PROTOCOL. MagMAX TM FFPE Total Nucleic Acid Isolation Kit

Transcription

1 PROTOCOL MagMAX TM FFPE Total Nucleic Acid Isolation Kit

2 For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. Limited Use Label License: Research Use Only The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser's activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California End Users are specifically not authorized to and are forbidden from reselling, transferring or distributing any products either as a stand alone product or as a component of another product. This product or portions thereof is manufactured and sold under license from GE Healthcare. End User Terms And Conditions Acceptance. These terms and conditions shall govern the purchase, use, transfer and acceptance of the products described in the purchase order, quotation or invoice, which products are sold and distributed by Applera Corporation to the buyer/transferee of such products (the End User ). The transfer/sale of products to the End User is expressly conditional upon End User's acceptance of these terms and conditions. Restrictions on Use. End Users are specifically not authorized to and are forbidden from reselling, transferring or distributing any products either as a stand alone product or as a component of another product. The right to use the products does not, in and of itself, include or carry any right of the End User to any GE Healthcare Bio-Sciences Corp.'s technology or intellectual property other than expressly provided herein. End Users may not attempt to reverse engineer parameters of any of GE Healthcare Bio-Sciences Corp. proprietary products or services. DISCLAIMER OF WARRANTIES. GE HEALTHCARE BIO-SCIENCES CORP. PROVIDES NO WARRANTIES TO END USER (STATUTORY OR IMPLIED), INCLUDING WITHOUT LIMITATION, AS TO PRODUCT QUALITY, CONDITION, DESCRIPTION, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, AND ALL SUCH WARRANTIES ARE HEREBY EXPRESSLY DISCLAIMED. GE HEAL THCARE BIO-SCIENCES CORP. HEREBY EXPRESSLY DISCLAIMS ANY WARRANTY REGARDING RESULTS OBTAINED THROUGH THE USE OF THE PRODUCTS, INCLUDING WITHOUT LIMITATION ANY CLAIM OF INACCURATE, INVALID OR INCOMPLETE RESULTS. Exclusion of Liability. GE Healthcare Bio-Sciences Corp. and its affiliates shall have no liability to an End User, including, without limitation, for any loss of use or profits, business interruption or any consequential, incidental, special or other indirect damages of any kind, regardless of how caused and regardless of whether an action in contract, tort, strict product liability or otherwise. TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TaqMan is a registered trademark of Roche Molecular Systems, Inc. TaqMan is a registered trademark of Roche Molecular Systems, Inc. Agilent and Bioanalyzer are trademarks of Agilent Technologies, Inc. NanoDrop is a registered trademark of NanoDrop Technologies LLC. LabChip is a registered trademark of Caliper Life Sciences Inc Life Technologies Corporation. All rights reserved. Part Number Rev. A 03/2011

3 Contents About This Guide Purpose Safety information Safety alert words SDSs PROTOCOL MagMAX FFPE Total Nucleic Acid Isolation Kit Product information Kit contents and storage Materials and equipment required but not included Factors affecting nucleic acid recovery and integrity Tissue fixation and embedding causes nucleic acid modifications The degree of nucleic acid fragmentation varies Workflow Isolate RNA Isolate DNA Prepare the wash solutions Isolate RNA Isolate RNA automatically on MagMAX Express Isolate RNA manually Isolate DNA Isolate DNA automatically on MagMAX Express Isolate DNA manually Assess Nucleic Acid Yield and Integrity Assess RNA yield Assess RNA quality Modify downstream applications Assess DNA yield Assess DNA quality Modify downstream applications Troubleshooting APPENDIX A Ordering Information, Related Documentation, and Support How to order Materials and equipment required but not included MagMAX TM FFPE Total Nucleic Acid Isolation Kit Protocol 3

4 Contents Related documentation Obtaining support APPENDIX B Safety Chemical safety General chemical safety SDSs Biological hazard safety MagMAX TM FFPE Total Nucleic Acid Isolation Kit Protocol

5 About This Guide Purpose The MagMAX TM FFPE Total Nucleic Acid Isolation Kit Protocol provides instructions and troubleshooting information for using the MagMAX TM FFPE Total Nucleic Acid Isolation Kit. Safety information Note: For general safety information, see this section and Appendix B, Safety on page 31. When a hazard symbol and hazard type appear by an instrument hazard, see the Safety Appendix for the complete alert on the instrument. Safety alert words Four safety alert words appear in Life Technologies user documentation at points in the document where you need to be aware of relevant hazards. Each alert word IMPORTANT, CAUTION, WARNING, DANGER implies a particular level of observation or action, as defined below: IMPORTANT! Indicates information that is necessary for proper instrument operation or accurate chemistry kit use. CAUTION! Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. WARNING! Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. DANGER! Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. SDSs The Safety Data Sheets (SDSs) for any chemicals supplied by Life Technologies or Invitrogen are available to you free 24 hours a day. For instructions on obtaining SDSs, see SDSs on page 32. IMPORTANT! For the SDSs of chemicals not distributed by Life Technologies or Invitrogen contact the chemical manufacturer. MagMAX TM FFPE Total Nucleic Acid Isolation Kit Protocol 5

