BiOstic FFPE Tissue RNA Isolation Kit (For use with formalin-fixed and paraffin-embedded tissues) Catalog No. Quantity 13250-50 50 preps Instruction Manual Please recycle Version: 02032014 1
Table of Contents Introduction... 3 Protocol Overview... 3 Flow Chart... 4 Equipment Required... 5 Kit Contents & Storage... 5 Precautions & Warnings... 5 Important Notes Before Starting... 6 Protocols: Experienced User Protocol... 7 Detailed Protocol (Describes what is happening at each step)... 9 Hints & Troubleshooting Guide... 12 Technical Guide..... 14 Contact Information... 15 Products Recommended For You... 16 2
Introduction The BiOstic FFPE Tissue RNA Isolation Kit is optimized for the extraction of RNA from formalin-fixed and paraffin-embedded tissues without the use of organic solvents. A proprietary buffer formulation results in complete dissolution of the wax during a 60 C heating step to release the tissue in conjunction with a Proteinase K lysis. A second 70 C heating step removes the cross-links that are inhibitory to RTqPCR. RNA from the melted samples is purified on our new Low Elution silica spin filters to provide a low volume, high concentration RNA eluent. Genomic DNA is removed in an on-column DNase digestion step utilizing our room temperature stable (RTS) DNase. RNA from the BiOstic FFPE Tissue RNA Isolation Kit has been successfully used in RT-qPCR applications. Protocol Overview BiOstic Low Elution spin column products utilize MO BIO Laboratories novel flat bottom spin column design, which provides improved sample processing and yields. The bucket configuration allows for enhanced sample flow and membrane drying after wash steps since the entire membrane is accessible to air flow. Silica technology provides a robust and fast way to purify nucleic acids without the use of organic solvents or cesium chloride gradients. The BiOstic FFPE Tissue RNA Isolation Kit is used to process 1 to 5 slices or up to 15 mg of paraffinembedded tissue per preparation. Tissue samples are initially melted away from the paraffin and then digested with Proteinase K at 60 C in an RNA-protective buffer. A 70 C heating step is used to remove cross-links in the RNA and ensures successful RT-qPCR. The sample is then mixed with a buffer containing chaotropic salt and 100% ethanol and bound to our Low Elution Spin Filters. Impurities are then washed from the column, genomic DNA is removed with an oncolumn DNase step and pure RNA is eluted in RNase-Free water. This kit is for research purposes only. Not for diagnostic use. Other Related Products Catalog No. Quantity BiOstic FFPE Tissue DNA Isolation Kit 12250-50 50 preps BiOstic Paraffin Removal Reagent 12251-50 2 x 25 ml RTS DNase Kit 15200-50 50 preps UltraClean Lab Cleaner 12095-250 12095-500 12095-1000 1 L bottle RNase-Free Gloves 1556-XS 1556-S 1556-M 1556-L 250 ml squeeze bottle 500 ml squeeze bottle X-Small (Bag of 150) Small (Bag of 150) Medium (Bag of 150) Large (Bag of 150) 3
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Equipment Required Microcentrifuge ( 16,000 x g) Pipettors (for volumes 4 600 µl) Water bath or heat block set to 60 C Water bath or heat block set to 70 C Vortex Reagents Required but Not Included: 100% ethanol (200 Proof, Molecular Biology Grade, no denaturants) Kit Contents Kit Catalog# 13250-50 Component Catalog# Amount Solution FR1 13250-50-1 17 ml Solution FR2 (Proteinase K) 13250-50-2 1.2 ml Solution FR3 13250-50-3 17 ml Solution FR4 13250-50-4 2 x 30 ml Solution FR5 13250-50-5 3 ml Solution FR6 13250-50-6 22 ml Solution FR7 13250-50-7 1.2 ml RTS DNase (RNase-Free) 13250-50-8 0.3 ml Low Elution Spin Filters 13250-50-SF 50 2 ml Collection Tubes 13250-50-T 150 Kit Storage Important: Store the Low Elution Spin Filters at 4ºC. NOTE: These Low Elution Spin Filters are for this kit only. Although they are visually comparable to other MO BIO Spin Filters, they are different and should not be used in other MO BIO protocols. Store them in their original colored bag to avoid confusion. Solution FR2 (Proteinase K) and the RTS DNase