HighPrep FFPE RNA 96. RNA isolation from FFPE (Formalin-Fix, Paraffin-Embeded) Tissue Magnetic beads based chemistry High-throughput capable PROTOCOL

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1 MAGBIO ACCELERATING genomic research HighPrep FFPE RNA 96 Catalog Nos. HPFF-R96, HPFF-R96X4 Manual Revision v3.00 RNA isolation from FFPE (Formalin-Fix, Paraffin-Embeded) Tissue Magnetic beads based chemistry High-throughput capable PROTOCOL Contents Product Description and Process... Kit Contents, Storage, Stability... Preparation of Reagents... FFPE RNA - 96 well format... Troubleshooting guide For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. MAGBIO GENOMICS, INC. DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL MAGBIO GENOMICS, INC. BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF,WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT MAGBIO GENOMICS, INC. IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. TRADEMARKS The trademarks mentioned herein are the property of or their respective owners.

2 Product Description The Highprep FFPE RNA 96 kit is a high quality, high throughput purification of total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The kit is xylene-free for safer processing and ease of use. Due to fixation and embedding procedures, nucleic acids in FFPE samples are heavily fragmented and modified by formaldehyde. While the HighPrep FFPE RNA 96 kit is optimized to minimize the effect of the formaldehyde modification, it is not recommended to use the RNA purified with this kit for downstream applications that requires full length RNA. Utilizing magnetic bead purification technology, the kit is high-throughput capable and can be adaped to most liquid handling workstations in the market. Workflow Overview The protocol is based on two methods to initially digest the FFPE sample. A non-xylene and a xylene method. The xylene approach will achieve higher yields but because xylene is toxic, it is up to the end user to determine if it is suitable for automation. The HighPrep FFPE RNA 96 kit utilizes magnetic beads technology to isolate total RNA from FFPE samples, therefore a magnetic processing device is required. Samples are first lysed in FFPE RTL Buffer following digestion with Pro K Solution. The lysate is then heated to denature the proteinase. FFPE RB Buffer and the MAG-S1 magnetic particles are added to bind the nucleic acid. Genomic DNA is removed by DNase. The RNA bound beads is washed twice and eluted with RNase-free water. Kit Contents and Storage HighPrep FFPE RNA 96 Kits Catalog No. HPFF-R96 HPFF-R96X4 STORAGE Number of Preps FFPE RTL Buffer 35 ml 140 ml C FFPE RB Buffer 20 ml 80 ml C FFPE RW1 Buffer 1 10 ml 40 ml C FFPE RW2 Buffer 1 25 ml 100 ml C DNase Digestion Buffer 10 ml 40 ml C DNase 150 μl 4 x 150 μl -20 C LPA 1.1 ml 4.4 ml C DEPC-Water 20 ml 40 ml C Pro K Solution 2 3 ml 12 ml C MAG-S1 Particles 2.2 ml 8.4 ml 2-8 C 1 Ethanol must be added prior to use. See Preparation of Reagents Stability All components are stable for 12 months when stored accordingly. 2 Pro K Solution comes in a ready to use Component is stable for 1 year when stored at C. For storage longer than 1 year, store at 2-8 C is recommended. 1

3 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). MSDS can be downloaded from the Product Resource tab when viewing the product kit. Preparation of Reagents Prepare the following components for each kit before use: Catalog No. HPFF-R96 Component Add 100% Isopropanol FFPE RW1 Buffer ml FFPE RW2 Buffer ml Add 100% Ethanol Storage Room Temp C Room Temp C Components are stable for 1 year when stored closed at room temperature Catalog No. HPFF-R96X4 Component Add 100% Isopropanol FFPE RW1 Buffer ml FFPE RW2 Buffer ml Add 100% Ethanol Storage Room Temp C Room Temp C Components are stable for 1 year when stored closed at room temperature 2

4 Protocol: FFPE RNA Kit - 96 plate format 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, please consult the appropriate material safety data sheets (MSDSs) from each product supplier. Nuclease-free 1.2 ml or 2 ml 96 well round-bottom plates Nuclease-free microplates Centrifuge with swing bucket rotor for 96 deep well plate capable of 4,000 x g Water bath or heat block capable of 37 C Water bath or heat block capable of 55 C Water bath or heat block capable of 80 C Sealing film Ethanol (100%) fresh Xylene (if using xylene digestion method) RNase-free filter pipette tips Magnetic separation device for 96-well plate Things to do before starting! The protocol is based on two methods to initially digest the FFPE sample. A non-xylene and a xylene method. The xylene approach will achieve higher yields but because xylene is toxic, it is up to the end user to determine if it is suitable for automation and use. Ensure FFPE RW1 Buffer and FFPE RW2 Buffer are prepared according to the instructions on page 2 and are at room temperature. Set water bath or heat block to 37 C, 55 C, 70 C and 80 C Protocol Choose either step 1 or step 2 of the digestion method. 1. NON XYLENE DIGESTION: a. Add 250 μl FFPE RTL Buffer to each well of the 96 well plate. b. Cut 2-5 paraffin sample sections between 5-10μm and add to the well containing the FFPE RTL Buffer immediately. Note: Do not use the first 2-3 sections from the sample block as more paraffin is present. c. Centrifuge at 4,000 x g for 2 min. d. Seal the sample plate with a sealing film. Incubate at 70 C for 1 hour. Gently shaking the plate 2-3 times to mix during the incubation. Note: Assure the tissue samples are submerged in the 3

