QIAzol™ Lysis Reagent - Applied Genomics Technology Center
Contents
1. i 2 ia N 7 D mel B oe 7 D H a EJ 4 7 2 d E 10 11 RNA in harvested tissues is not protected until the tissues are treated with RNAlater 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 Place the tip of the TissueRuptor disposable probe into the GlAzol Lysis Reagent and operate the TissueRuptor at full speed until the tissue lysate is uniformly homogeneous usually 20 40 s Note To avoid damage to the TissueRuptor and disposable probe during operation make sure the tip of the probe remains submerged in the QIAzol lysis Reagent Note Incomplete homogenization leads to significantly reduced RNA yields Homogenization with the TissueRuptor or Tissuelyser generally results in higher RNA yields than with other methods Optional For samples containing a relatively high content of fot proteins polysaccharides or extracellular material centrifuge the homogenate at 12 000 x g for 10 min at 4 C to remove insoluble material Carefully transfer the supernatant to a new tube and proceed to step 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 Add 0 2 ml chloroform per 1 ml QIAzol Lysis Reagent pipetted in2. 362 7737 QIAGEN ences Sample amp Assay Technologies
3. not dry the RNA using a vacuum lt n N 2I 2 9 E xw Q co vC 9 gt oN J 9 c E 14 Redissolve the RNA in an appropriate volume of RNase free water Clean up the RNA using the RNeasy MinElute Cleanup Kit or RNeasy Mini Midi or Maxi Kit We recommend RNA cleanup to remove contaminating phenol The RNeasy MinElute Cleanup Kit and RNeasy Mini Midi and Maxi Kits allow cleanup of up to 45 pg 100 pg 1 mg and 6 mg total RNA respectively For details refer to the RNA cleanup protocol in the handbook supplied with these kits GlAzol Handbook 01 2009 13 Protocol Lysis and Homogenization of Fatty Tissues Using the Tissuelyser This protocol is intended for fatty tissues but can also be used with all other types of tissue Important points before starting E Ensure that you are familiar with operating the Tissuelyser by referring to the operating instructions and Tissuelyser Handbook For other bead mills refer to suppliers guidelines E If using GlAzol lysis Reagent for the first time read Important Notes page 9 ra ES o E 1 zn E Fresh frozen or RNAlater Allprotect stabilized tissues can be used If freezing tissues flash freeze in liquid nitrogen and immediately transfer to 70 C where they can be stored for several months Do not allow tissues to thaw during weighing or handling prior to disruption in GlAzol Lysis Reagent Homogenized tissue lysates from step 3 can also be
4. not reuse the stainless steel beads Note Incomplete homogenization leads to significantly reduced RNA yields Homogenization with the Tissuelyser or TissueRuptor generally results in higher RNA yields than with other methods Optional For samples containing a relatively high content of fat proteins polysaccharides or extracellular material centrifuge the homogenate at 12 000 x g for 10 min at 4 C to remove insoluble material Carefully transfer the supernatant to a new tube and proceed to step 4 4 Place the tubes containing the homogenates on the benchtop at room temperature 15 25 C for 5 min This step promotes dissociation of nucleoprotein complexes Centrifuge the rack of collection microtubes at 6000 x g for 1 min at 15 25 C to collect residual liquid from the caps of the tubes 5 Add 0 2 ml chloroform per 1 ml GlAzol Lysis Reagent pipetted in step 3 Securely cap the tubes containing the homogenates and shake vigorously for 15 s Thorough mixing is important for subsequent phase separation 6 Place the tubes containing the homogenates on the benchtop at room temperature for 2 3 min GlAzol Handbook 01 2009 15 2 o EN 5 P d B om wn Ly ra ES onl o E 1 4 E Centrifuge at A 12 000 x g or 6 6000 x g for 15 min at 4 C After centrifugation the sample separates into 3 phases an upper colorless aqueous phase containing RNA a white interphase and a lower red organi
5. step 1 Securely cap the tube containing the homogenate and shake it vigorously for 15 s Thorough mixing is important for subsequent phase separation Place the tube containing the homogenate on the benchtop at room temperature for 2 3 min Centrifuge at 12 000 x g for 15 min at 4C 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 is approximately 60 of the volume of the QIAzol Lysis Reagent pipetted in step 1 Transfer the upper aqueous phase to a new tube Add 0 5 ml isopropanol per 1 ml GlAzol Lysis Reagent pipetted in step 1 Mix thoroughly by vortexing Place the tube on the benchtop at room temperature for 10 min Centrifuge at 12 000 x g for 10 min at 4 C Carefully aspirate and discard the supernatant The RNA pellet is often visible as a gel like or white pellet at the bottom of the tube GlAzol Handbook 01 2009 12 Add at least 1 ml of 75 ethanol per 1 ml GlAzol Lysis Reagent pipetted in step 1 Centrifuge at 7500 x g for 5 min at 4 C If the RNA pellet floats or sticks to the side of the tube bring it to the bottom of the tube by centrifuging at 12 000 x g for 5 min at 4 C 13 Remove the supernatant completely and briefly air dry the RNA pellet Do