6 About This Guide Safety information 6 MagMAX TM FFPE Total Nucleic Acid Isolation Kit Protocol

7 PROTOCOL MagMAX FFPE Total Nucleic Acid Isolation Kit Product information This protocol describes the use of the MagMAX FFPE Total Nucleic Acid Isolation Kit ( Total Kit ). The MagMAX Total Nucleic Acid Isolation Kit has been designed to extract nucleic acids from formaldehyde- or paraformaldehyde-fixed, paraffin-embedded (FFPE) tissues. Up to two 10-µm sections can be processed per isolation in either manual plate or automated format. All isolations using this kit can be completed in less than one day, and when using the high-throughput automated format, each procedure can be completed with about 30 minutes of hands-on time. Unlike previous procedures, no deparaffinization steps are required before the FFPE sections are subjected to a rigorous Protease digestion with an incubation time tailored for recovery of either RNA or DNA. Nucleic acids are purified using a rapid magnetic bead methodology that includes an on-bead nuclease treatment and are eluted into the low salt buffer provided. The recovered nucleic acids are suitable for downstream applications such as microarray analysis, quantitative real-time RT-PCR, sequencing, and mutation screening. However, sample fixation and storage typically cause nucleic acid fragmentation and modification. The procedure does not affect nucleic acid fragmentation, but some chemical modifications may remain after the procedure. As a result, certain downstream procedures may require modification for best results. Kit contents and storage The MagMAX FFPE Total Nucleic Acid Isolation Kit (Part no ) contains the following components: Kit contents Box Component Quantity Storage conditions 1 Nucleic acid binding beads 2 ml 2 8 C Digestion Buffer 15 ml Ambient RNA Digestion Additive 3 ml Ambient DNA Digestion Additive 3 ml Ambient Binding Buffer 40 ml Ambient DNase Buffer 20 ml Ambient Wash Solution 1 Concentrate 24 ml Ambient (Add 24 ml of 100% Isopropanol) Wash Solution 2 Concentrate (Add 48 ml of 100% Ethanol) 12 ml Ambient MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 7

8 Materials and equipment required but not included Kit contents Box Component Quantity Storage conditions 1 (cont.) Elution Buffer 7 ml Ambient Nuclease-free Water 18 ml Ambient Processing Plate 1 each Ambient Elution Plate 1 each Ambient 2 Protease 400 µl -15 to -25 C DNase 500 µl -15 to -25 C RNase A 120 µl -15 to -25 C Add the amount and type of alcohol listed in the protocol and on the bottle. Do not freeze beads. Materials and equipment required but not included For the SDS of any chemical not distributed by Life Technologies or Invitrogen, contact the chemical manufacturer. Before handling any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions. Item Source Ethanol Isopropanol (ACS reagent grade or equivalent) Lab equipment (pipettors, pipette tips, vortexer, microcentrifuge, tubes, and conicals) Heat Source (water bath, heat block, incubator, etc.) Centrifuge capable of spinning 96-well deep well plates Note: The procedures in the this document were developed with the Eppendorf Microtome and related supplies Titer plate shaker Note: The procedures in this document are optimized for use with the Thermo Scientific Barnstead/Lab-Line Titer Plate Shaker. MagMAX Express 96 Deep Well Magnetic Particle Processor (BR- Basic Research Customer) Major Laboratory Suppliers Part no MagMAX Express 96 Standard Plates Part no MagMAX Express 96 Deep Well Plates Part no MagMAX Express 96 Deep Well Tip Combs Part no Magnetic stand for 96-well plates: Magnetic Stand-96 (24-magnet block) 96-Well Magnetic-Ring Stand (96-ring magnet block) Part no. AM10027 Part no. AM MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

9 Factors affecting nucleic acid recovery and integrity Item Plate covers: MicroAmp Optical Adhesive Film MicroAmp Clear Adhesive Film Source Part no Part no Factors affecting nucleic acid recovery and integrity Tissue fixation and embedding causes nucleic acid modifications The degree of nucleic acid fragmentation varies During the tissue fixation process, formaldehyde reacts primarily with the nitrogen atoms of lysine, arginine, and histidine, resulting in extensive cross-linking of the protein matrix within each cell. Some of these crosslinks extend to the nucleic acid species present, primarily through the exocyclic nitrogens of adenine and cytidine. The subsequent embedding process further accelerates this chemistry by heating the sample, often in the formalin solution prior to the soak in hot paraffin. The resulting extensive web of protein-protein and protein-nucleic acid crosslinks tends to physically entrap nucleic acid species, even those that are not chemically crosslinked. Protease treatment breaks down this web and frees the nucleic acid. Even after Protease treatment, some of the nucleic acid bases will still be crosslinked to amino acids, peptides, or other biomolecules. There may also be other, unidentified, formaldehyde-induced modifications. Any sites of modification on the nucleic acid chain provide a block to polymerases using it as a template. Nucleic acid modifications also contribute to the fragmentation of RNA (and of DNA to a lesser extent) over time, even while stored in the block of paraffin. For this reason, the average size of RNA obtained from FFPE samples can vary widely. One key factor that contributes to RNA quality appears to be removal of all excess formaldehyde during the original embedding procedure. Another is storage of blocks without cut faces, which prevents damage from atmospheric oxygen, water and other environmental factors such as light and infestation (fungus, insects, etc.). With FFPE samples stored a relatively short time, broad peaks reminiscent of 18S and 28S rrnas will be seen on electrophoretic analysis. Even with these samples, the extent of modification is apparent from the broadness of the peaks and a diminished function as a template for polymerases; quantitative PCR (RT or standard) will often show an increase in the cycle threshold of 5 6 cycles (with equivalent mass inputs) compared to RNA or DNA from unfixed tissue. In older FFPE samples, the RNA is typically already highly fragmented, often with peak sizes of only 100 nt or so. In these cases, a good prep will merely obtain these fragments. DNA recovered from FFPE samples tends to be much less fragmented, but still exhibit a broad size distribution, and has diminished capacity to serve as a template for polymerases. Workflow This procedure consists of two workflows: one for isolating RNA, the other for isolating DNA. Each of those workflows contains two sub-flows, to isolate the target nucleic acids automatically or manually. MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 9

10 Workflow Isolate RNA Prepare the wash solutions (page 12) Add 24 ml of 100% isopropanol to Wash Solution 1 (page 12) Add 48 ml of 100% ethanol to Wash Solution 2 (page 12) Isolate RNA (page 12) Isolate RNA automatically on MagMAX Express 96 (page 12) OR Isolate RNA manually (page 15) Prepare the samples (page 12) Prepare the samples (page 15) Run the samples on MagMAX Express 96 (page 13) Bind RNA to beads (page 16) Use or store the samples (page 14) Wash the beads (page 16) Treat with DNase (page 16) Wash and elute RNA (page 17) Use or store the samples (page 17) 10 MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

11 Workflow Isolate DNA Prepare the wash solutions (page 12) Add 24 ml of 100% isopropanol to Wash Solution 1 (page 12) Add 48 ml of 100% ethanol to Wash Solution 2 (page 12) Isolate DNA (page 18) Isolate DNA automatically on MagMAX Express 96 (page 18) OR Isolate DNA manually (page 20) Prepare the samples (page 18) Prepare the samples (page 20) Run the samples on MagMAX Express 96 (page 19) Bind DNA to beads (page 21) Use or store the samples (page 20) Wash the beads (page 21) Treat with RNase (page 22) Wash and elute DNA (page 22) Use or store the samples (page 23) MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 11

