QIAGEN RNeasy Microarray Tissue RNA purification kit Handbook
Below you will find brief information for RNA purification kit RNeasy Microarray Tissue. This handbook provides a comprehensive guide on the RNeasy Microarray Tissue Kit, which is designed for high-quality total RNA purification from various types of tissue for microarray analysis. The kit utilizes QIAzol Lysis Reagent for efficient tissue disruption and homogenization, followed by RNeasy spin columns for RNA purification and contaminant removal. It’s a valuable resource for researchers working with RNA from tissue samples for advanced applications like microarray analysis.
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Second Edition
RNeasy
®
Microarray Tissue
Handbook
For purification of total RNA from all types of tissue for microarray analysis
Sample & Assay Technologies
QIAGEN Sample and Assay Technologies
QIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result.
QIAGEN sets standards in:
■
Purification of DNA, RNA, and proteins
■
Nucleic acid and protein assays
■ microRNA research and RNAi
■
Automation of sample and assay technologies
Our mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit www.qiagen.com.
QIAGEN is a member of the Forest Stewardship Council (FSC). For the production of printed materials, including handbooks, QIAGEN has a policy to select suppliers that comply with FSC standards for printing processes and well-managed forests.
Contents
Kit Contents
Storage
Quality Control
Product Use Limitations
Product Warranty and Satisfaction Guarantee
Technical Assistance
Safety Information
Introduction
Principle and procedure
Automated purification
Equipment and Reagents to Be Supplied by User
Important Notes
Determining the amount of starting material
Handling and storing starting material
Disrupting and homogenizing starting material
Protocol
■
Purification of Total RNA from Tissues
Troubleshooting Guide
Appendix A: General Remarks on Handling RNA
Appendix B: Storage, Quantification, and Determination of Quality of RNA 27
Appendix C: Optional On-Column DNase Digestion with the RNase-Free DNase Set 30
References
Ordering Information
31
32
15
21
25
11
11
12
13
5
6
5
5
7
7
8
10
4
4
4
RNeasy Microarray Tissue Handbook 11/2009
3
4
Kit Contents
RNeasy Microarray Tissue Mini Kit
Catalog no.
Number of preps
RNeasy Mini Spin Columns (each in a 2 ml Collection Tube)
Collection Tubes (1.5 ml)
Collection Tubes (2 ml)
QIAzol ® Lysis Reagent*
Buffer RW1*
Buffer RPE † (concentrate)
RNase-Free Water
Handbook
(50)
73304
50
50
50
50
50 ml
45 ml
11 ml
10 ml
1
†
* Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 6 for safety information.
Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a working solution.
Note: QIAzol Lysis Reagent is delivered separately.
Storage
The RNeasy Microarray Tissue Mini Kit should be stored dry at room temperature
(15–25°C). All components are stable for at least 9 months under these conditions.
QIAzol Lysis Reagent can be stored at room temperature or at 2–8°C.
Quality Control
In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of
RNeasy Microarray Tissue Mini Kit is tested against predetermined specifications to ensure consistent product quality.
RNeasy Microarray Tissue Handbook 11/2009
Product Use Limitations
The RNeasy Microarray Tissue Mini Kit is intended for molecular biology applications.
This product is neither intended for the diagnosis, prevention, or treatment of a disease, nor has it been validated for such use either alone or in combination with other products.
Therefore, the performance characteristics of this product for clinical use (i.e., diagnostic, prognostic, therapeutic, or blood banking) are unknown.
Product Warranty and Satisfaction Guarantee
QIAGEN guarantees the performance of all products in the manner described in our product literature. The purchaser must determine the suitability of the product for its particular use. Should any product fail to perform satisfactorily due to any reason other than misuse, QIAGEN will replace it free of charge or refund the purchase price. We reserve the right to change, alter, or modify any product to enhance its performance and design. If a QIAGEN ® product does not 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 RNeasy Microarray Tissue Mini Kit 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).
RNeasy Microarray Tissue Handbook 11/2009
5
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.
CAUTION: DO NOT add bleach or acidic solutions directly to the sample-preparation waste.
QIAzol Lysis Reagent contains guanidine thiocyanate and Buffer RW1 contains a small amount of guanidine thiocyanate. Guanidine salts can form highly reactive compounds when combined with bleach. If liquid containing these buffers is spilt, clean with suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1%
(v/v) sodium hypochlorite.
The following risk and safety phrases apply to the components of the RNeasy
Microarray Tissue Mini Kit.
QIAzol Lysis Reagent
Contains phenol, guanidine thiocyanate: toxic, corrosive. Risk and safety phrases:*
R23/24/25-32-34-48/20/21/22-68, S24/25-26-36/37/39-45
Buffer RW1
Contains ethanol: flammable. Risk phrase:* R10
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
* R10: Flammable; R23/24/25: Toxic by inhalation, in contact with skin and if swallowed; R32: Contact with acids liberates very toxic gas; R34: Causes burns; R48/20/21/22: Harmful: danger of serious damage to health by prolonged exposure through inhalation, in contact with skin and if swallowed; R68:
Possible risk of irreversible effects; S24/25: Avoid contact with skin and eyes; S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice; S36/37/39: Wear suitable protective clothing, gloves and eye/face protection; S45: In case of accident or if you feel unwell seek medical advice immediately (show the label where possible).
6
RNeasy Microarray Tissue Handbook 11/2009
Introduction
The RNeasy Microarray Tissue Mini Kit provides the optimal solution for purifying RNA for microarray analysis. QIAzol Lysis Reagent ensures efficient phenol/guanidinebased lysis of even difficult-to-lyse tissues, while trusted RNeasy spin columns allow fast and convenient purification of intact RNA without any phenol contamination. The result is highly pure RNA that can be efficiently reverse transcribed, ensuring reliable results in microarray analysis and avoiding the need for unnecessary repetition of experiments.* The purified RNA is of the same quality as that achieved with other
RNeasy Kits, and is therefore also ready to use for other applications, such as real-time
RT-PCR.
Traditional methods of purifying RNA for microarray analysis involve acid-phenol/ chloroform extraction followed by precipitation of RNA from the aqueous phase, redissolving of the RNA, and cleanup of the RNA using silica-membrane technology.
With the RNeasy Microarray Tissue Mini Kit, RNA is purified directly from the aqueous phase using an RNeasy spin column. No precipitation and redissolving of RNA is required, which saves time, prevents potential loss of RNA, and avoids variability in
RNA yields.