are stable at room temperature (up to 6 months). For prolonged storage (up to 2 years), place the Solution FR2 and RTS DNase at 4ºC. Store all other reagents and kit components at room temperature (15-30 C). Precautions Please wear gloves when using this product. Avoid all skin contact with kit reagents. In case of contact, wash thoroughly with water. Do not ingest. See Material Safety Data Sheets for emergency procedures in case of accidental ingestion or contact. All MSDS information is available upon request (760-929- 9911) or at www.mobio.com. Reagents labeled flammable should be kept away from open flames and sparks. WARNING: Solution FR4 contains isopropanol. It is flammable. 5
Important Notes Before Starting This protocol describes how to extract RNA from FFPE tissues starting with 1 to 5 slices or up to 15 mg of tissue. Be certain that you have all the components of this kit in hand before starting the protocol. The RTS DNase and the Low Elution Spin Filters are stored at 4 C. MO BIO recommends that first time users of this kit perform a set of test runs with representative samples before working with important research test samples. Try a range of FFPE tissue input quantities (e.g., 5, 10, and 15 mg of tissue) to assess kit performance and to familiarize yourself with the protocol workflow. RNA yield and quality from FFPE tissue will vary based on the tissue source, the age of the sample, the thickness of the slice, the length of time it was fixed, and the ratio of wax to tissue in each slice. This protocol uses three (3) 2 ml Collection Tubes and one Low Elution Spin Filter per prep for the RNA extraction. To speed processing and make transfers more convenient, label the caps of the Collection Tubes and Spin Filters in advance. The Low Elution Spin Filters are for this kit only and must be stored at 4 C. Although they are visually comparable to other MO BIO Spin Filters, they are different and should not be used in other MO BIO protocols. All steps in the protocol, other than the specified heating steps, are carried out at room temperature. Prepare two heat blocks (or water baths). One at 60ºC and one at 70ºC. Solution FR5 and the RTS DNase for the on column DNase digestion step (Step 13) can be combined just prior to starting the protocol or during the heating steps by adding 4 µl RTS DNase and 46 µl of Solution FR5 per prep. Mix gently by pipetting. Do not vortex to mix. You will need 1.8 ml of 100% ethanol per prep which is not included in the kit. The ethanol needs to be Absolute Ethanol - 200 Proof, Molecular Biology Grade, no denaturants. Wear RNase-Free Gloves (1556) at all times and remove RNase from the work area using Lab Cleaner (12095) for RNase Removal. Both of these products are available from MO BIO. Please see the Products recommended for you section at the end of this manual. 6
Experienced User Protocol Please wear gloves at all times. Be certain you have all kit components in hand before starting the protocol. The RTS DNase and the Low Elution Spin Filters are stored at 4 C. 1. To the tube containing 1-5 FFPE tissue slices (up to 15 mg) add 300 µl of Solution FR1 and 20 µl of Solution FR2. Vortex for 20 seconds at high speed to bring buffers into contact with the paraffin slice(s) that may be stuck on the side of the tube. Briefly centrifuge at 2000 x g for 5 seconds to bring the solution and wax down into the tube. Note: If some of the wax is still stuck on the side of the tube, it will fall down into the solution during the first heating step. 2. Heat the sample at 60ºC for 15 minutes. 3. Transfer the samples to the heat block set to 70ºC and heat for 15 minutes. Note: It is optimal for the recovered RNA to be processed expeditiously. However, if a second heat block is not available, place the samples at room temperature, turn the heat block to 70ºC and wait for it to reach temperature. Be sure the temperature has stabilized at 70ºC before transferring the samples back to the heat block. 