12 Prepare the wash solutions Prepare the wash solutions Prepare the wash solutions by adding the following solvents to the bottles as received, and shaking to mix. Wash Solution 1: add 24 ml of 100% isopropanol Wash Solution 2: add 48 ml of 100% ethanol Isolate RNA Isolate RNA automatically on MagMAX Express 96 Confirm that your MagMAX Express 96 Deep Well Magnetic Particle Processor has installed the DW FFPE RNA protocol. If not, obtain and install the appropriate protocol to your MagMAX Express 96: 1. to request the DW FFPE RNA protocol. Alternatively, contact Invitrogen Technical Support by going to: 2. Install the protocol. a. Transfer the protocol to a computer attached to the MagMAX Express 96 instrument. b. Install the protocol to your MagMAX Express 96 instrument as explained in the MagMAX Express 96 Deep Well Magnetic Particle Processor User Guide (Part no. N07849). Prepare the samples 1. Cut µm sections from each sample, then transfer the tissue sections to a MagMAX deep well 96-well plate. Use one well for each sample. IMPORTANT! You can use up to 2 sections per sample if needed. Do not use more than 2 sections, however, as this may result in low yield or other problems. 2. Add 150 µl of Digestion Buffer to each sample well. Push each tissue section into the liquid with a syringe plunger from a 1-mL serological syringe. 3. Add 4 µl of Protease and 30 µl of RNA Digestion Additive to each sample well. 4. Cover the plate with an adhesive plate cover and incubate the covered plate in a water bath or a 96-well heat block. Use the following temperatures and times, in order: a. 60 C for 45 minutes b. 80 C for 30 minutes IMPORTANT! Check the liquid temperature in the plate (using water in an empty well) to verify that the samples are heated properly. You may need to set the incubator temperature to C for the 60 C incubation, and to 95 C for the 80 C incubation. 12 MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

13 Isolate RNA 5. While samples are digesting, prepare plates for the MagMAX Express 96 Deep Well Magnetic Particle Processor using the volumes and reagents listed below: Plate ID Position Reagent Final volume in well Plate type Digestion/Binding Plate 1 Digested sample Binding reagents Nucleic Acid Binding Beads 820 µl Deep Well Wash 1 2 Wash Solution 1 (with isopropanol) 400 µl Deep Well Wash 2_1 3 Wash Solution 2 (with ethanol) 150 µl Standard DNase Plate 4 DNase Mix (196 µl DNase Buffer + 4 µl DNase) 200 µl Deep Well Wash 2_2 5 Wash Solution 2 (with ethanol) 150 µl Standard Wash 2_3 6 Wash Solution 2 (with ethanol) 150 µl Standard Elution 7 Elution Buffer 70 µl Standard Tip Plate 8 Tip Comb (Deep Well) Deep Well 6. When the incubation is complete, tap the plate to remove the liquid from the cover, then add 200 µl of Binding Buffer and 420 µl of 100% isopropanol to each sample well. Note: To save time, you can combine the reagents beforehand in a binding mix, then add 620 µl of the mixture to each sample well. In this case, add at least 5% to the calculated volumes to allow for losses and avoid bubbles in the pipette tips. For example: Reagents One reaction Volume 96 reactions (with overage) Binding Buffer 200 µl µl Isopropanol 420 µl µl Total 620 µl µl 7. Spin the plate for approximately 5 seconds (optional) and let it cool for 5 minutes at room temperature. 8. Add 20 µl of Nucleic Acid Binding Beads to each sample well, then with the prepared isolation plates (step 5) move to the MagMAX Express 96 Deep Well instrument for isolation. Run the samples on MagMAX Express 96 Refer to the MagMAX Express 96 User Guide (Part no. N07849) for instructions on loading and running the instrument. 1. Load the MagMAX Express 96, then start the protocol. a. Turn on the power to the MagMAX Express 96. MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 13

14 Isolate RNA b. Place the MagMAX Express 96 Deep Well Tip Comb into the MagMAX Express Deep Well 96-Well Plate. c. Using the keypad or MagMAX Express Software, select the DW FFPE RNA protocol. d. If using the keypad, press Start. Alternatively, begin the run as instructed by the MagMAX Express Software. e. If the lid is in place, open the sliding door. f. Load the plates into the loading station as prompted by the instrument. Press Start after loading each plate. 2. When prompted, remove plate from instrument then add 150 µl of Binding Buffer µl of 100% Isopropanol (as a master mix with 5% overage) to each well. 3. When the MagMAX Express 96 has completed the protocol, remove all plates from the loading station as prompted by the instrument. Press start after removing each plate. IMPORTANT! Cover the elution plate (the first plate removed), which contains the purified RNA, as soon as it is removed from the MagMAX Express 96. To prevent evaporation, do not allow the sample to sit uncovered at room temperature for an extended time. 4. When the MagMAX Express 96 displays END_OF_RUN, press stop. 5. Shut down the MagMAX Express 96. Use or store the samples Store the elution plate at 4 C for up to 24 hours. If the samples must be stored for over 24 hours, store them at -20 to -80 C. 14 MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