Principle and procedure
The RNeasy Microarray Tissue Mini Kit integrates phenol/guanidine-based sample lysis and silica-membrane purification of total RNA. QIAzol Lysis Reagent, included in the kit, is a monophasic solution of phenol and guanidine thiocyanate, designed to facilitate lysis of all types of tissue and inhibit RNases. The high lysis efficiency of the reagent and the subsequent removal of contaminants by organic phase extraction enables use of larger amounts of tissue with RNeasy spin columns. RNA can be purified from up to
50 mg of human or animal tissue (or up to 100 mg of brain or adipose tissue) per
RNeasy Mini spin column.
†
Tissue samples are homogenized in QIAzol Lysis Reagent. After addition of chloroform, the homogenate is separated into aqueous and organic phases by centrifugation. RNA partitions to the upper, aqueous phase while DNA partitions to the interphase and proteins to the lower, organic phase or the interphase.
The upper, aqueous phase is collected, and ethanol is added to provide appropriate binding conditions. The sample is then applied to an RNeasy spin column, where the total RNA binds to the membrane, and phenol and other contaminants are efficiently washed away. High-quality RNA is then eluted in RNase-free water (see flowchart, page 9).
†
* For microarray analysis of cultured cells, we recommend purifying RNA using either the RNeasy
Microarray Tissue Mini Kit or the RNeasy Mini Kit (cat. no. 74104).
To ensure optimal RNA yields, the binding capacity of the RNeasy spin column must not be exceeded. For details, see the protocol (page 15).
RNeasy Microarray Tissue Handbook 11/2009
7
With the RNeasy Microarray Tissue Mini Kit, all RNA molecules longer than
200 nucleotides are purified. The procedure provides an enrichment for mRNA since most RNAs <200 nucleotides (such as 5.8S rRNA, 5S rRNA, and tRNAs, which together comprise 15–20% of total RNA) are selectively excluded. The size distribution of the purified RNA is comparable to that obtained by centrifugation through a CsCl gradient or cushion, where small RNAs do not sediment efficiently. For purification of small RNA, including microRNA, from tissues and cells, we recommend using miRNeasy Kits (see ordering information, page 34).
Automated purification
Purification of RNA can be fully automated on the QIAcube ® . The innovative QIAcube uses advanced technology to process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into your laboratory workflow.
Sample preparation using the QIAcube follows the same steps as the manual procedure
(i.e., bind, wash, and elute), enabling you to continue using the RNeasy Microarray
Tissue Mini Kit for purification of high-quality RNA. For more information about the automated procedure, see the relevant protocol sheet available at www.qiagen.com/MyQIAcube.
The QIAcube is preinstalled with protocols for purification of plasmid DNA, genomic
DNA, RNA, viral nucleic acids, and proteins, plus DNA and RNA cleanup. The range of protocols available is continually expanding, and additional QIAGEN protocols can be downloaded free of charge at www.qiagen.com/MyQIAcube.
8
The QIAcube.
RNeasy Microarray Tissue Handbook 11/2009
RNeasy Microarray Tissue Procedure
RNeasy Microarray Tissue Handbook 11/2009
9
Equipment and Reagents to Be Supplied by User
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.
■
Chloroform
■
Ethanol (70% and 96–100%)*
■
Sterile, RNase-free pipet tips
■
Equipment for tissue disruption and homogenization (see page 13): we recommend either the TissueRuptor ® with TissueRuptor Disposable Probes or a
TissueLyser system (see ordering information, pages 32–33)
■
For stabilization of RNA in tissues (see page 12): RNA later ® RNA Stabilization
Reagent or Allprotect Tissue Reagent (see ordering information, page 32) or liquid nitrogen and dry ice
■
1.5 ml or 2 ml microcentrifuge tubes
■
Microcentrifuge(s) (with rotor for 2 ml tubes) for centrifugation at 4°C and at room temperature (15–25°C)
* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.
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RNeasy Microarray Tissue Handbook 11/2009
Important Notes
Determining the amount of starting material
It is essential to use the correct amount of starting material in order to obtain optimal
RNA yield and purity. The maximum amount that can be used is determined by:
■
The type of tissue and its RNA content
■
The volume of QIAzol Lysis Reagent required for efficient lysis
■
The RNA binding capacity of the RNeasy spin column
When processing samples containing high amounts of RNA, less than the maximum amount of starting material shown in Table 1 should be used, so that the RNA binding capacity of the RNeasy spin column is not exceeded.
When processing samples containing low amounts of RNA, the maximum amount of starting material shown in Table 1 can be used. However, even though the RNA binding capacity of the RNeasy spin column is not reached, the maximum amount of starting material must not be exceeded. Otherwise, lysis will be incomplete and cellular debris may interfere with the binding of RNA to the RNeasy spin column membrane, resulting in lower RNA yield and purity.
More information on using the correct amount of starting material is given in the protocol. Table 2 shows expected RNA yields from various sources.
Table 1. RNeasy spin column specifications
Specification
Maximum binding capacity
Maximum loading volume
RNA size distribution
Minimum elution volume
Maximum amount of starting tissue
RNeasy Mini spin column
100 µg RNA
700 µl
RNA >200 nucleotides
30 µl
≤100 mg
Note: If the binding capacity of the RNeasy spin column is exceeded, RNA yields will not be consistent and may be reduced. If lysis of the starting material is incomplete,
RNA yields will be lower than expected, even if the binding capacity of the RNeasy spin column is not exceeded.
RNeasy Microarray Tissue Handbook 11/2009
11
Table 2. Typical yields of total RNA with the RNeasy Microarray Tissue Mini Kit
Mouse/rat tissue (10 mg)
Adipose tissue
Yield of total RNA (µg)*
0.5–2.5
Brain 5–20
Heart 5–25
Intestine 10–60
Kidney 5–40
Liver 15–80
Lung 5–15
Muscle 5–35
Skin 2–5
Spleen 15–100
* Amounts can vary due to factors such as species and developmental stage (especially with adipose tissues, large variations are possible due to developmental stage and location of the tissue). Since the RNeasy procedure enriches for mRNA and other RNA species >200 nucleotides, the total RNA yield does not include 5S rRNA, tRNA, and other low-molecular-weight RNAs, which make up 15–20% of total cellular
RNA.