4. Centrifuge the samples at 13,000 x g for 1 minute and transfer the digested lysate to a new 2 ml Collection Tube (provided). Note: After centrifugation, a thin top wax layer may be visible in the tube. Aspirate the lysate (approximately 300 µl) from below this layer. Avoid carryover of the waxy layer. 5. Add 300 µl of Solution FR3. 6. Add 600 µl of 100% Absolute ethanol (user provided). Mix by vortexing or pipetting. 7. Load 600 µl onto the Low Elution Spin Filter. Centrifuge at 13,000 x g for 30 seconds. Note: Low Elution Spin Filters are not interchangeable with other MO BIO Spin Filters. 8. Decant the flow-through, put the column back into the same 2 ml Collection Tube and load the remaining volume of lysate/bind to the Low Elution Spin Filter. Centrifuge at 13,000 x g for 30 seconds. 9. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and load with 600 µl of Solution FR4. Centrifuge for 30 seconds at 13,000 x g. 10. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and load with 600 µl of 100% Absolute ethanol (user provided). Centrifuge for 30 seconds at 13,000 x g. 11. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and dry spin the column in the centrifuge for 2 minutes at 16,000 x g. 12. Discard the 2 ml Collection Tube and transfer the Low Elution Spin Filter to a clean 2 ml Collection Tube (provided). 13. Add 50 µl of the RTS DNase / Solution FR5 directly on top of the membrane in the Low Elution Spin Filter and incubate for 15 minutes at room temperature. Note: The RTS DNase / Solution FR5 is 4 µl RTS DNase added to 46 µl of Solution FR5 per prep. Mix gently by pipetting. Do not vortex to mix. 14. Add 400 µl of Solution FR6 to the Low Elution Spin Filter and centrifuge at 13,000 x g for 30 seconds. Note: The Solution FR6 is added on top of the RTS DNase / Solution FR5. Do not centrifuge between steps 13 and 14. 15. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and load with 600 µl of Solution FR4. Centrifuge 30 seconds at 13,000 x g. 16. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and load with 600 µl of 100% Absolute ethanol (user provided). Centrifuge for 30 seconds at 13,000 x g. 7
17. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and dry spin the column in the centrifuge for 2 minutes at 16,000 x g. 18. Discard the 2 ml Collection Tube and transfer the Low Elution Spin Filter to a clean 2 ml Collection Tube (provided). 19. Elute the purified RNA with 20 µl of Solution FR7 (RNase-Free water). Place the FR7 buffer directly on top of the membrane and incubate for 3 minutes at room temperature to maximize RNA recovery. Centrifuge for 2 minutes at 16,000 x g. 20. Remove and discard the Low Elution Spin Filter and close the cap of the 2 ml Collection Tube. The eluted RNA in the 2 ml Collection Tube should be stored at -80 C until ready for use. Thank you for choosing the BiOstic FFPE Tissue RNA Isolation Kit! 8
Detailed Protocol (Describes what is happening at each step) Please wear gloves at all times. Be certain you have all kit components in hand before starting the protocol. The RTS DNase and the Low Elution Spin Filters are stored at 4 C. 1. To the tube containing 1-5 FFPE tissue slices (up to 15 mg) add 300 µl of Solution FR1 and 20 µl of Solution FR2. Vortex for 20 seconds at high speed to bring buffers in contact with paraffin slice(s) that may be stuck on the side of the tube. Briefly centrifuge at 2000 x g for 5 seconds to bring the solution and wax down into the tube. Note: If some of the wax is still stuck on the sides, it will fall down into the solution during the first heating step. What s happening: The Solution FR1 is a high activity dissolving buffer that breaks down wax and enhances Proteinase K activity. Solution FR2 (Proteinase K) breaks the RNA-protein cross-links and releases the RNA from the wax. 2. Heat the sample at 60ºC for 15 minutes. What s happening: The digestion step at 60ºC melts the wax and promotes optimal Proteinase K activity. RNA is very stable in the Solution FP1 / FP2 (Proteinase K). 