15 Isolate RNA Isolate RNA manually IMPORTANT! The procedures in this section are optimized for use with the Thermo Scientific Barnstead/Lab-Line Titer Plate Shaker. Speeds and times (for example, shake at speed 8 for 1 minute ) are based on that instrument. If you use a different titer plate shaker, in all cases adjust the shaker speed to avoid splashing and contamination on the plate. Prepare the samples 1. Cut µm sections from each sample, then transfer the tissue sections to the 96-well processing plate. Use one well for each sample. IMPORTANT! You can use up to 2 sections per sample if needed. Do not use more than 2 sections, however, as this may result in low yield or other problems. 2. Add 150 µl of Digestion Buffer to each sample well. Push each tissue section down into the liquid with a syringe plunger from a 1-mL serological syringe. 3. Add 4 µl of Protease and 30 µl of RNA Digestion Additive to each sample well. 4. Cover the plate with an adhesive plate cover, then place the plate into a heated water bath or a 96-well heat block and incubate. Use the following temperatures and times, in order: a. 60 C for 45 minutes b. 80 C for 30 minutes IMPORTANT! Check the liquid temperature in the plate (using water in an empty well) to verify that the samples are heated properly. You may need to set the incubator temperature to C for the 60 C incubation, and to 95 C for the 80 C incubation. Note: While samples are incubating, begin heating Elution Buffer to 50 C. 5. When incubation is complete, tap the plate to collect liquid from the cover, then add 620 µl of Binding Mix (200 µl Binding Buffer µl 100% Isopropanol) to each sample well. Note: To save time, you can combine the reagents beforehand in a binding mix, then add 620 µl of the mixture to each sample well. In this case, add at least 5% to the calculated volumes to allow for losses and avoid bubbles in the pipette tips. For example: Reagents One reaction Volume 96 reactions (with overage) Binding Buffer 200 µl µl Isopropanol 420 µl µl Total 620 µl µl 6. Spin the plate for approximately 5 seconds (optional) and let it cool for 5 minutes at room temperature. MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 15

16 Isolate RNA Bind RNA to beads 1. Add 20 µl of Nucleic Acid Binding Beads to each sample well, then place the plate on a titer shaker at speed 3 4 for 3 minutes. 2. Remove the plate from the shaker. Pipet the samples up and down carefully times. Place the plate on a magnetic stand for 3 5 minutes (or until the samples are clear). 3. Remove the supernatant from each sample well, then remove the sample plate from the magnetic stand. Wash the beads 1. Add 150 µl of Wash Solution 1 (see Prepare the wash solutions on page 12) to each sample well. Place the plate on a shaker at speed 8 for 1 minute. 2. Transfer the plate from the shaker to a magnetic stand for 1 2 minutes (or until the samples are clear). 3. Remove the supernatant from each sample well, then remove the plate from the magnetic stand. 4. Add 150 µl of Wash Solution 2 (see Prepare the wash solutions on page 12) to each sample then shake at speed 8 for 1 minute. 5. Transfer the plate from the vortex shaker to a magnetic stand for 1 2 minutes (or until the solution is clear). 6. Remove the supernatant from each sample well. Shake plate uncovered at speed 9 for 2 minutes to dry the beads. Treat with DNase 1. Prepare the DNase mix: Combine 96 µl of DNase Buffer + 4 µl DNase in a master mix with 5% overage. Add 100 µl of this mixture to each sample, then mix at speed 8 for 2 minutes. 2. Cover the plate and place it in a heat bath at 37 C for 20 minutes. 3. Transfer the plate from the heat bath to a vortex shaker. Shake at speed 8 for 5 10 minutes. 4. Once treatment is complete, add 75 µl of Binding Buffer followed by 175 µl of 100% Isopropanol. Note: To save time, you can combine the reagents beforehand in a binding mix, then add 250 µl of the mixture to each sample well. In this case, add at least 5% to the calculated volumes to allow for losses and avoid bubbles in the pipette tips. For example: 16 MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

17 Isolate RNA Reagents One reaction Volume 96 reactions (with overage) Binding Buffer 75 µl 7560 µl Isopropanol 175 µl µl Total 250 µl µl 5. Return the plate to the shaker. Shake at speed 6 7 for 3 minutes. 6. Remove the plate from the shaker and pipet samples up and down times to mix. 7. Place the plate on a magnetic stand for 3 4 minutes (or until the solution is clear). 8. Remove the supernatant. Wash and elute RNA 1. Add 150 µl of Wash Solution 2 to each sample, then shake at speed 8 for 1 minute. 2. Transfer the plate from the shaker to a magnetic stand for 1 2 minutes (or until the solution is clear). 3. Remove the supernatant from each sample well, then remove the plate from the magnetic stand. 4. Repeat steps Shake the plate uncovered at speed 9 for 2 minutes to dry the beads. 6. Add 70 µl of heated Elution Buffer (heated to 50 C). 7. Shake the plate at speed 9 for 1 minute. If this does not break up the beads, pipet the Elution Buffer up and down gently 5 7 times. 8. Incubate the plate at 37 C for 4 minutes. 9. Transfer the plate from the heat bath to a vortex shaker. Shake the plate at speed 9 for 4 minutes. 10. Place the plate on a magnetic stand for 2 3 minutes (or until the solution is clear). 11. Pipet the eluate from each sample well to a clean elution plate. Use or store the samples Store the elution plate at 4 C for up to 24 hours. If the samples must be stored for over 24 hours, store them at -20 to -80 C. MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 17

18 Isolate DNA Isolate DNA Isolate DNA automatically on MagMAX Express 96 Confirm that your MagMAX Express 96 Deep Well Magnetic Particle Processor has installed the DW FFPE DNA Protocol. If not, download and install the appropriate protocol to your MagMAX Express 96: 1. to request the DW FFPE DNA protocol. Alternatively, contact Invitrogen Technical Support by going to: 2. Install the protocol. a. Transfer the protocol to a computer attached to the MagMAX Express 96 instrument. b. Install the protocol to your MagMAX Express 96 instrument as explained in the MagMAX Express 96 Deep Well Magnetic Particle Processor User Guide (Part no. N07849). Prepare the samples 1. Cut µm sections from each sample, then transfer the tissue sections to a MagMAX deep well 96-well plate. Use one well for each sample. IMPORTANT! You can use up to 2 sections per sample if needed. Do not use more than 2 sections, however, as this may result in low yield or other problems. 2. Add 150 µl of Digestion Buffer to each sample well. Push the tissue sections into the liquid with a syringe plunger from a 1-mL serological syringe. 3. Add 4 µl of Protease and 30 µl of DNA Digestion Additive to each sample well. 4. Cover the plate with an adhesive plate cover and incubate in a water bath or a 96- well heat block. Use the following temperatures and times, in order: a. 60 C for 60 minutes b. 80 C for 30 minutes IMPORTANT! Check the liquid temperature in the plate (using water in an empty well) to verify that the samples are heated properly. You may need to set the incubator temperature to C for the 60 C incubation, and to 95 C for the 80 C incubation. 5. While samples are digesting, prepare plates for the MagMAX Express 96 Deep Well Magnetic Particle Processor using the volumes and reagents listed below: Plate ID Position Reagent Final volume in well Plate type Digestion/Binding Plate 1 Digested sample Binding reagents Nucleic Acid Binding Beads 820 µl Deep Well Wash 1 2 Wash Solution 1 (with isopropanol) 400 µl Deep Well 18 MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