Handling and storing starting material
RNA in harvested tissue is not protected until the sample is treated with RNA later RNA
Stabilization Reagent, flash-frozen, or disrupted and homogenized in the presence of
RNase-inhibiting or denaturing reagents. Otherwise, unwanted changes in the gene expression profile will occur. It is therefore important that tissue samples are immediately frozen in liquid nitrogen and stored at –70°C, or immediately immersed in RNA later RNA Stabilization Reagent at room temperature. An alternative to RNAlater
RNA Stabilization Reagent is Allprotect Tissue Reagent, which provides immediate stabilization of DNA, RNA, and protein in tissue samples at room temperature.
Note: RNAlater RNA Stabilization Reagent cannot be used to stabilize RNA in adipose tissue due to the high abundance of fat, but can be used to stabilize RNA in other fatty tissues such as brain. Allprotect Tissue Reagent can stabilize adipose and brain tissue.
The procedures for tissue harvesting and RNA protection should be carried out as quickly as possible. Frozen tissue samples should not be allowed to thaw during handling or weighing.
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RNeasy Microarray Tissue Handbook 11/2009
Disrupting and homogenizing starting material
Efficient disruption and homogenization of the starting material is an absolute requirement for all total RNA purification procedures. Disruption and homogenization are 2 distinct steps:
■
Disruption: Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the RNA contained in the sample. Incomplete disruption results in significantly reduced RNA yields.
■
Homogenization: Homogenization is necessary to reduce the viscosity of the lysates produced by disruption. Homogenization shears high-molecular-weight genomic DNA and other high-molecular-weight cellular components to create a homogeneous lysate. Incomplete homogenization results in inefficient binding of
RNA to the RNeasy spin column membrane and therefore significantly reduced
RNA yields.
Disruption and homogenization of tissue samples can be carried out rapidly and efficiently using either the TissueRuptor (for processing samples individually) or a
TissueLyser system (for processing multiple samples simultaneously). Disruption and homogenization with TissueRuptor and TissueLyser systems generally results in higher
RNA yields than with other methods.
Disruption and homogenization using the TissueRuptor
The TissueRuptor is a rotor–stator homogenizer that thoroughly disrupts and simultaneously homogenizes single tissue samples in the presence of lysis buffer in
15–90 seconds, depending on the toughness and size of the sample. The blade of the
TissueRuptor disposable probe rotates at a very high speed, causing the sample to be disrupted and homogenized by a combination of turbulence and mechanical shearing.
For guidelines on using the TissueRuptor, refer to the TissueRuptor Handbook. For other rotor–stator homogenizers, refer to suppliers’ guidelines.
Disruption and homogenization using TissueLyser systems
In bead-milling, tissues can be disrupted by rapid agitation in the presence of beads and lysis buffer. Disruption and simultaneous homogenization occur by the shearing and crushing action of the beads as they collide with the cells. Two bead mills are available from QIAGEN: the TissueLyser LT for low- to medium-throughput disruption, and the TissueLyser II for medium- to high-throughput disruption.
The TissueLyer LT disrupts and homogenizes up to 12 samples at the same time. The instrument needs to be used in combination with the TissueLyser LT Adapter, which holds
12 x 2 ml microcentrifuge tubes containing stainless steel beads of 5 mm or 7 mm mean diameter. For guidelines on using the TissueLyser LT, refer to the TissueLyser LT
Handbook.
RNeasy Microarray Tissue Handbook 11/2009
13
The TissueLyser II disrupts and homogenizes up to 48 tissue samples simultaneously when used in combination with the TissueLyser Adapter Set 2 x 24, which holds
48 x 2 ml microcentrifuge tubes containing stainless steel beads of 5 mm mean diameter. For guidelines on using the TissueLyser II, refer to the TissueLyser Handbook.
If using other bead mills for sample disruption and homogenization, refer to suppliers’ guidelines.
Note: Tungsten carbide beads react with QIAzol Lysis Reagent and must not be used to disrupt and homogenize tissues.
The TissueLyser II can also disrupt and homogenize up to 192 tissue samples simultaneously when used in combination with the TissueLyser Adapter Set 2 x 96, which holds 192 x 1.2 ml microtubes containing stainless steel beads of 5 mm mean diameter. In this case, we recommend using the RNeasy 96 Universal Tissue Kit, which provides high-throughput RNA purification from all types of tissue, including fatty tissues, in 96-well format and is based on the same technology as the RNeasy
Microarray Tissue Mini Kit. For ordering information, see page 34.
14
RNeasy Microarray Tissue Handbook 11/2009
Protocol: Purification of Total RNA from Tissues
Determining the correct amount of starting material
It is essential to use the correct amount of tissue in order to obtain optimal RNA yield and purity. With the RNeasy Microarray Tissue Mini Kit, a maximum of 100 mg brain or adipose tissue can generally be processed. For these tissues, the RNA binding capacity of the RNeasy Mini spin column and the lysing capacity of QIAzol Lysis
Reagent will not be exceeded by these amounts. For other tissues, a maximum of 50 mg tissue can generally be used. For tissues with high RNA content such as liver, spleen, and thymus, we recommend using no more than 30 mg tissue to ensure optimal RNA yields and to avoid exceeding the binding capacity of the RNA spin column. Average
RNA yields from various tissues are given in Table 2 (page 12).
If there is no information about the nature of your starting material, we recommend starting with no more than 30 mg tissue. Depending on RNA yield and purity, it may be possible to use up to 100 mg tissue in subsequent preparations.
Do not overload the RNeasy spin column, as this will significantly reduce RNA yield and quality.
Weighing tissue is the most accurate way to quantify the amount of starting material.
As a guide, a 4 mm cube (64 mm 3 ) of most animal tissues weighs 70–85 mg.
Important points before starting
■
If using the RNeasy Microarray Tissue Mini Kit for the first time, read “Important
Notes” (page 11).
■
If working with RNA for the first time, read Appendix A (page 25).
■
If using a TissueRuptor or TissueLyser system, ensure that you are familiar with operating it by referring to the supplied user manual (operating instructions) and handbook.
■
To freeze tissue for long-term storage (several months), flash-freeze in liquid nitrogen, and immediately transfer to –70°C. Do not allow tissues to thaw during weighing or handling prior to disruption in QIAzol Lysis Reagent. Homogenized tissue lysates from step 3 can also be stored at –70°C for several months. Incubate frozen lysates at 37°C in a water bath until completely thawed and salts are dissolved before continuing with step 4. Avoid prolonged incubation, which may compromise RNA integrity.