5 e. Remove sample plate from incubator and let cool for 5 min at room temperature. f. Tap the plate to collect the liquid from the sealing film and remove film. Add 20 μl Pro K Solution to each sample well. Seal the sample plate and incubate overnight at 55 C. Occasiaonally mix the plate by gently shaking the plate. 2. XYLENE DIGESTION METHOD: a. Add 1 ml xylene to each well of the 96 well plate. b. Cut 2-5 paraffin sample sections between 5-10μm and add to the well containing the xylene. Note: Do not use the first 2-3 sections from the sample block as more paraffin is present. c. Seal the plate and incubate at room temperature for 2 min. Vortex for 30 seconds. d. Centrifuge at 4,000 x g for 5 min at room temperature or until a tight pellet. e. Remove the supernatant by pipetting. Do not remove any of the pellet. f. Add 1 ml ethanol (100%) to the pellet, and mix by vortexing. g. Centrifuge at 4,000 x g for 5 min. Proceed to step 3. h. Remove the supernatant by pipetting. Do no remove any of the pellet. i. Repeat steps 2f - 2h for second ethanol wash. Remove any residual ethanol with a fine pipet tip. j. Incubate the plate at room temperature (15-25 C) or up to 37 C. Incubate for 10 min or until all residual ethanol has evaporated. k. Add 250 μl FFPE RTL Buff er and 20 μl Pro K Solution. Pipet mix 10 times. Seal the sample plate and incubate overnight at 55 C. 3. Incubate at 80 C for 15 min. 4. Centrifuge at 4,000 x g for 5 min. The paraffin will form a thin layer on top of the lysate 5. Transfer 200 μl cleared lysate below the paraffin from each well into a new 96-well roundwell plate. Try not to transfer the paraffin material from the top layer. Suggestion: Use a 1 ml pipette tip or large orifice tip to penetrate the paraffin layer. 6. Add 200 μl FFPE RB Buffer, 20 μl MAG-S1 particles, and 430 μl ethanol (100%) to each sample well. Pipette mix times. Note: If expecting low RNA content from sample or if mirna is the target, add 10 μl LPA! Shake thoroughly the MAG-S1 particles to fully resuspend before use. 7. Incubate at room temperature for 5-10 min. 4

6 8. Place the sample plate on the magnetic separation device until the beads clear the 9. Aspirate and discard the cleared supernatant. Do not disturb the magnetized beads. 10. Remove the plate from the magnetic separation device. 11. Add 400 μl FFPE RW1 Buffer to each sample well. Resuspend the beads by pipette mixing times. (See Preparation of Reagents on page 2 for FFPE RW1 Buffer) 12. Place the sample plate on the magnetic separation device until the beads clear the 13. Aspirate and discard the cleared supernatant. Do not disturb the magnetized beads. 14. Remove the plate from the magnetic separation device. Add 73.5 μl DNase Digestion Buffer and 1.5 μl RNase-free DNase. Pipette mix times. 15. Incubate the sample plate at 37 C for 15 min. Note: Do not exceed 15 min. 16. Add 225 μl FFPE RW2 Buffer to each sample well. Resuspend the beads by pipette mixing times. (See Preparation of Reagents on page 2 for FFPE RW2 Buffer) 17. Incubate the sample plate at room temperature for 3-5 min. 18. Place the sample plate on the magnetic separation device until the beads clear the 19. Aspirate and discard the cleared supernatant. Do not disturb the magnetized beads. 20. Remove the sample plate from the magnetic separation device. Add 400 μl FFPE RW1 Buffer. Resuspend the beads by pipette mixing times. 21. Place the sample plate on the magnetic separation device until the beads clear the 22. Aspirate and discard the cleared supernatant. Do not disturb the magnetized beads. 23. Repeat steps for a second wash. 24. Leave the plate on the magnetic separation device for 5-10 min to air dry the magnetized beads. It is critical to completely remove all liquid from each well since ethanol carryover in the eluate may interfere with some downstream applications. Do not over dry the beads. 25. Add μl DEPC Water. Pipette mix 30 times for complete resuspension. 26. Incubate at room temperature for 10 min. 27. Place the sample plate on the magnetic separation device until the beads clear the 28. Pipet the cleared supernatant containing purified RNA into a new nuclease-free 96 well microplate and seal with a sealing filem. Store the purified RNA at -80 C. 5

7 Troubleshooting guide Please use this guide to troubleshoot any problems that may arise. For further assistance, please contact technical support via: Phone: (in US), outside US, Symptoms Possible Causes Comments Low RNA Yields MAG-S1 particles do not completely clear from solution RNA degraded during sample storage. Incomplete resuspension of Mag-S1 particles. Loss of MAG-S1 particles during operation. FFPE RW1 Buffer and FFPE RW2 Buffer were not prepared correctly. Binding was incomplete. Too short of magnetizing time. Ensure that the sample is properly stored. Process the sample immediately after collection or removal from storage. Resuspend MAG-S1 particles by pipette mixing or vortexing vigorously before use. Avoid disturbing the MAG-S1 particles during aspiration of supernatant. Prepare buffers accordingly. See Preparation of Reagents on page 2. Ensure that the samples are mixed well before collecting the beads. Increase pipette mixing or vortex/shake plate to ensure complete mixing. Increase collection time on the magnet. Problems in downstream applications Carryover of the magnetic beads in the elution Degraded RNA Bead collection time is too short. During incubation at 37 C, do not incubate samples over 15 min. Carryover beads will not affect downstream applications. To remove carryover magnetic beads, magnetize the magnetic beads and transfer the eluate containing the RNA to a new plate. Ordering Information Product Description Catalog No. Preps HighPrep FFPE RNA 96 HPFF-R96 96 HighPrep FFPE RNA 96 (96 x 4) HPFF-R96X

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