3. Transfer the samples to the heat block set to 70ºC and heat for 15 minutes. Note: It is optimal for the recovered RNA to be processed expeditiously. However, if a second heat block is not available, place the samples at room temperature, turn the block to 70ºC and wait for it to reach temperature. Be sure the temperature has stabilized at 70ºC before transferring the samples back to the heat block. What s happening: The digestion step at 70ºC removes the cross links that are inhibitory to RT- qpcr. 4. Centrifuge the samples at 13,000 x g for 1 minute and transfer the digested lysate to a new 2 ml Collection Tube (provided). Note: After centrifugation, a thin top wax layer may be visible in the tube. Aspirate the lysate (approximately 300 µl) from below this layer. Avoid carryover of the waxy layer. What s happening: The centrifugation step sends the heavier lysate to the bottom of the tube while the lighter waxy material remains on top making for easier transfer of the lysate. 5. Add 300 µl of Solution FR3. What s happening: Solution FR3 is the chaotropic salt buffer required for RNA binding to the silica filter membrane. 6. Add 600 µl of 100% Absolute ethanol (user provided). Mix by vortexing or pipetting. What s happening: The 100% ethanol is needed for optimal binding of RNA to the spin filter membrane. 7. Load 600 µl onto the Low Elution Spin Filter. Centrifuge at 13,000 x g for 30 seconds. Note: Low Elution Spin Filters are not interchangeable with other MO BIO Spin Filters. 9
8. Decant the flow-through, put the column back into the same 2 ml Collection Tube and load the remaining volume of lysate/bind to the Low Elution Spin Filter. Centrifuge at 13,000 x g for 30 seconds. What s happening: The RNA is bound to the spin filter membrane. 9. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and load with 600 µl of Solution FR4. Centrifuge for 30 seconds at 13,000 x g. What s happening: The Solution FR4 is an isopropanol based wash buffer for removal of protein and other non-aqueous contaminants from the spin filter membrane. 10. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and load with 600 µl of 100% Absolute ethanol (user provided). Centrifuge for 30 seconds at 13,000 x g. What s happening: The ethanol wash removes salt and residual isopropanol from the spin filter membrane prior to the On Column DNase digestion. 11. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and dry spin the column in the centrifuge for 2 minutes at 16,000 x g. What s happening: Residual ethanol is removed from the spin filter membrane. 12. Discard the 2 ml Collection Tube and transfer the Low Elution Spin Filter to a clean 2 ml Collection Tube (provided). 13. Add 50 µl of the RTS DNase / Solution FR5 directly on top of the membrane in the Low Elution Spin Filter and incubate for 15 minutes at room temperature. Note: The RTS DNase / Solution FR5 is 4 µl RTS DNase added to 46 µl of Solution FR5 per prep. Mix gently by pipetting. Do not vortex to mix. What s happening: DNase I is mixed with high activity digestion buffer and is used to completely remove genomic DNA from the spin filter membrane. 14. Add 400 µl of Solution FR6 to the Low Elution Spin Filter and centrifuge at 13,000 x g for 30 seconds. Note: The Solution FR6 is added on top of the RTS DNase / Solution FR5. Do not centrifuge between steps 13 and 14. What s happening: Solution FR6 is a wash buffer used to inactivate DNase I and wash away residual enzyme and digested DNA while allowing RNA to remain tightly bound to the spin filter membrane. 15. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and load with 600 µl of Solution FR4. Centrifuge for 30 seconds at 13,000 x g. What s happening: Solution FR4 is an isopropanol based wash buffer used to remove residual salt and contaminants on the spin filter membrane. 16. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and load with 600 µl of Absolute 100% ethanol (user provided). Centrifuge for 30 seconds at 13,000 x g. 10