■
Generally, DNase digestion is not required since integrated QIAzol and RNeasy technologies efficiently remove most of the DNA without DNase treatment. If desired, residual DNA can be removed by optional on-column DNase-digestion using the RNase-Free DNase Set (see Appendix C, page 30).
RNeasy Microarray Tissue Handbook 11/2009
15
■
QIAzol Lysis Reagent and Buffer RW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach. See page 6 for safety information.
■
Except for phase separation (step 7), all protocol and centrifugation steps should be performed at room temperature (15–25°C). During the procedure, work quickly.
Things to do before starting
■
Buffer RPE is supplied as a concentrate. Before using for the first time, add
4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a working solution.
■
If performing optional on-column DNase digestion, prepare DNase I stock solution as described in Appendix C (page 30).
Procedure
1.
If using a TissueLyser system, add one stainless steel bead (5 mm mean diameter) per 2 ml microcentrifuge tube (not supplied).* If working with tissues that are not stabilized in RNA later or Allprotect Reagent, place the tubes on dry ice.
2.
Excise the tissue sample from the animal or remove it from storage. Determine the amount of tissue. Do not use more than 100 mg. Proceed immediately to step 3.
Weighing tissue is the most accurate way to determine the amount.
If the tissue sample was stored in RNA later or Allprotect Reagent, remove it from the reagent using forceps and be sure to remove any excess reagent or crystals that may have formed.
RNA in harvested tissues is not protected until the tissues are treated with RNA later or Allprotect Reagent, flash-frozen, or disrupted and homogenized in step 3.
Frozen tissues should not be allowed to thaw during handling. The relevant procedures should be carried out as quickly as possible.
3.
Disrupt the tissue and homogenize the lysate using the TissueRupter (follow step 3a), TissueLyser LT (follow step 3b), or TissueLyser II (follow step 3c).
See “Disrupting and homogenizing starting material”, page 13, for more details on disruption and homogenization.
Note: Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy spin column. Homogenization with TissueRupter and TissueLyser systems generally results in higher RNA yields than with other methods.
* When disrupting tough or very tough samples with the TissueLyser LT, we recommend using one or two
7 mm stainless steel beads.
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RNeasy Microarray Tissue Handbook 11/2009
3a. Disruption and homogenization using the TissueRuptor:
■
Place the tissue in a suitably sized vessel containing 1 ml QIAzol Lysis
Reagent.
Note: Use a suitably sized vessel with sufficient extra headspace to accommodate foaming, which may occur during homogenization.
Generally, round-bottomed tubes allow more efficient disruption and homogenization than conical-bottomed tubes.
■
Place the tip of the disposable probe into the vessel and operate the
TissueRuptor at full speed until the lysate is uniformly homogeneous (usually
20–40 s). Proceed to step 4.
Note: To avoid damage to the TissueRuptor and disposable probe during operation, make sure the tip of the probe remains submerged in the buffer.
Foaming may occur during homogenization, especially of brain tissue. If this occurs, let the homogenate stand at room temperature for 2–3 min until the foam subsides before continuing with the procedure.
3b. Disruption and homogenization using the TissueLyser LT:
■
■
Keep the tubes prepared in step 1 on dry ice for at least 15 min (however, keep the insert of the TissueLyser LT Adapter at room temperature). Then place the tissues in the tubes, and keep the tubes on dry ice for another 15 min.
If working with RNA later or Allprotect stabilized tissues, it is not necessary to place the tubes on dry ice.
Place the tubes in the insert of the TissueLyser LT Adapter, and incubate at room temperature for 2 min. Then immediately add 1 ml QIAzol Lysis Reagent per tube.
■
■
■
Do not incubate for longer than 2 min, otherwise frozen tissues will thaw, resulting in potential RNA degradation.
Place the tubes in the TissueLyser LT Adapter.
Operate the TissueLyser LT for 2–5 min at 50 Hz.
The time depends on the tissue being processed and can be extended until the tissue is completely homogenized.
Carefully pipet the lysates into new microcentrifuge tubes (not supplied).
Proceed to step 4.
Do not reuse the stainless steel beads.
3c. Disruption and homogenization using the TissueLyser II:
■
■
Place the tissues in the tubes prepared in step 1.
If the tubes were stored on dry ice, place them at room temperature. Then immediately add 1 ml QIAzol Lysis Reagent per tube.
RNeasy Microarray Tissue Handbook 11/2009
17
■
■
■
Place the tubes in the TissueLyser Adapter Set 2 x 24.
Operate the TissueLyser II for 2 min at 20 Hz.
The time depends on the tissue being processed and can be extended until the tissue is completely homogenized.
Disassemble the adapter set, rotate the rack of tubes so that the tubes nearest to the TissueLyser II are now outermost, and reassemble the adapter set.
Operate the TissueLyser II for another 2 min at 20 Hz.
Rearranging the tubes allows even homogenization.
Carefully pipet the lysates into new microcentrifuge tubes (not supplied).
■
Proceed to step 4.
Do not reuse the stainless steel beads.
4.
Place the tube containing the homogenate on the benchtop at room temperature
(15–25°C) for 5 min.
This step promotes dissociation of nucleoprotein complexes.
5.
Add 200 µl chloroform. Securely cap the tube containing the homogenate, and shake it vigorously for 15 s.
Thorough mixing is important for subsequent phase separation.
6.
Place the tube containing the homogenate on the benchtop at room temperature for 2–3 min.
7.
Centrifuge at 12,000 x g for 15 min at 4°C. After centrifugation, heat the centrifuge to room temperature (15–25°C) if the same centrifuge will be used in the later steps of this procedure.
After centrifugation, the sample separates into 3 phases: an upper, colorless, aqueous phase containing RNA; a white interphase; and a lower, red, organic phase. For tissues with an especially high fat content, an additional, clear phase may be visible below the red, organic phase. The volume of the aqueous phase should be approximately 600 µl.
8.
Transfer the upper, aqueous phase to a new tube (not supplied). Add 1 volume
(usually 600 µl) of 70% ethanol, and mix thoroughly by pipetting up and down.
Do not centrifuge. Proceed immediately to step 9.
Note: The volume of lysate may be less than 600 µl due to loss during homogenization and centrifugation.
Precipitates may be visible after addition of ethanol. Resuspend precipitates completely by vigorous shaking, and proceed immediately to step 9.
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RNeasy Microarray Tissue Handbook 11/2009
9.