6. stored at 70 C for at least 1 month 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 2 o EN 5 P d cm wn Ey B6 Inthe procedure below A refers to use of the Tissuelyser Adapter Set 2 x 24 with 5 mm diameter stainless steel beads for 100 mg tissue and refers to use of the Tissuelyser Adapter Set 2 x 96 with 5 mm diameter stainless steel beads for 75 mg tissue Procedure 1 Add A one stainless steel bead 5 mm mean diameter per 2 ml microcentrifuge tube or 6 one stainless steel bead 5 mm mean diameter per collection microtube Place the tubes on dry ice The tubes do not need to be placed on dry ice if the tissue samples are stabilized in RNAlater or Allprotect Reagent 2 Excise the tissue samples from the animal or remove them from storage Determine the amount of each tissue Place each tissue into a tube from step 1 Weighing tissue is the most accurate way to determine the amount If the tissue samples were stored in RNAlater or Allprotect Reagent remove them from the reagent using forceps and be sure to remove any excess reagent or crystals that may have formed RNAlater RNA Stabilization Reagent cannot be used with adipose tissue due to the high abundance of fat but can be used with other fatty tissues such as brain 14 GlAzol Handbook 0
7. 00 x g For stabilization of RNA in tissues see page 9 RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent see ordering information page 20 or liquid nitrogen Equipment for tissue disruption and homogenization see page 9 We recommend one of the following B _ TissueRuptor with TissueRuptor Disposable Probes W Tissuelyser with the following accessories Tissuelyser Adapter Set 2 x 24 Tissuelyser Single Bead Dispenser 5 mm Stainless Steel Beads 5 mm W Tissuelyser with the following accessories Tissuelyser Adapter Set 2 x 96 Tissuelyser 5 mm Bead Dispenser 96 well Stainless Steel Beads 5 mm Collection Microtubes racked Collection Microtube Caps For ordering information see page 20 Kit for RNA cleanup after the GlAzol procedure m RNeasy MinElute Cleanup Kit for cleanup of up to 45 pg RNA m RNeasy Mini Kit for cleanup of up to 100 pg RNA m RNeasy Midi Kit for cleanup of up to 1 mg RNA m RNeasy Maxi Kit for cleanup of up to 6 mg RNA For ordering information see page 21 GlAzol Handbook 01 2009 Important Notes Handling and storing starting material RNA in harvested tissue is not protected until the sample is treated with RNAlater 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 immed
8. 1 2009 RNA in harvested tissues is not protected until the tissues are treated with RNAlater 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 Remove the tubes from the dry ice Add GlAzol Lysis Reagent to each tube 1 ml GlAzol Lysis Reagent per 100 mg tissue is required The volume of tissue should not exceed 10 of the volume of GlAzol Lysis Reagent A Place the tubes in the Tissuelyser Adapter Set 2 x 24 Operate the Tissuelyser for 2 min at 20 Hz Disassemble the adapter set rotate the rack of tubes so that the tubes nearest to the Tissuelyser are now outermost and reassemble the adapter set Operate the TissueLyser for another 2 min at 20 Hz lt a wn Am n oO eg 2g S 8 x a E Close the collection microtubes using the collection microtube caps Place the rack of tubes in the Tissuelyser Adapter Set 2 x 96 Operate the Tissuelyser for 2 min at 20 Hz Disassemble the adapter set rotate the rack of tubes so that the tubes nearest to the TissueLyser are now outermost and reassemble the adapter set Operate the TissueLyser for another 2 min at 20 Hz The time and frequency depend on the tissue being processed and can be increased until the tissue is completely homogenized e g up to 2 x 5 min at 25 Hz Rearranging the tubes allows even homogenization Do