19 Isolate DNA Plate ID Position Reagent Final volume in well Plate type Wash 2_1 3 Wash Solution 2 (with ethanol) 150 µl Standard RNase Plate 4 RNase Mix (199 µl Nuclease-free Water + 1 µl RNase) 200 µl Deep Well Wash 2_2 5 Wash Solution 2 (with ethanol) 150 µl Standard Wash 2_3 6 Wash Solution 2 (with ethanol) 150 µl Standard Elution 7 Elution Buffer 70 µl Standard Tip Plate 8 Tip Comb (Deep Well) Deep Well 6. When incubation is complete, tap the plate to collect liquid from the cover, then add 620 µl of Binding Mix (200 µl Binding Buffer µl 100% Isopropanol) to each sample well. Note: To save time, you can combine the reagents beforehand in a binding mix, then add 620 µl of the mixture to each sample well. In this case, add at least 5% to the calculated volumes to allow for losses and avoid bubbles in the pipette tips. For example: Reagents One reaction Volume 96 reactions (with overage) Binding Buffer 200 µl µl Isopropanol 420 µl µl Total 620 µl µl 7. Spin the plate for approximately 5 seconds (optional) and let it cool for 5 minutes at room temperature. 8. Add 20 µl of Nucleic Acid Binding Beads to each sample well, then with the prepared isolation plates (step 5) move to the MagMAX Express 96 Deep Well instrument for isolation. Run the samples on MagMAX Express 96 Refer to the MagMAX Express 96 User Guide (Part no. N07849) for instructions on loading and running the instrument. 1. Load the MagMAX Express 96, then start the protocol. a. Power on the MagMAX Express 96. b. Place the MagMAX Express 96 Deep Well Tip Comb into the MagMAX Express Deep Well 96-Well Plate. c. Using the keypad or MagMAX Express Software, select the DW FFPE DNA protocol. d. If using the keypad, press Start. Alternatively, begin the run as instructed by the MagMAX Express Software. e. If the lid is in place, open the sliding door. MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 19

20 Isolate DNA f. Load the plates into the loading station as prompted by the instrument. Press Start after loading each plate. 2. Remove the plate from the instrument when prompted, then add 200 µl of Binding Buffer µl of 100% Isopropanol (as a master mix with 5% overage) to each well. 3. When the MagMAX Express 96 has completed the protocol, remove all plates from the loading station as prompted by the instrument. Press Start after removing each plate. IMPORTANT! Cover the elution plate (the first plate removed), which contains the purified DNA, as soon as it is removed from the MagMAX Express 96. To prevent evaporation, do not allow the sample to sit uncovered at room temperature for an extended time. 4. When the MagMAX Express 96 displays END_OF_RUN, press Stop. 5. Shut down the MagMAX Express 96. Use or store the samples Store the elution plate at 4 C for up to 24 hours. If the samples must be stored for over 24 hours, store them at -20 to -80 C. Isolate DNA manually IMPORTANT! The procedures in this section are optimized for use with the Thermo Scientific Barnstead/Lab-Line Titer Plate Shaker. Speeds and times (for example, shake at speed 8 for 1 minute ) are based on that instrument. If you use a different titer plate shaker, in all cases adjust the shaker speed to avoid splashing and contamination on the plate. Prepare the samples 1. Cut µm sections from each sample, then transfer the tissue sections to the 96-well processing plate provided in the kit. Use one well for each sample. IMPORTANT! You can use up to 2 sections per sample if needed. Do not use more than 2 sections, however, as this may result in low yield or other problems. 2. Add 150 µl of Digestion Buffer to each sample well. Push each tissue section down into the liquid with a syringe plunger from a 1-mL serological syringe. 3. Add 4 µl of Protease and 30 µl of DNA Digestion Additive to each sample well. 4. Cover the plate with an adhesive plate cover, then place the plate into a heated water bath or a 96-well heat block and incubate. Use the following temperatures and times, in order: a. 60 C for 60 minutes 20 MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

21 Isolate DNA b. 80 C for 30 minutes IMPORTANT! Check the liquid temperature in the plate (using water in an empty well) to verify that the samples are heated properly. You may need to set the incubator temperature to C for the 60 C incubation, and to 95 C for the 80 C incubation. Note: While the samples are incubating, begin heating the Elution Buffer to 80 C. 5. When incubation is complete, tap the plate to collect liquid from the cover, then add 620 µl of Binding Mix (200 µl Binding Buffer µl 100% Isopropanol) to each sample well. Note: To save time, you can combine the reagents beforehand in a binding mix, then add 620 µl of the mixture to each sample well. In this case, add at least 5% to the calculated volumes to allow for losses and avoid bubbles in the pipette tips. For example: Reagents One reaction Volume 96 reactions (with overage) Binding Buffer 200 µl µl Isopropanol 420 µl µl Total 620 µl µl 6. Spin the plate for approximately 5 seconds (optional) and let it cool for 5 minutes at room temperature. Bind DNA to beads 1. Add 20 µl of Nucleic Acid Binding Beads to each sample well, then place the plate on a titer shaker at speed 3 4 for 3 minutes. 2. Remove the plate from the shaker. Pipet the beads up and down carefully times. Place the plate on a magnetic stand for 3 5 minutes (or until the samples are clear). 3. Remove the supernatant from each sample well, then remove the sample plate from the magnetic stand. Wash the beads 1. Add 150 µl Wash Solution 1 (see Prepare the wash solutions on page 12) to each sample well. Place the plate on a vortex shaker at speed 8 for 1 minute. 2. Transfer the plate from the vortex shaker to a magnetic stand for 1 2 minutes (or until the samples are clear). 3. Remove the supernatant from each sample well, then remove the plate from the magnetic stand. 4. Add 150 µl Wash Solution 2 (see Prepare the wash solutions on page 12) to each sample then shake at speed 8 for 1 minute. MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 21