Transfer up to 700 µl of the sample to an RNeasy Mini spin column placed in a
2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at
ⱖ
8000 x g (
ⱖ
10,000 rpm) at room temperature (15–25°C). Discard the flowthrough.*
Reuse the collection tube in step 10.
10. Repeat step 9 using the remainder of the sample. Discard the flow-through.*
Reuse the collection tube in step 11.
Optional: If performing optional on-column DNase digestion (see “Important points before starting”), follow steps C1–C4 (page 30) after performing this step.
11. Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at
ⱖ
8000 x g (
ⱖ
10,000 rpm) to wash the membrane. Discard the flow-through.*
Reuse the collection tube in step 12.
After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely.*
Skip this step if performing optional on-column DNase digestion (page 30).
12. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at
ⱖ
8000 x g (
ⱖ
10,000 rpm) to wash the membrane. Discard the flow-through.
Reuse the collection tube in step 13.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to
Buffer RPE before use (see “Things to do before starting”, page 16).
13. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at
ⱖ
8000 x g (
ⱖ
10,000 rpm) to wash the membrane.
The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions.
Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.
14. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min.
Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column after step 13.
* Flow-through contains QIAzol Lysis Reagent or Buffer RW1 and is therefore not compatible with bleach.
See page 6 for safety information.
RNeasy Microarray Tissue Handbook 11/2009
19
15. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add
30–50 µl RNase-free water directly to the spin column membrane. Close the lid gently. To elute the RNA, centrifuge for 1 min at
ⱖ
8000 x g (
ⱖ
10,000 rpm).
16. Repeat step 15 using another volume of RNase-free water, or using the eluate from step 15 (if high RNA concentration is required). Reuse the collection tube from step 15.
If using the eluate from step 15, the RNA yield will be 15–30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.
20
RNeasy Microarray Tissue Handbook 11/2009
Troubleshooting Guide
This troubleshooting guide may be helpful in solving any problems that may arise. For more information, see also the Frequently Asked Questions page at our Technical
Support Center: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN
Technical Services are always happy to answer any questions you may have about either the information and protocol in this handbook or sample and assay technologies
(for contact information, see back cover or visit www.qiagen.com).
Comments and suggestions
Phases do not separate completely
a) No chloroform added or chloroform not pure b) Homogenate not sufficiently mixed before centrifugation c) Organic solvents in samples used for RNA purification
Make sure to add chloroform that does not contain isoamyl alcohol or other additives.
After addition of chloroform (step 5), the homogenate must be vigorously shaken. If the phases are not well separated, shake the tube vigorously for at least 15 s, and repeat the incubation and centrifugation in steps 6 and 7.
Make sure that the starting sample does not contain organic solvents (e.g., ethanol,
DMSO), strong buffers, or alkaline reagents.
These can interfere with the phase separation.
Clogged RNeasy spin column
a) Inefficient disruption and/or homogenization b) Too much starting material
See “Disrupting and homogenizing starting material” (page 13) for details on disruption and homogenization methods.
Increase g-force and centrifugation time if necessary.
In subsequent preparations, reduce the amount of starting material (see page 11 and protocol, page 15) and/or increase the homogenization time.
In subsequent preparations, reduce the amount of starting material. It is essential to use the correct amount of starting material
(see page 11 and protocol, page 15).
RNeasy Microarray Tissue Handbook 11/2009
21
Comments and suggestions
c) Centrifugation temperature too low Except for phase separation (step 7), all centrifugation steps should be performed at
15–25°C. Some centrifuges may cool to below 20°C even when set at 20°C. This can cause formation of precipitates that can clog the RNeasy spin column. If this happens, set the centrifugation temperature to 25°C. Warm the ethanol-containing lysate to 37°C before transferring to the
RNeasy spin column.
Low RNA yield
a) Insufficient disruption and homogenization b) Too much starting material
See “Disrupting and homogenizing starting material” (page 13) for details on disruption and homogenization methods.
In subsequent preparations, reduce the amount of starting material. It is essential to use the correct amount of starting material
(see page 11 and protocol, page 15).
c) RNA still bound to RNeasy spin column membrane d) Centrifugation temperature too low Except for phase separation (step 7), all centrifugation steps should be performed at
15–25°C. Some centrifuges may cool to below 20°C even when set at 20°C. This can cause formation of precipitates that can clog the RNeasy spin column. If this happens, set the centrifugation temperature to 25°C. Warm the ethanol-containing lysate to 37°C before transferring to the
RNeasy spin column.
Low or no recovery of RNA
Repeat RNA elution, but incubate the
RNeasy spin column on the benchtop for
10 min with RNase-free water before centrifuging.
RNase-free water incorrectly dispensed
Add RNase-free water to the center of the
RNeasy spin column membrane to ensure that the membrane is completely covered.
22
RNeasy Microarray Tissue Handbook 11/2009
Comments and suggestions
Low A
260
/ A
280 value
a) Not enough QIAzol Lysis Reagent In subsequent preparations, reduce the used for homogenization amount of starting material and/or increase the volume of QIAzol Lysis Reagent and the homogenization time.
b) Sample not incubated for 5 min after homogenization c) Water used to dilute RNA for
A
260
/ A
280 measurement
Place the sample at room temperature
(15–25°C) for 5 min after homogenization, as indicated in the protocol (step 4). This step is important to promote dissociation of nucleoprotein complexes.
Use 10 mM Tris·Cl, pH 7.5, not RNase-free water, to dilute the sample before measuring purity (see Appendix B, page 27).
RNA degraded
a) Inappropriate handling of starting material b) RNase contamination
For frozen tissue samples, ensure that they were flash-frozen immediately in liquid nitrogen and properly stored at –70°C.
Perform the RNeasy procedure quickly, especially the first few steps.
See Appendix A (page 25) and “Handling and storing starting material” (page 12).
Although all RNeasy buffers have been tested and are guaranteed RNase-free,
RNases can be introduced during use. Be certain not to introduce any RNases during the RNeasy procedure or later handling. See
Appendix A (page 25) for general remarks on handling RNA.
Do not put RNA samples into a vacuum dryer that has been used in DNA preparation where RNases may have been used.
DNA contamination in downstream experiments
a) Phase separation performed at too high a temperature
The phase separation (step 7) should be performed at 4°C to allow optimal phase separation and removal of genomic DNA from the aqueous phase. Make sure that the centrifuge does not heat above 10°C during the centrifugation.
