini Kit
fatty tissues
(e.g., adipose
tissue and
brain)
Table 2. Kits for RNA purification from animal or human tissues using
magnetic particles or 96-well plates
Sample type Kit
Kit format
Page
Magnetic particles; up to 10 mg
tissue; automated on EZ1
Advanced (16 samples per run)
or EZ1 Advanced XL (114
samples per run)*
17
MagAttract RNA
Magnetic particles; up to 10 mg
Tissue Mini M48 Kit tissue; automated on BioRobot
M48 (648 samples per run)
17
17
17
MagAttract RNA
Universal Tissue
M48 Kit
Magnetic particles; up to 50 mg
tissue; automated on BioRobot
M48 (648 samples per run)
17
RNeasy 96
Universal Tissue
Kits
miRNeasy 96 Kit
Any type of
tissue
Also automatable on BioRobot Gene Expression Real-Time RT-PCR and BioRobot 8000
(no longer available).
Table 3. Kits for RNA purification from plant tissues, bacteria, and yeast
Sample type
Kit
Kit format
Page
Plant tissue
(e.g., leaf)
19
RNeasy 96 Kit
Bacteria
RNeasy Protect
(Gram-postive Bacteria Mini Kit
and -negative) RNeasy Protect
Bacteria Midi Kit
21
21
Yeast
22
Kit format
Page
DNeasy Blood
& Tissue Kit
DNeasy 96
Blood & Tissue
Kit
QIAamp DNA
Mini Kit
23
Reagent Pack,
96-well plate; automated on QIAxtractor
DX, and
Plasticware Pack,
DX
23
Kit format
Page
25
25
MagAttract 96 DNA
Plant Core Kit
25
25
25
* No longer available.
Kit
AllPrep DNA/RNA
Micro Kit
Spin column; up to 5 mg
tissue
AllPrep DNA/RNA
Mini Kit
Spin column; up to 30 mg 17
tissue; automatable on
QIAcube
AllPrep DNA/RNA
96 Kit
96-well plate; up to 10 mg 17
tissue
10
Kit format
Page
17
11
Kit for purification of DNA, RNA, and/or protein (see ordering information
on pages 3237 or visit www.qiagen.com)
12
Important Notes
General remarks on disruption and homogenization
Efficient disruption and homogenization of the starting material is an absolute
requirement for all nucleic acid purification procedures. Disruption and
homogenization are 2 distinct steps.
Cellular disruption is one of the most critical steps in nucleic acid purification.
Disruption in lysis buffer alone, without physical shearing, may result in nucleic
acid degradation by endogenous DNases and RNases. Incomplete disruption
prevents the lysis buffer, which inactivates nucleases, from contacting nucleic
acids within the intact cells. Furthermore, cellular debris that is not disrupted
can result in decreased yield and increases the risk of clogging the purification
column. After sample disruption, there should be no visible particulates (except
when disrupting materials containing hard, noncellular components, such as
connective tissue, bone, or woody plant tissue). QIAGEN kits and protocols
contain recommendations for the most appropriate method of sample
disruption and homogenization to maximize the yield and quality of your DNA,
RNA, and protein preparation.
13
Consistency of sample
Samples are disrupted and homogenized in lysis buffer. For animal and
human tissues that are either freshly isolated or frozen in liquid nitrogen,
the sample tube and tissue sample need to be precooled on dry ice. For
animal and human tissues that are stabilized in Allprotect Tissue Reagent
or RNAlater RNA Stabilization Reagent, no precooling is required.
Samples are precooled on dry ice for at least 30 minutes and then
disrupted and homogenized without lysis buffer. Lysis buffer is added after
disruption and homogenization. When precooling samples, the insert of
the TissueLyser LT Adapter as well as the sample tubes, each containing the
correct amount of sample and a grinding bead, are placed on dry ice.
Important: When using the TissueLyser LT Adapter, do not freeze the adapter
and sample tubes in liquid nitrogen, as this may result in breakage of the tubes.
Note: After disrupting and homogenizing tissues using the TissueLyser LT, some
debris may stick to the lids of the sample tubes. We therefore recommend a
brief centrifugation before opening the sample tubes.
Bead selection
For human, animal, and plant tissues, the optimal beads to use are 57 mm
(mean diamter) stainless steel beads. When disrupting tough tissue samples, we
recommend using 7 mm beads instead of 5 mm beads to improve disruption
efficiency. For disruption of cells, the optimal beads to use are 0.10.6 mm
(mean diameter) glass beads for bacteria, and 0.5 mm glass beads for yeast
and unicellular animal cells. It is essential that glass beads are pretreated
before use by washing in concentrated nitric acid.* Pretreated (acid-washed)
beads can be purchased from many vendors of biological supplies (e.g.,
Sigma, cat. nos. G1145, G1277, and G8772). Disruption parameters for
samples not addressed in this handbook must be determined empirically.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and
protective goggles. For more information, consult the appropriate material safety data sheets
(MSDSs), available from the product supplier.