22 Isolate DNA 5. Transfer the plate from the vortex shaker to a magnetic stand for 1 2 minutes (or until the solution is clear). 6. Remove the supernatant from each sample well. Place plate on shaker uncovered and shake at speed 9 for 2 minutes to dry the samples. Treat with RNase 1. Prepare the RNase mix: For each sample, combine 99 µl of Nuclease-free Water + 1 µl RNase in a master mix with 5% overage. Add 100 µl to each sample, then mix at speed 8 for 2 minutes. 2. Cover the plate and place it in a heat bath at 37 C for minutes. 3. Transfer the plate from the heat bath to a shaker. Shake at speed 8 for 5 10 minutes. 4. Once treatment is complete, add 100 µl of Binding Buffer followed by 200 µl of 100% Isopropanol. Note: To save time, you can combine the reagents beforehand in a binding mix, then add 300 µl of the mixture to each sample well. In this case, add at least 5% to the calculated volumes to allow for losses and avoid bubbles in the pipette tips. For example: Reagents One reaction Volume 96 reactions (with overage) Binding Buffer 100 µl µl Isopropanol 200 µl µl Total 300 µl µl 5. Return the plate to the shaker. Shake at speed 6 7 for 3 minutes. 6. Remove the plate from the shaker and pipet the samples up and down times to mix. 7. Place the plate on a magnetic stand for 3 4 minutes or until the solution is clear. 8. Remove the supernatant. Wash and elute DNA 1. Add 150 µl of Wash Solution 2 to each sample, then shake at speed 8 for 1 minute. 2. Transfer the plate from the shaker to a magnetic stand for 1 2 minutes (or until the solution is clear). 3. Remove the supernatant from each sample well, then remove the plate from the magnetic stand. 4. Repeat steps Place the plate on the shaker uncovered and shake at speed 9 for 2 minutes to dry the samples. 22 MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

23 Assess Nucleic Acid Yield and Integrity 6. Add 70 µl of heated Elution Buffer (80 C). 7. Shake the plate at speed 9 for 1 minute. If this does not break up the beads, pipet up and down gently 5 7 times. 8. Incubate the plate at 80 C for 4 minutes. IMPORTANT! As with the digestion incubations, ensure that the heat block or water bath is set so that the liquid in the plate is heated to the specified temperature. This may require that the incubator be set as high as 95 C. 9. Transfer the plate from the heat bath to a vortex shaker. Shake the plate at speed 9 for 4 minutes. 10. Place the plate on a magnetic stand for 3 8 minutes or until the solution is clear. 11. Pipet the eluate from each sample well to a clean elution plate. Use or store the samples Store the elution plate at 4 C for up to 24 hours. If the samples must be stored for over 24 hours, store them at -20 to -80 C. Assess Nucleic Acid Yield and Integrity Assess RNA yield Note: The expected RNA yield can vary greatly depending on the tissue type and the method used for fixing and storing the sample. Spectrophotometry The concentration of an RNA solution can be determined by measuring its absorbance at 260 nm. We recommend using the NanoDrop 1000/8000 Spectrophotometer. Measure 2 µl of the RNA sample directly. Alternatively, the RNA concentration can be determined by diluting an aliquot of the preparation in TE (10 mm Tris-HCl, ph 8, 1 mm EDTA) and reading the absorbance in a traditional spectrophotometer at 260 nm. With a path length of 1 cm, an A260 of 1 is equivalent to 40 µg RNA/mL. Calculate the RNA concentration (µg/ml) as follows: A260 X dilution factor X 40 µg/ml = µg RNA/mL Note: Any contaminating DNA in the RNA prep will lead to an overestimation of RNA yield, since all nucleic acids absorb at 260 nm. Fluorometry If a fluorometer or a fluorescence microplate reader is available, Molecular Probes' RiboGreen fluorescence-based assay for RNA quantitation is a convenient and sensitive way to measure RNA concentration. Follow the manufacturer's instructions for using RiboGreen. MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 23

24 Assess Nucleic Acid Yield and Integrity Assess RNA quality Spectrophotometry The A260/A280 ratio of the RNA is an indication of its purity. The RNA isolated with this protocol should have an A260/A280 ratio of Agarose gel electrophoresis You can also assess the quality of your RNA sample by agarose gel electrophoresis. Protocols are available at Note: Typically, RNA recovered from FFPE samples will appear smeared. In higher quality preparations, two broad bands representing 18S and 28S rrna will be seen; only a smear will be visible in lower quality preparations. Microfluidics analysis The Agilent 2100 Bioanalyzer, in conjunction with an RNA LabChip Kit, provides a powerful and sensitive method to assess RNA integrity. To use this system, follow the instructions for RNA analysis provided with the RNA LabChip Kit. As with agarose gel electrophoresis, higher quality preparations will show a mass range mound with peaks corresponding to the 18S (and sometimes 28S) rrna. Poorer quality preparations will range in size from nt, with a peak size of nt. Recovery of mirna Fragmentation of FFPE RNA seems to have a size endpoint of ~80 nt, and modifications affect only about 1% of the nucleotides. Thus, mirna molecules, which are nt, are basically untouched by the fixation/embedding process. Using sample sections of 10 µm thickness will minimize loss of mirna. Modify downstream applications The purity of the recovered nucleic acid is sufficient for most applications. Nucleic acid fragmentation and some chemical modifications, caused by sample fixation and storage, will remain after the procedure. Therefore, some downstream procedures may need to be modified for best results. Real-time PCR The RNA recovered with the MagMAX FFPE Total Nucleic Acid Isolation Kit can be used in real-time PCR analysis. Because the RNA extracted from fixed tissues is likely to be degraded, plan to analyze small amplicons. RNA from FFPE samples always requires more PCR cycles to produce the same signal as RNA from frozen tissues from the same source. This is probably due to nucleic acid modifications incurred by the fixation process. Array analysis While recovered RNA can be amplified for use in array analysis using, for example, the MessageAmp Premier Kit (P/N AM1792), there are several caveats. Since amplification procedures prime from the 3' end of mrnas, the fragmentation and presence of formaldehyde-induced lesions on the RNA recovered with this kit will create a population of targets that are weighted to the extreme 3' ends of the messages. By using appropriate probes on the arrays, or by applying a filter to the data so that 24 MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