RNeasy Microarray Tissue Handbook 11/2009
23
Comments and suggestions
b) Interphase contamination of aqueous phase
Contamination of the aqueous phase with the interphase results in an increased DNA content in the RNA eluate. Make sure to transfer the aqueous phase without interphase contamination.
c) Not enough QIAzol Lysis Reagent In subsequent preparations, reduce the used for homogenization amount of starting material and/or increase the volume of QIAzol Lysis Reagent and the homogenization time.
d) e)
Organic solvents in samples used for RNA purification
No treatment
Make sure that the starting sample does not contain organic solvents (e.g., ethanol,
DMSO), strong buffers, or alkaline reagents.
These can interfere with the phase separation.
Perform optional on-column DNase digestion using the RNase-Free DNase Set
(Appendix C, page 30) at step 10 of the protocol.
RNA does not perform well in downstream experiments
a) Salt carryover during elution b) Ethanol carryover
Ensure that Buffer RPE is at 20–30°C.
During the second wash with Buffer RPE (step
13), be sure to dry the RNeasy spin column membrane by centrifuging at ⱖ
8000 x g
( ⱖ
10,000 rpm) for 2 min at 15–25°C. After centrifugation, carefully remove the column from the collection tube so that the column does not contact the flow-through.
Otherwise, carryover of ethanol will occur.
To eliminate any chance of possible ethanol carryover, place the RNeasy Mini spin column in a new 2 ml collection tube and perform the optional 1-min centrifugation step as described in step 14 of the protocol.
24
RNeasy Microarray Tissue Handbook 11/2009
Appendix A: General Remarks on Handling RNA
Handling RNA
Ribonucleases (RNases) are very stable and active enzymes that generally do not require cofactors to function. Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA, do not use any plasticware or glassware without first eliminating possible RNase contamination. Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure. In order to create and maintain an RNase-free environment, the following precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA.
General handling
Proper microbiological, aseptic technique should always be used when working with
RNA. Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination. Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment. Change gloves frequently and keep tubes closed whenever possible. Keep purified RNA on ice when aliquots are pipetted for downstream applications.
To remove RNase contamination from bench surfaces, nondisposable plasticware, and laboratory equipment (e.g., pipets and electrophoresis tanks), use of RNaseKiller
(cat. no 2500080) from 5 PRIME (www.5prime.com) is recommended. RNase contamination can alternatively be removed using general laboratory reagents. To decontaminate plasticware, rinse with 0.1 M NaOH, 1 mM EDTA* followed by RNasefree water (see ”Solutions”, page 26), or rinse with chloroform* if the plasticware is chloroform-resistant. To decontaminate electrophoresis tanks, clean with detergent
(e.g., 0.5% SDS),* rinse with RNase-free water, rinse with ethanol (if the tanks are ethanol-resistant), and allow to dry.
Disposable plasticware
The use of sterile, disposable polypropylene tubes is recommended throughout the procedure. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases.
* 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.
RNeasy Microarray Tissue Handbook 11/2009
25
Glassware
Glassware should be treated before use to ensure that it is RNase-free. Glassware used for RNA work should be cleaned with a detergent,* thoroughly rinsed, and oven baked at 240°C for at least 4 hours (overnight, if more convenient) before use. Autoclaving alone will not fully inactivate many RNases. Alternatively, glassware can be treated with
DEPC* (diethyl pyrocarbonate), as described in “Solutions” below.
Solutions
Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong, but not absolute, inhibitor of RNases. It is commonly used at a concentration of
0.1% to inactivate RNases on glass or plasticware or to create RNase-free solutions and water. DEPC inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution. Let the solution incubate for 12 hours at 37°C. Autoclave for 15 minutes to remove any trace of DEPC. DEPC will react with primary amines and cannot be used directly to treat
Tris* buffers. DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO
2
. When preparing Tris buffers, treat water with DEPC first, and then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation. Carbethoxylated RNA is translated with very low efficiency in cell-free systems. However, its ability to form DNA:RNA or
RNA:RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified. Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100°C for 15 minutes.
Note: RNeasy buffers are guaranteed RNase-free without using DEPC treatment and are therefore free of any DEPC contamination.
* 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.
26
RNeasy Microarray Tissue Handbook 11/2009
Appendix B: Storage, Quantification, and
Determination of Quality of RNA
Storage of RNA
Purified RNA may be stored at –20°C or –70°C in RNase-free water. Under these conditions, no degradation of RNA is detectable after 1 year.
Quantification of RNA
The concentration of RNA should be determined by measuring the absorbance at
260 nm (
A
260
) in a spectrophotometer (see “Spectrophotometric quantification of RNA” below). For small amounts of RNA, however, it may be difficult to determine amounts photometrically. Small amounts of RNA can be quantified using the QIAxcel ® system
(www.qiagen.com/QIAxcel) or Agilent fluorometric quantification.
® 2100 Bioanalyzer, quantitative RT-PCR, or
Spectrophotometric quantification of RNA
To ensure significance, A
260 readings should be greater than 0.15. An absorbance of
1 unit at 260 nm corresponds to 44 µg of RNA per ml ( A
260
=1
→ 44 µg/ml). This relation is valid only for measurements at a neutral pH. Therefore, if it is necessary to dilute the RNA sample, this should be done in a buffer with neutral pH.* As discussed below (see “Purity of RNA”, page 28), the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity.
When measuring RNA samples, be certain that cuvettes are RNase-free, especially if the RNA is to be recovered after spectrophotometry. This can be accomplished by washing cuvettes with 0.1 M NaOH, 1 mM EDTA,* followed by washing with RNasefree water (see “Solutions”, page 26). Use the buffer in which the RNA is diluted to zero the spectrophotometer. An example of the calculation involved in RNA quantification is shown below:
Volume of RNA sample = 100 µl
Dilution = 10 µl of RNA sample + 490 µl of 10 mM Tris·Cl,* pH 7.0
(1/50 dilution)
Measure absorbance of diluted sample in a 1 ml cuvette (RNase-free)
A
260
= 0.2
* 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.