This is not a complete list of suppliers and does not include many important vendors of
biological supplies.
14
Figure 1. Loading the TissueLyser LT Adapter. Ensure that the TissueLyser LT Adapter is
balanced by loading samples tubes as shown above.
15
16
Ensure that you are familiar with operating the TissueLyser LT by referring
to the TissueLyser LT User Manual.
Procedure
1. Place 2 ml microcentrifuge tubes containing 1 stainless steel bead
(5 mm mean diameter) on dry ice for at least 15 min. Keep the insert
of the TissueLyser LT Adapter at room temperature (1525C).
2. Transfer up to 30 mg fresh or frozen tissue to the precooled tubes
and incubate for another 15 min on dry ice.
If handling tissue samples stabilized with RNAlater RNA Stabilization
Reagent or Allprotect Tissue Reagent, cooling on dry ice is not necessary.
3. Place the tubes into the insert of the TissueLyser LT Adapter, and
incubate at room temperature for 2 min to avoid freezing of lysis
buffer in step 4.
Do not incubate for longer than 2 min, otherwise the tissue will thaw,
resulting in potential RNA degradation.
17
18
Ensure that you are familiar with operating the TissueLyser LT by referring
to the TissueLyser LT User Manual.
If using a QIAGEN kit for RNA purification, read the supplied handbook
carefully before starting.
Soft, fresh tissues from plants such as Nicotiana and Arabidopsis can often
also be disrupted and homogenized in lysis buffer. Hard tissues (e.g.,
woody plant materials) may require freezing and disruption under frozen
conditions.
Procedure
1. Place 2 ml microcentrifuge tubes containing 1 stainless steel bead
(5 mm mean diameter) into the insert of the TissueLyser LT Adapter.
Incubate on dry ice for at least 30 min.
2. Determine the amount of fresh plant material. Do not use more than
100 mg per sample.
Weighing tissue is the most accurate way to determine the amount.
3. Transfer the weighed tissue to the precooled tubes and incubate for
another 30 min on dry ice.
Alternatively, plant tissues can be flash-frozen in liquid nitrogen prior to
transfer to the precooled tubes. In this case, the additional 30 min
incubation on dry ice is not necessary.
Note: Do not freeze the adapter and tubes in liquid nitrogen, as this may
lead to breakage of the tubes.
4. Place the precooled insert with sample tubes into the base of the
TissueLyser LT Adapter, which is attached to the TissueLyser LT. Place
the lid of the TissueLyser LT Adapter over the insert, and screw the
knob until the lid is securely fastened.
19
20
Ensure that you are familiar with operating the TissueLyser LT by referring
to the TissueLyser LT User Manual.
If using an RNeasy Protect Bacteria Kit for RNA purification, read the
supplied handbook carefully before starting.
Procedure
1. Pellet the bacterial cells by centrifugation. Immediately add the
appropriate volume of lysis buffer (e.g., Buffer RLT) to each sample
and vortex vigorously.
2. Transfer each sample to 2 ml microcentrifuge tubes containing 25
50 mg acid-washed glass beads (150600 m mean diameter).
3. Place the tubes into the insert of the TissueLyser LT Adapter.
4. Place the insert with sample tubes into the base of the TissueLyser LT
Adapter, which is attached to the TissueLyser LT. Place the lid of the
TissueLyser LT Adapter over the insert, and screw the knob until the
lid is securely fastened.
5. Operate the TissueLyser LT for 5 min at 50 Hz.
The duration of disruption and homogenization depends on the sample
being processed and can be extended until no debris is visible.
6. Proceed with RNA purification.
21
Ensure that you are familiar with operating the TissueLyser LT by referring
to the TissueLyser LT User Manual.
If using the RNeasy Mini Kit for RNA purification, read the supplied
handbook carefully before starting.
Procedure
1. Pellet the yeast cells by centrifugation. Immediately add the
appropriate volume of lysis buffer (e.g., Buffer RLT) to each sample
and vortex vigorously.
2. Transfer each sample to 2 ml microcentrifuge tubes containing
approximately 600 l acid-washed glass beads (450550 m mean
diameter).
3. Place the tubes into the insert of the TissueLyser LT Adapter.
4. Place the insert with sample tubes into the base of the TissueLyser LT
Adapter, which is attached to the TissueLyser LT. Place the lid of the
TissueLyser LT Adapter over the insert, and screw the knob until the
lid is securely fastened.