25 Assess Nucleic Acid Yield and Integrity only 3' end-most probes are analyzed, biologically relevant information can be obtained. Even with these compensatory steps, older samples (archived for 10 yr) will often be so highly fragmented that the number of expressed genes present will be drastically reduced. Assess DNA yield Assess DNA quality DNA yield and quality depends on the tissue and fixing procedure. The concentration of a DNA solution can be determined by measuring its absorbance at 260 nm. We recommend using the NanoDrop 1000/8000 Spectrophotometer. Measure 2 µl of the DNA sample directly. Alternatively, determine the DNA concentration by diluting an aliquot of the preparation in TE and reading the absorbance at 260 nm in a traditional spectrophotometer. With a path length of 1 cm, an A260 of 1 is equivalent to 50 µg of double-stranded DNA/mL. Calculate the DNA concentration (µg/ml) as follows: A260 X dilution factor X 50 µg/ml = µg DNA/mL Spectrophotometry The A260/A280 ratio of the DNA is an indication of its purity. The DNA isolated with this protocol has an A260/A280 ratio of Quantitative real-time PCR vs a DNA standard The ability of recovered DNA to function as a template for PCR can be measured using the TaqMan DNA Template Reagents Kit (Part No ) and the TaqMan RNase P Detection Reagents Kit (Part No ). For further information, see the Application Note DNA Genotyping from Human FFPE Samples-Reliable and Reproducible; search for stock number 137AP04-01 at Agarose gel electrophoresis The DNA obtained from this procedure will always be a population of sheared fragments. You can assess the average size of this population using standard electrophoretic methods. This will require the use of ~ µg of DNA, which may be a sizeable fraction of the yield for some samples. Microfluidics analysis An instrument such as the Agilent 2100 Bioanalyzer requires a much smaller amount of the recovered DNA than does agarose gel electrophoresis. The recovered DNA typically appears as a broad smear, covering a size range up to thousands of base pairs, with the modal size varying from less than 100 ~3000 bp, depending on the age and condition of the original sample. Modify downstream applications DNA recovered with the kit can be used in PCR analysis and other downstream applications; however, the same caveats apply as for analysis of RNA. Modifications from the fixation and embedding process occur on average every bp; therefore, we recommend analysis of amplicons around 100 bp or smaller. MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 25

26 Troubleshooting Troubleshooting Observation Possible Cause Recommended Action Nucleic acids appear fragmented Low yield Overheating during Protease digestion Do not extend the 30-min incubation at 80 C more than 5 min. Make sure the temperature of the incubation device does not fluctuate significantly. RNA is degraded after purification. Tissues can vary enormously in their RNA content and in extraction efficiency. Very fibrous tissues, such as muscle, will tend to form a more tightlycrosslinked web upon fixation, so relative recovery will be lower. Tissue was not digested. Too much tissue was added to the digestion reaction. Too much digestion additive in the reaction. The binding bead mix was stored incorrectly The amount of Nucleic Acid Binding Beads added was not sufficient. The bead pellet was lost during the binding or washing steps. Use best practices for handling RNA to avoid degradation by nucleases. For problematic tissues: After adding binding mix, remove samples to a fresh plate before proceeding with isolation. Increase sample mixing by pipetting the samples up and down before proceeding with automated protocol or increase the number of up and down mixes for the manual protocol. For DNA, try incubating at 60 C overnight instead of the shorter incubation. Ensure that the entire section within the deepwell plate is fully immersed in the Digestion Buffer. Note: The rubber end of a plunger from a 1-mL serological syringe is best suited for ensuring the entire sample is immersed in the Digestion Buffer. Ensure that the water bath or heat block is heating the sample at the correct temperature. When using a heat block, be sure to check the temperature of the liquid in the 96-well plate, not just the block itself. Use no more than 2 tissue sections, 10 µm thick. Use no more than 30 µl of digestion additive. Store the binding beads at room temperature for immediate use, or at 2 8 C for the long term. IMPORTANT! Do not freeze the binding beads. Vortex the tube containing the binding beads thoroughly, immediately before use. When removing the supernatant, angle the pipette tip(s) away from the beads. Place the plate on the magnetic stand for the recommended times. 26 MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

27 Troubleshooting Observation Possible Cause Recommended Action Low Yield, continued Reduced downstream functionality The Nucleic Acid Binding Beads were added before the isopropanol. Binding was incomplete. The volume of isopropanol was incorrect. Nucleic acid is still bound to the beads or beads remain clumped after elution. Samples did not go through the rebinding step The elution conditions were incorrect. Add the binding beads after adding the isopropanol and Binding Buffer. Ensure that the samples are mixed well before collecting the beads. Pipetting up and down after the initial plate shaking ensures complete mixing. Use the volumes called for in the protocol. Increase the volume of Elution Buffer. Increase the speed of the shaker used during the elution step. Pipet up/down to break apart the bead pellets. Verify that the samples were mixed with the correct volumes of Binding Buffer and isopropanol after the nuclease treatment. Use only the MagMAX Elution Buffer. For the manual protocol, always preheat the Elution Buffer to the recommended temperature. For RNA: Use random primers or gene-specific primers in the RT reaction. Use smaller amplicons for real-time PCR or increase mass input into the reactions. For DNA: Use qpcr primer/probe sets with smaller amplicons. Increase the incubation time to overnight at 60 C to improve functionality for downstream applications requiring larger PCR amplicons. Contamination DNA contamination of RNA Either reduce the sample size, or do an additional DNase treatment after RNA isolation (e.g., using the Ambion TURBO DNA-free Kit, Part no. AM1907). Always use the provided DNase and DNase Buffer when working with the binding beads. RNA contamination of DNA Paraffin residue carryover Salt carryover Alcohol carryover Do an additional RNase treatment after DNA isolation (e.g., using RNase A (RPA grade), Part no. AM2272). Trim away excess paraffin from the sample prior to digestion. After adding the binding mix, remove samples to a fresh plate before proceeding with isolation. Verify that the correct type and volume of alcohol is added to each wash concentrate. For the manual procedure, verify that the sample is dry before adding the Elution Buffer. MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol 27