9. January 2009 GlAzol Handbook For efficient lysis of fatty tissues and all other types of tissue before RNA purification QIAGEN GIAGEN Sample and Assay Technologies GIAGEN 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 qgiagen com Contents Kit Contents Shipping and Storage Quality Control Product Use Limitations Product Warranty and Satisfaction Guarantee Technical Assistance Safety Information Introduction Equipment and Reagents to Be Supplied by User Important Notes Handling and storing starting material Disrupting and homogenizing starting material Protocols E Lysis and Homogenization of Fatty Tissues Using the TissueRuptor E Lysis and Homogenization of Fatty Tissues Using the TissueLyser Troubleshooting Guide Ordering Information GlAzol Handbook 01 2009 000 NO aa i BR SS Kit Contents GlAzol Lysis Reagent 200 ml Catalog no 79306 GlAzol lysis Reagent 200 ml Handbook 1 Contains a guanidine salt Not compatib
10. al 800 988 0325 800 988 0327 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 02 33430 420 Fax 02 33430 426 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 1544 7145 Fax 1544 7146 Technical 1544 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 65 67775366 Fax 65 67785177 Technical 65 67775366 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 e e e e UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 e e USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800
11. c 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 is approximately 60 of the volume of the QIAzol Lysis Reagent pipetted in step 3 Transfer the upper aqueous phase to new tubes Add 0 5 ml isopropanol per 1 ml GlAzol Lysis Reagent pipetted in step 3 Mix thoroughly by vortexing Place the tubes on the benchtop at room temperature for 10 min Centrifuge at 12 000 x g for 10 min at 4 C Carefully aspirate and discard the supernatants The RNA pellet is often visible as a geHike or white pellet at the bottom of the tube Add at least 1 ml of 75 ethanol per 1 ml QIAzol Lysis Reagent pipetted in step 3 Centrifuge at 7500 x g for 5 min at 4 C If the RNA pellet floats or sticks to the side of the tube bring it to the bottom of the tube by centrifuging at 12 000 x g for 5 min at 4 C Remove the supernatants completely and briefly air dry the RNA pellets Do not dry the RNA using a vacuum Redissolve the RNA in an appropriate volume of RNase free water Clean up the RNA using the RNeasy Micro Mini Midi or Maxi Kit We recommend RNA cleanup to remove contaminating phenol The RNeasy MinElute Cleanup Kit and RNeasy Mini Midi and Maxi Kits allow cleanup of up to 45 pg 100 pg 1 mg and 6 mg total RNA respectively For details refer to the RNA cleanup protocol in the handbook supplied with these kits G
12. except as described in the GlAzol Handbook and additional protocols available at www qiagen com 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 This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated 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 2002 2009 QIAGEN all rights reserved www qiagen com Australia Orders 03 9840 9800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 0086 2 1 3865 3865 Fax 0086 21 3865 3965 Technic
13. fugation Contamination of the aqueous phase with the interphase results in an increased DNA content in the purified RNA Make sure to transfer the aqueous phase without interphase contamination In subsequent preparations reduce the amount of starting material and or increase the volume of QIAzol Lysis Reagent and the homogenization time Treat the RNA sample with DNase and then clean up the RNA using an RNeasy Kit Alternatively carry out RNA cleanup and on column DNase digestion using an RNeasy Kit For details see the handbook supplied with the RNeasy Kit Ordering Information Product Contents Cat no GlAzol Lysis Reagent 200 ml 200 ml GlAzol Lysis Reagent 79306 Accessories Allprotect Tissue For stabilization of DNA RNA and 76405 Reagent 100 ml protein in 50 x 200 mg tissue samples 100 ml Allprotect Tissue Reagent Allprotect Reagent Pump RNAlater RNA Stabilization For stabilization of RNA in 76104 Reagent 50 ml 25 x 200 mg tissue samples 50 ml RNAlater RNA Stabilization Reagent RNAlater RNA Stabilization For stabilization of RNA in 76106 Reagent 250 ml 125 x 200 mg tissue samples 250 ml RNAlater RNA Stabilization Reagent RNAlater TissueProtect For stabilization of RNA in 76154 Tubes 50 x 1 5 ml 50 x 150 mg tissue samples 50 screw top tubes containing 1 5 ml RNAlater RNA Stabilization Reagent each RNAlater TissueProtect Tubes For stabilization of RNA in 76163 20 x 5 ml 20 x 500 mg tissue
14. gh molecular weight genomic DNA and other high molecular weight cellular components to create a homogeneous lysate Incomplete homogenization results in 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 the Tissuelyser for processing multiple samples simultaneously Disruption and homogenization with the TissueRuptor or Tissuelyser generally results in higher RNA yields than with other methods GlAzol Handbook 01 2009 9 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 the TissueLyser 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 cel