5. Operate the TissueLyser LT for 5 min at 50 Hz.
The duration of disruption and homogenization depends on the sample
being processed and can be extended until no debris is visible.
6. Proceed with RNA purification.
22
Ensure that you are familiar with operating the TissueLyser LT by referring
to the TissueLyser LT User Manual.
If using a QIAGEN kit for DNA purification, read the supplied handbook
and appropriate supplementary protocol carefully before starting.
Procedure
1. Place 2 ml microcentrifuge tubes containing 1 stainless steel bead
(5 mm mean diameter) on dry ice for at least 15 min. Keep the insert
of the TissueLyser LT Adapter at room temperature (1525C).
2. Transfer up to 25 mg fresh or frozen tissue to the precooled tubes
and incubate for another 15 min on dry ice.
3. Place the tubes into the insert of the TissueLyser LT Adapter, and
incubate at room temperature for 2 min to avoid freezing of lysis
buffer in step 4.
Do not incubate for longer than 2 min, otherwise the tissue will thaw,
resulting in potential DNA degradation.
4. Immediately add the appropriate volume of lysis buffer (e.g., Buffer
ATL) to each tube.
5. Place the insert with sample tubes into the base of the TissueLyser LT
Adapter, which is attached to the TissueLyser LT. Place the lid of the
TissueLyser LT Adapter over the insert, and screw the knob until the
lid is securely fastened.
6. Operate the TissueLyser LT for 40 s at 30 Hz.
Note: Depending on the type of tissue, exceeding this homogenization time
and intensity may lead to significant fragmentation of genomic DNA.
However, for tough samples, it may be necessary to exceed this
homogenization time and/or intensity to improve disruption efficiency.
If working with fibrous tissues, cutting the tissue into smaller pieces before
starting disruption will improve disruption efficiency.
23
24
Ensure that you are familiar with operating the TissueLyser LT by referring
to the TissueLyser LT User Manual.
If using a QIAGEN kit for DNA purification, read the supplied handbook
carefully before starting.
Procedure
1. Place 2 ml microcentrifuge tubes containing 1 stainless steel bead
(5 mm mean diameter) into the insert of the TissueLyser LT Adapter.
Incubate on dry ice for at least 30 min.
2. Determine the amount of fresh plant material. Do not use more than
100 mg per sample.
Weighing tissue is the most accurate way to determine the amount.
3. Transfer the weighed tissue to the precooled tubes and incubate for
another 30 min on dry ice.
Alternatively, plant tissues can be flash-frozen in liquid nitrogen prior to
transfer to the precooled tubes. In this case, the additional 30 min
incubation on dry ice is not necessary.
Note: Do not freeze the adapter and tubes in liquid nitrogen, as this may
lead to breakage of the tubes.
4. Place the precooled insert with sample tubes into the base of the
TissueLyser LT Adapter, which is attached to the TissueLyser LT. Place
the lid of the TissueLyser LT Adapter over the insert, and screw the
knob until the lid is securely fastened.
25
26
Ensure that you are familiar with operating the TissueLyser LT by referring
to the TissueLyser LT User Manual.
Procedure
1. Place 2 ml microcentrifuge tubes containing 1 stainless steel bead
(5 mm mean diameter) on dry ice for at least 15 min. Keep the insert
of the TissueLyser LT Adapter at room temperature (1525C).
2. Transfer up to 30 mg fresh or frozen tissue to the precooled tubes
and incubate for another 15 min on dry ice.
If handling tissue samples stabilized with Allprotect Tissue Reagent, cooling
on dry ice is not necessary.
3. Place the tubes into the insert of the TissueLyser LT Adapter, and
incubate at room temperature for 2 min to avoid freezing of lysis
buffer in step 4.
Do not incubate for longer than 2 min, otherwise the tissue will thaw,
resulting in potential protein degradation.
4. Immediately add the appropriate volume of lysis buffer (e.g.,
Mammalian Cell Lysis Buffer) to each tube.
5. Place the insert with sample tubes into the base of the TissueLyser LT
Adapter, which is attached to the TissueLyser LT. Place the lid of the
TissueLyser LT Adapter over the insert, and screw the knob until the
lid is securely fastened.
6. Operate the TissueLyser LT for 25 min at 50 Hz.
The duration of disruption and homogenization depends on the tissue being
processed and can be extended until no tissue debris is visible.
TissueLyser LT Handbook 05/2009
27
28
29
The QIAcube.
30
Capability
EZ1 Advanced
EZ1 Advanced XL
QIAsymphony SP
BioRobot Universal
System
BioSprint 96
31
Ordering Information
Product
Contents
Cat. no.
TissueLyser LT
85600
TissueLyser LT Adapter,
12-Tube
69980
990381
69989
69990
69965
69967
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Notes
38
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