RNeasy Microarray Tissue Handbook 11/2009
27
Concentration of RNA sample = 44 µg/ml x A
260 x dilution factor
= 44 µg/ml x 0.2 x 50
= 440 µg/ml
Total amount = concentration x volume in milliliters
= 440 µg/ml x 0.1 ml
= 44 µg of RNA
Purity of RNA
The ratio of the readings at 260 nm and 280 nm ( A
260
/ A
280
) provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum, such as protein. However, the A
260
/ A
280 ratio is influenced considerably by pH. Since water is not buffered, the pH and the resulting A
260
/ A
280 ratio can vary greatly. Lower pH results in a lower A
260
/ A
280 ratio and reduced sensitivity to protein contamination.* For accurate values, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Pure
RNA has an A
260
/ A
280 ratio of 1.9–2.1
† in 10 mM Tris·Cl, pH 7.5. Always be sure to calibrate the spectrophotometer with the same solution used for dilution.
For determination of RNA concentration, however, we recommend dilution of the sample in a buffer with neutral pH since the relationship between absorbance and concentration ( A
260 reading of 1 = 44 µg/ml RNA) is based on an extinction coefficient calculated for RNA at neutral pH (see “Spectrophotometric quantification of RNA”, page 27).
DNA contamination
No currently available purification method can guarantee that RNA is completely free of DNA, even when it is not visible on an agarose gel. While RNeasy Kits will remove the vast majority of cellular DNA, trace amounts may still remain, depending on the amount and nature of the sample.
If desired, DNase digestion of the isolated RNA with RNase-free DNase can be performed to remove residual DNA. A protocol for optional on-column DNase digestion using the RNase-Free DNase Set is provided in Appendix C (page 30). The DNase is efficiently washed away in subsequent wash steps.
†
* Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.
Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with some spectrophotometers.
28
RNeasy Microarray Tissue Handbook 11/2009
Integrity of RNA
The integrity and size distribution of total RNA purified with the RNeasy Microarray
Tissue Mini Kit can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining* or by using the QIAxcel system or Agilent 2100 Bioanalyzer. The respective ribosomal RNAs should appear as sharp bands or peaks. The apparent ratio of 28S rRNA to 18S rRNA should be approximately 2:1. If the ribosomal bands or peaks of a specific sample are not sharp, but appear as a smear towards smaller sized
RNAs, it is likely that the sample suffered major degradation either before or during
RNA purification.
The Agilent 2100 Bioanalyzer also provides an RNA Integrity Number (RIN) as a useful measure of RNA integrity. Ideally, the RIN should be close to 10, but in many cases
(particularly with tissue samples), RNA quality is greatly influenced by how well the original sample was preserved.
* 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.
RNeasy Microarray Tissue Handbook 11/2009
29
Appendix C: Optional On-Column DNase Digestion with the RNase-Free DNase Set
The RNase-Free DNase Set (cat. no. 79254) provides efficient on-column digestion of
DNA during RNA purification. The DNase is efficiently removed in subsequent wash steps.
Note: Standard DNase buffers are not compatible with on-column DNase digestion.
Use of other buffers may affect the binding of RNA to the RNeasy membrane, reducing
RNA yield and integrity.
Lysis and homogenization of the sample and binding of RNA to the RNeasy membrane are performed according to the standard protocol. After washing with a reduced volume of Buffer RW1, the RNA is treated with DNase I while bound to the RNeasy membrane. The DNase I is removed by a second wash with Buffer RW1. Washing with
Buffer RPE and elution of RNA are then performed according to the standard protocol.
Important points before starting
■
Generally, DNase digestion is not required since integrated QIAzol and RNeasy technologies efficiently remove most of the DNA without DNase treatment.
■
Do not vortex the reconstituted DNase I. DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube.
Things to do before starting
■
Prepare DNase I stock solution before using the RNase-Free DNase Set for the first time. Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNasefree water provided. To avoid loss of DNase I, do not open the vial. Inject RNasefree water into the vial using an RNase-free needle and syringe. Mix gently by inverting the vial. Do not vortex.
■
For long-term storage of DNase I, remove the stock solution from the glass vial, divide it into single-use aliquots, and store at –20°C for up to 9 months. Thawed aliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots after thawing.
Procedure
Prepare and load samples onto the RNeasy spin column as indicated in steps 1–10 of the protocol on pages 15–19. Instead of performing step 11, follow steps C1–C4 below.
30
RNeasy Microarray Tissue Handbook 11/2009
C1. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at
ⱖ
8000 x g (
ⱖ
10,000 rpm) to wash the membrane. Discard the flow-through.*
Reuse the collection tube in step C4.
C2. Add 10 µl DNase I stock solution (see above) to 70 µl Buffer RDD. Mix by gently inverting the tube, and centrifuge briefly to collect residual liquid from the sides of the tube.
Buffer RDD is supplied with the RNase-Free DNase Set.
Note: DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube. Do not vortex.
C3. Add the DNase I incubation mix (80 µl) directly to the RNeasy spin column membrane, and place on the benchtop (20–30°C) for 15 min.
Note: Be sure to add the DNase I incubation mix directly to the RNeasy spin column membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the O-ring of the spin column.
C4. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at
ⱖ
8000 x g (
ⱖ
10,000 rpm). Discard the flow-through.*
Continue with step 12 of the protocol on page 19.
References
QIAGEN maintains a large, up-to-date online database of scientific publications utilizing QIAGEN products. Comprehensive search options allow you to find the articles you need, either by a simple keyword search or by specifying the application, research area, title, etc.
For a complete list of references, visit the QIAGEN Reference Database online at www.qiagen.com/RefDB/search.asp or contact QIAGEN Technical Services or your local distributor.
* Flow-through contains Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety information.
RNeasy Microarray Tissue Handbook 11/2009
31
Ordering Information
Product Contents Cat. no.