15. he aqueous phase by addition of isopropanol The RNA is then pelleted and washed with ethanol before being redissolved in RNase free water We recommend cleanup of the redissolved RNA using RNeasy Kits which are based on silicoz membrane technology in order to remove any contaminating phenol The presence of residual phenol can result in overestimation of RNA yield and inhibition of enzymatic action in downstream applications The removal of contaminants by RNA cleanup also improves the stability of the RNA during storage RNA purified using QIAzol Lysis Reagent may contain residual amounts of genomic DNA that can affect sensitive downstream applications such as real time RT PCR Genomic DNA contamination in the RNA sample can be removed by adding DNase After DNase digestion the RNA sample can be cleaned up using RNeasy Kits to remove the DNase Alternatively DNase digestion can be carried out during RNA cleanup using RNeasy Kits Details about DNase digestion are provided in the handbook supplied with RNeasy Kits GlAzol Handbook 01 2009 7 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 Isopropanol 75 ethanol RNase free water Refrigerated laboratory centrifuge or microcentrifuge capable of 12 0
16. iately frozen in liquid nitrogen and stored at 70 C or immediately immersed in RNAlater RNA Stabilization Reagent at room temperature An alternative to RNAlafer 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 After disruption and homogenization in QlAzol Lysis Reagent samples can be stored at 70 C for at least 1 month 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 MH 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 MH Homogenization Homogenization is necessary to reduce the viscosity of the lysates produced by disruption Homogenization shears hi
17. itrogen and immediately transfer to 70 C where they can be stored for several months Do not allow tissues to thaw during weighing or handling prior to disruption in GlAzol Lysis Reagent Homogenized tissue lysates from step 3 can also be stored at 70 C for at least 1 month 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 Procedure 1 Add QlAzol Lysis Reagent to an appropriate vessel for disruption and homogenization and subsequent centrifugation 1 ml GlAzol Lysis Reagent per 100 mg tissue is required The volume of tissue should not exceed 10 of the volume of GlAzol Lysis Reagent Generally round bottomed tubes allow more efficient disruption and homogenization than conical bottomed tubes 2 Excise the tissue sample from the animal or remove it from storage Determine the amount of tissue and place it into the QIAzol Lysis Reagent Proceed immediately to step 3 Weighing tissue is the most accurate way to determine the amount If the tissue sample was stored in RNAlater or Allprotect Reagent remove it from the reagent using forceps and be sure to remove any excess reagent or crystals that may have formed RNAlater RNA Stabilization Reagent cannot be used with adipose tissue due to the high abundance of fat but can be used with other fatty tissues such as brain GlAzol Handbook 01 2009 11
18. lAzol Handbook 01 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 giagen 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 protocols in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Comments and suggestions Phases do not separate completely a No chloroform added or Make sure to add chloroform that does not chloroform not pure contain isoamyl alcohol or other additives b Homogenate not sufficiently After addition of chloroform step 5 the mixed before centrifugation 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 c Organic solvents in samples Make sure that the starting sample does not used for RNA purification contain organic solvents e g ethanol DMSO strong buffers or alkaline reagents These can interfere with the phase separation RNA difficult to dissolve a RNA pellet overdried Airdry RNA pellets instead of using a vacuum If necessary dissolve the RNA in a larger volume of RNase free water or allow more time for the RNA to dissolve b Too m