28 Troubleshooting Observation Possible Cause Recommended Action Contamination, continued The nucleic acid yield varies from well to well. Other contaminants Variability of FFPE tissue samples The Nucleic Acid Binding Beads were not fully resuspended/dispersed. Sample evaporation Precipitate with ethanol to further purify the recovered nucleic acids. 1. Add ammonium acetate to a final concentration of M and mix well. 2. Add 4 volumes of ethanol, and mix again. 3. Chill at -20 C or lower for at least 30 min. To maximize mirna recovery, chill at -20 C for at least 12 hours. 4. Centrifuge at 16,000 x g for min to pellet the nucleic acids. 5. Wash the pellet twice with 80% ethanol. 6. Resuspend the nucleic acids in Nuclease-free Water or TE (10 mm Tris-HCl, ph 8, 1 mm EDTA). Process replicate sections for each tissue. Resuspend the binding beads at temperatures above 20 C. For the manual procedure, ensure that the beads are fully dispersed by pipetting up/down a minimum of 10 times. If the samples are stored for too long and evaporation occurs, immediately resuspend the samples using water to the correct volume. Cover the plate immediately after transferring the eluate, or removing the plate from the automated platform. Note: The elution plates are not meant for longterm storage or archiving. If samples need to be archived, we recommend transferring samples to a more appropriate container. 28 MagMAX FFPE Total Nucleic Acid Isolation Kit Protocol

29 APPENDIX A Appendix A Ordering Information, Related Documentation, and Support How to order For information on the MagMAX FFPE Total Nucleic Acid Isolation Kit, go to the Invitrogen website at: Select Products & Services > Kits & Assays > Nucleic Acid Purification Kits & Reagents > Total Nucleic Acid Purification Kits > MagMAX FFPE Total Nucleic Acid Isolation Kit. Item Source MagMAX FFPE Total Nucleic Acid Isolation Kit, 96 reactions Part no Materials and equipment required but not included For the SDS of any chemical not distributed by Life Technologies or Invitrogen, contact the chemical manufacturer. Before handling any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions. Item Source Ethanol Isopropanol (ACS reagent grade or equivalent) Lab equipment (pipettors, pipette tips, vortexer, microcentrifuge, tubes, and conicals) Heat Source (water bath, heat block, incubator, etc.) Centrifuge capable of spinning 96-well deep well plates Note: The procedures in the this document were developed with the Eppendorf Microtome and related supplies Titer plate shaker Note: The procedures in this document are optimized for use with the Thermo Scientific Barnstead/Lab-Line Titer Plate Shaker. MagMAX Express 96 Deep Well Magnetic Particle Processor (BR- Basic Research Customer) Major Laboratory Suppliers Part no MagMAX Express 96 Standard Plates Part no MagMAX Express 96 Deep Well Plates Part no MagMAX TM FFPE Total Nucleic Acid Isolation Kit Protocol 29

30 Appendix A Ordering Information, Related Documentation, and Support Related documentation Item Source MagMAX Express 96 Deep Well Tip Combs Part no Magnetic stand for 96-well plates: Magnetic Stand-96 (24-magnet block) 96-Well Magnetic-Ring Stand (96-ring magnet block) Plate covers: MicroAmp Optical Adhesive Film MicroAmp Clear Adhesive Film Part no. AM10027 Part no. AM10050 Part no Part no Related documentation The following related documents are available: Document Part number MagMAX FFPE Total Nucleic Acid Isolation Kit Quick Reference Card MagMAX FFPE DNA Isolation Kit Protocol MagMAX FFPE DNA Isolation Kit Quick Reference Card Portable document format (PDF) versions of these documents are available on the Life Technologies web site at or on the Invitrogen website at Obtaining support For the latest services and support information for all locations, go to: At these sites, you can: Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities. Search through frequently asked questions (FAQs). Submit a question directly to Technical Support. Order user documents, SDSs, certificates of analysis, and other related documents. Download PDF documents. Obtain information about customer training. Download software updates and patches. 30 MagMAX TM FFPE Total Nucleic Acid Isolation Kit Protocol

31 APPENDIX B Appendix B Safety This appendix covers: Chemical safety General chemical safety SDSs Biological hazard safety Chemical safety General chemical safety Chemical hazard warning WARNING! CHEMICAL HAZARD. Before handling any chemicals, refer to the Safety Data Sheet (SDS) provided by the manufacturer, and observe all relevant precautions. Chemical safety guidelines To minimize the hazards of chemicals: Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See About SDSs on page 32.) Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the SDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the SDS. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer s cleanup procedures as recommended in the SDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. MagMAX TM FFPE Total Nucleic Acid Isolation Kit Protocol 31

32 Appendix B Safety Chemical safety SDSs About SDSs Chemical manufacturers supply current Safety Data Sheets (SDSs) with shipments of hazardous chemicals to new customers. They also provide SDSs with the first shipment of a hazardous chemical to a customer after an SDS has been updated. SDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely. Each time you receive a new SDS packaged with a hazardous chemical, be sure to replace the appropriate SDS in your files. Obtaining SDSs The SDS for any chemical supplied by Life Technologies or Invitrogen is available to you free 24 hours a day. To obtain SDSs: 1. Go to one of these web sites: Click Support, then select SDS. 3. In the Keyword Search field, enter the chemical name, product name, SDS part number, or other information that appears in the SDS of interest. Select the language of your choice, then click Search. 4. Find the document of interest, right-click the document title, then select any of the following: Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note: For the SDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer. 32 MagMAX TM FFPE Total Nucleic Acid Isolation Kit Protocol

33 Appendix B Safety Chemical safety Biological hazard safety General biohazard WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the following: U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (found at: Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR ; nara/cfr/waisidx_01/ 29cfr1910a_01.html). Your company s/institution s Biosafety Program protocols for working with/ handling potentially infectious materials. Additional information about biohazard guidelines is available at: MagMAX TM FFPE Total Nucleic Acid Isolation Kit Protocol 33