RNeasy Microarray
Tissue Mini Kit (50)
50 RNeasy Mini Spin Columns,
Collection Tubes, QIAzol Lysis
Reagent, RNase-Free Reagents and
Buffers
73304
Accessories
Allprotect Tissue
Reagent (100 ml)
RNA later RNA Stabilization
Reagent (50 ml)
RNA
Reagent (250 ml)
RNA later RNA Stabilization later TissueProtect
Tubes (50 x 1.5 ml)
For stabilization of DNA, RNA, and protein in 50 x 200 mg tissue samples: 100 ml Allprotect Tissue
Reagent, Allprotect Reagent Pump
For stabilization of RNA in
25 x 200 mg tissue samples:
50 ml RNA later RNA Stabilization
Reagent
For stabilization of RNA in
125 x 200 mg tissue samples:
250 ml RNA later RNA Stabilization
Reagent
For stabilization of RNA in
50 x 150 mg tissue samples:
50 screw-top tubes containing
1.5 ml RNA later RNA Stabilization
Reagent each
76405
76104
76106
76154
RNA later TissueProtect
Tubes (20 x 5 ml)
For stabilization of RNA in
20 x 500 mg tissue samples:
20 screw-top tubes containing
5 ml RNA later RNA Stabilization
Reagent each
QIAzol Lysis Reagent (200 ml) 200 ml QIAzol Lysis Reagent
TissueRuptor
76163
79306
Handheld rotor–stator homogenizer, 9001271*
5 TissueRuptor Disposable Probes 9001272 †
9001273 ‡
9001274 §
§
‡
†
* 120 V, 60 Hz (for North America and Japan)
235 V, 50/60 Hz (for Europe, excluding UK and Ireland)
235 V, 50/60 Hz (for UK and Ireland)
235 V, 50/60 Hz (for Australia)
32
RNeasy Microarray Tissue Handbook 11/2009
Ordering Information
Product Contents Cat. no.
TissueRuptor Disposable
Probes (25)
TissueLyser LT
TissueLyser LT Adapter,
12-Tube
Sample Tubes RB (2 ml)
TissueLyser II
TissueLyser Adapter
Set 2 x 24
25 nonsterile plastic disposable probes for use with the TissueRuptor
990890
Compact bead mill, 100–240 V AC, 85600
50–60 Hz; requires the TissueLyser
LT Adapter, 12-Tube (available separately)
Adapter for disruption of up to
12 samples in 2 ml microcentrifuge tubes on the TissueLyser LT
69980
1000 safe-lock microcentrifuge tubes 990381
(2 ml) for use with the TissueLyser LT
85300 Bead mill, 100–120/220–240 V,
50/60 Hz; requires the TissueLyser
Adapter Set 2 x 24 or TissueLyser
Adapter Set 2 x 96 (available separately)
2 sets of Adapter Plates and 2 racks for use with 2 ml microcentrifuge tubes on the TissueLyser II
69982
TissueLyser Single-Bead
Dispenser, 5 mm
Stainless Steel Beads,
5 mm (200)
Stainless Steel Beads,
7 mm (200)
RNase-Free DNase Set (50)
Collection Tubes (2 ml)
TissueLyser Adapter
Set 2 x 96
For dispensing individual beads
(5 mm diameter)
69965
Stainless Steel Beads, suitable for use 69989 with TissueLyser systems
Stainless Steel Beads, suitable for use 69990 with TissueLyser systems
79254 For 50 RNA minipreps: 1500 units
RNase-Free DNase I, RNase-Free
Buffer RDD, and RNase-Free Water
1000 x 2 ml Collection Tubes
2 sets of Adapter Plates for use with
Collection Microtubes (racked) on the TissueLyser II
19201
69984
Collection Microtubes (racked) Nonsterile polypropylene tubes
(1.2 ml), 960 in racks of 96
19560
RNeasy Microarray Tissue Handbook 11/2009
33
Ordering Information
Product Contents Cat. no.
Collection Microtube
Caps (120 x 8)
Nonsterile polypropylene caps for collection microtubes (1.2 ml),
960 in strips of 8
Related products
RNeasy 96 Universal Tissue Kit — for high-throughput RNA purification from any type of animal tissue
RNeasy 96 Universal
Tissue Kit (4)*
†
For 4 x 96 total RNA preps:
4 RNeasy 96 Plates, Collection
Microtubes, Elution Microtubes CL,
Caps, S-Blocks, AirPore Tape Sheets,
QIAzol Lysis Reagent, RNase-Free
Reagents and Buffers
19566
74881
miRNeasy Kits — for purification of microRNA and total RNA from a wide range of animal tissues and cells
miRNeasy Mini Kit (50) For 50 preps: 50 RNeasy Mini Spin 217004
Columns, Collection Tubes (1.5 ml and 2 ml), QIAzol Lysis Reagent,
RNase-Free Reagents and Buffers miRNeasy 96 Kit (4) † For 4 x 96 preps: 4 RNeasy 96 plates, Collection Microtubes
(racked), Elution Microtubes CL,
Caps, S-Blocks, AirPore Tape
Sheets, QIAzol Lysis Reagent,
RNase-Free Reagents and Buffers
217061
For up-to-date licensing information and product-specific disclaimers, see the respective
QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.
Visit www.qiagen.com/geneXpression to find out more about standardized solutions for gene expression analysis — from RNA preparation to real-time RT-PCR
†
* Larger kit size available; see www.qiagen.com/RNA.
Requires use of the QIAGEN 96-Well-Plate Centrifugation System with refrigeration capability (TissueLyser
II recommended for disruption and homogenization; QIAvac 96 optional).
34
RNeasy Microarray Tissue Handbook 11/2009
Trademarks: QIAGEN
®
, QIAcube
®
, QIAxcel
®
, QIAzol
®
, RNeasy
®
, TissueRuptor
®
(QIAGEN Group); Agilent
®
(Agilent Technologies, Inc.). “RNA later
®
” is a trademark of
AMBION, Inc., Austin, Texas and is covered by various U.S. and foreign patents.
QIAzol Lysis Reagent is a subject of US Patent No. 5,346,994 and foreign equivalents.
Limited License Agreement
Use of this product signifies the agreement of any purchaser or user of the RNeasy Microarray Tissue Mini Kit to the following terms:
1.
The RNeasy Microarray Tissue Mini Kit may be used solely in accordance with the RNeasy Microarray Tissue Handbook and for use with components contained in the Kit only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the
RNeasy Microarray Tissue Handbook and additional protocols available at www.qiagen.com.
2.
Other than expressly stated licenses, QIAGEN makes no warranty that this Kit and/or its use(s) do not infringe the rights of third-parties.
3.
This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4.
QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5.
The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and/or its components.
For updated license terms, see www.qiagen.com.
© 2009 QIAGEN, all rights reserved.
www.qiagen.com
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Fax 65-67785177
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Spain
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Sweden
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Switzerland
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Orders 055-254-22-11
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UK
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Technical 800-DNA-PREP (800-362-7737)
1057659 11/2009
Sample & Assay Technologies

Public link updated
The public link to your chat has been updated.
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Key features
- Fast and convenient purification
- High-quality RNA for microarray analysis
- Efficient lysis of various tissues
- No phenol contamination
- Optional DNase digestion
- Automated purification with QIAcube