19. lace the sample at room temperature 15 25 C for 5 min after homogenization as indicated in the protocol This step is important to promote dissociation of nucleoprotein complexes Check for residual pellet Be sure to wash any RNA from the side of the tube especially if the tube is made of glass See also RNA difficult to dissolve above Use 10 mM Tris Cl pH 7 5 not RNase free water to dilute the sample before measuring purity 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 18 GlAzol Handbook 01 2009 Comments and suggestions RNA degraded Inappropriate handling of starting material For frozen tissue samples ensure that they were flash frozen immediately in liquid nitrogen and properly stored at 70 C Perform the QlAzol procedure quickly especially the first few steps See Handling and storing starting material page 9 DNA contamination in downstream experiments a Phase separation performed at too high a temperature b Interphase contamination of aqueous phase c Not enough QIAzol Lysis Reagent used for homogenization d No DNase treatment GlAzol Handbook 01 2009 The phase separation step 7 should be performed at 4 C Make sure that the centrifuge does not heat above 10 C during the centri
20. le with disinfectants containing bleach See page 6 for safety information Shipping and Storage GlAzol lysis Reagent is shipped at ambient temperature It can be stored at room temperature 15 25 C or at 2 8 C GlAzol lysis Reagent is stable for at least 12 months under these conditions Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of GlAzol Lysis Reagent is tested against predetermined specifications to ensure consistent product quality Product Use Limitations GlAzol lysis Reagent 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 the product for clinical use i e diagnostic prognostic therapeutic or blood banking are unknown All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines 4 GlAzol Handbook 01 2009 Product Warranty and Satisfaction Guarantee GIAGEN 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 pe
21. ll as to the researchers at GIAGEN 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 giagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com GlAzol Handbook 01 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 GIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste GlAzol Lysis Reagent contains guanidine thiocyanate which can form highly reactive compounds when combined with bleach If liquid containing this reagent 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 QIAzol Lysis Reagent GlAzol Lysis Reagent Contains phenol guanidine thiocyana
22. ls The Tissuelyser 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 The Tissuelyser 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 For guidelines on using the Tissuelyser refer to the Tissuelyser Handbook For other bead mills refer to suppliers guidelines Note Tungsten carbide beads react with GlAzol Lysis Reagent and must not be used to disrupt and homogenize tissues 10 GlAzol Handbook 01 2009 Protocol Lysis and Homogenization of Fatty Tissues Using the TissueRuptor This protocol is intended for fatty tissues but can also be used with all other types of tissue lt n N S a o S E x Q co 5 2 gt oN 9 c E Important points before starting E Ensure that you are familiar with operating the TissueRuptor by referring to the TissueRuptor User Manual and TissueRuptor Handbook For other rotor stator homogenizers refer to suppliers guidelines E If using QIAzol Lysis Reagent for the first time read Important Notes page 9 WE Fresh frozen or RNAlater Allprotect stabilized tissues can be used If freezing tissues flash freeze in liquid n
23. rform 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 giagen 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 GlAzol Lysis Reagent or QIAGEN products in general please do not hesitate to contact us GIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as we
24. s and Buffers RNeasy Midi Kit 10 10 RNeasy Midi Spin Columns 75142 Collection Tubes RNase Free Reagents and Buffers RNeasy Maxi Kit 12 12 RNeasy Maxi Spin Columns 75162 Collection Tubes RNase Free Reagents and Buffers 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 Larger kit size available see www giagen com RNA a o gt GlAzol Handbook 01 2009 21 Ordering Information Product Contents Cat no Related products RNeasy Lipid Tissue Kits for purification of total RNA from fatty tissues and all other types of tissue RNeasy Lipid Tissue 50 RNeasy Mini Spin Columns 74804 Mini Kit 50 Collection Tubes GlAzol Lysis Reagent RNase Free Reagents and Buffers RNeasy Lipid Tissue 10 RNeasy Midi Spin Columns 75842 Midi Kit 10 Collection Tubes GlAzol Lysis Reagent RNase Free Reagents and Buffers RNeasy Fibrous Tissue Kits for purification of total RNA from fiber rich tissues RNeasy Fibrous Tissue 50 RNeasy Mini Spin Columns 74704 Mini Kit 50 Collection Tubes Proteinase K RNase Free DNase I RNase Free Reagents and Buffers RNeasy Fibrous Tissue 10 RNeasy Midi Spin Columns 75742 Midi Kit 10 Collection Tubes Proteinase K RNase Free DNase I RNase Free Reagents and Buffers RNeasy Plus Mini Kit for purification of total RNA from cultured cells and tiss
25. samples 20 screw top tubes containing 5 ml RNAlater RNA Stabilization Reagent each Tissuelyser II Universal laboratory mixer mill 85300 Tissuelyser Adapter Set 2 x 24 2 sets of Adapter Plates and 2 racks 69982 for use with 2 ml microcentrifuge tubes on the Tissuelyser Tissuelyser Adapter Set 2 x 96 2 sets of Adapter Plates for use with 69984 Collection Microtubes racked on the Tissuelyser Collection Microtubes racked Nonsterile polypropylene tubes 19560 1 2 ml 960 in racks of 96 20 GlAzol Handbook 01 2009 Ordering Information Product Contents Cat no Collection Microtube Caps Nonsterile polypropylene caps for 19566 120 x 8 collection microtubes 1 2 ml 960 in strips of 8 Tissuelyser Single Bead For dispensing individual beads 69965 Dispenser 5 mm 5 mm diameter Tissuelyser 5 mm Bead For dispensing 96 beads 5 mm 69975 Dispenser 96 Well diameter in parallel Stainless Steel Beads Stainless Steel Beads suitable for 69989 5 mm 200 use with the TissueLyser system TissueRuptor Handheld rotor stator homogenizer 9001271 5 TissueRuptor Disposable Probes 9001272 9001273 9001274 TissueRuptor Disposable 25 nonsterile plastic disposable 990890 Probes 25 probes for use with the TissueRuptor RNeasy MinElute 50 RNeasy MinElute Spin Columns 74204 Cleanup Kit 50 Collection Tubes RNase Free Reagents and Buffers RNeasy Mini Kit 50 50 RNeasy Mini Spin Columns 74104 Collection Tubes RNase Free Reagent
26. te toxic corrosive Risk and safety phrases R23 24 25 32 34 48 20 21 22 68 S24 25 26 36 37 39 45 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 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 36 37 39 Wear suitable protective clothing gloves and eye face protection 45 In case of accident or if you feel unwell seek medical advice immediately show the label where possible 6 GlAzol Handbook 01 2009 Introduction GlAzol Lysis Reagent is a monophasic solution of phenol and guanidine thiocyanate designed to facilitate lysis of fatty tissues and inhibit RNases Tissue samples are disrupted and 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 RNA is precipitated from t
27. uch isopropanol in Be sure to wash the RNA pellet with 75 RNA pellet ethanol as described in the protocol to remove isopropanol If necessary dissolve the RNA in a larger volume of RNase free water or allow more time for the RNA to dissolve Low RNA yield a Insufficient disruption and See Disrupting and homogenizing starting homogenization material page 9 for details on disruption and homogenization methods GlAzol Handbook 01 2009 17 Comments and suggestions b RNA pellet incompletely dissolved Low A Asso value a Not enough QIAzol lysis Reagent used for homogenization b Contamination of aqueous phase with phenol c Sample not incubated for 5 min after homogenization d RNA pellet incompletely dissolved eJ Water used to dilute RNA for Ao o A280 measurement In subsequent preparations reduce the amount of starting material and or increase the volume of QIAzol Lysis Reagent and the homogenization time Check for residual pellet Be sure to wash any RNA from the side of the tube especially if the tube is made of glass See also RNA difficult to dissolve above In subsequent preparations reduce the amount of starting material and or increase the volume of QIAzol Lysis Reagent and the homogenization time When removing the aqueous phase be sure not to carry over any of the other phases After the QIAzol procedure clean up the RNA by following an RNeasy RNA cleanup protocol P
28. ues using gDNA Eliminator columns RNeasy Plus Mini Kit 50 50 RNeasy Mini Spin Columns 74134 50 gDNA Eliminator Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers Allprotect Tissue Reagent RNAlater RNA Stabilization Reagent the TissueRuptor the Tissuelyser Il and RNeasy Kits are intended for molecular biology applications These products are neither intended for the diagnosis prevention or treatment of a disease nor have they been validated for such use either alone or in combination with other products Visit www qiagen com geneXpression to find out more about standardized solutions for gene expression analysis from RNA preparation to real time RT PCR 22 GlAzol Handbook 01 2009 Trademarks QIAGEN QIAzol MinElute RNeasy TissueRuptor QIAGEN Group RNAlater is a trademark of AMBION Inc Austin Texas and is covered by various U S and foreign patents GlAzol 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 GlAzol Lysis Reagent to the following terms 1 GlAzol Lysis Reagent may be used solely in accordance with the GlAzol 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
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