What s happening: The ethanol is used to remove any final residual salts and isopropanol on the spin filter membrane in preparation for the release and elution of the bound RNA. 17. Decant the flow-through, put the column back into the same 2 ml Collection Tube, and dry spin the column in the centrifuge for 2 minutes at 16,000 x g. What s happening: Residual ethanol is removed from the spin filter membrane in preparation for the release and elution of the bound RNA. 18. Discard the 2 ml Collection Tube and transfer the Low Elution Spin Filter to a clean 2 ml Collection Tube (provided). 19. Elute the purified RNA with 20 µl of Solution FR7 (RNase-Free water). Place the FR7 buffer directly on top of the membrane and incubate for 3 minutes at room temperature to maximize RNA recovery. Centrifuge for 2 minutes at 16,000 x g. What s happening: Solution FR7 is highly pure water used to elute the RNA from the spin filter membrane. 20. Remove and discard the Low Elution Spin Filter and close the cap of the 2 ml Collection Tube. The eluted RNA in the 2ml Collection Tube should be stored at -80 C until ready for use. Thank you for choosing the BiOstic FFPE Tissue RNA Isolation Kit! 11
Hints and Troubleshooting Guide Low Yields or RNA Degradation If the yield or integrity of RNA obtained is lower than expected, the following reasons may apply: RNA yield and quality from FFPE tissue will vary based on the tissue source, the age of the sample, the thickness of the slice, the length of time it was fixed, and the ratio of wax to tissue in each slice. RNA will not always run correctly on non-denaturing gels and may show smearing due to RNA secondary structure. Run RNA on a denaturing gel according to the Protocol for Formaldehyde Gel Electrophoresis. Alternatively, to visualize RNA on a non-denaturing agarose gel, heat denature the RNA prior to loading by performing the steps for RNA Sample Preparation under the section below called Protocol for Formaldehyde Gel Electrophoresis. RNA should only be heated in water or in a loading buffer that contains EDTA to chelate divalent cations that can cause hydrolysis of RNA. The 260/280 ratio is a good indicator of RNA quality as the absorbance at 260 will increase as RNA is digested into smaller fragments and single nucleotides. A ratio above 2.3 may indicate RNA degradation. If you are using the Agilent BioAnalyzer to visualize RNA, make sure to perform the heat denaturation step prior to loading the sample into the chip to obtain accurate RNA profiles. RNA Floats Out of Well When Loaded on a Gel Residual Ethanol may be in the final sample. To ensure complete drying of the membrane after the ethanol wash, centrifuge the Spin Filter in a clean 2 ml Collection Tube for an additional minute after the final wash step. If you live in a humid climate, you may experience increased difficulty with drying the membrane in the centrifuge. Increase the centrifugation times by another minute. RNA Has Low 260/280 Ratio The ratio for pure RNA should be 1.9-2.1. A 260/280 reading below 1.6 may have significant protein contamination. A low ratio can occur when the sample is measured in water. The low ph of water can influence the 280 reading and cause reduced sensitivity to protein contamination*. Remeasure the 260/280 diluting the RNA for measurement in 10 mm Tris, ph 7.5. Low 260/280 ratios may occur if excess tissue was used. Do not exceed 15 mg of tissue. *Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of ph and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474. 12
Hints and Troubleshooting Guide cont. Concentrating the RNA Your final volume will be 20 µl. If this is too dilute for your purposes, add 2 µl of 3 M Sodium Acetate and mix. Then add 100 µl of 100% cold ethanol. Mix and incubate at -70 C for 15 minutes or -20 C for 2 hours to overnight. Centrifuge at 16,000 x g for 10-15 minutes at 4 C. Decant all liquid. Briefly dry residual ethanol in a speed vac or ambient air. Avoid over-drying the pellet or resuspension may be difficult. Resuspend precipitated RNA in desired volume of RNase-Free water. Storing RNA RNA is eluted in RNase-Free water (Solution FR7) and should be used immediately or stored at -20 C or -80 C to avoid degradation. RNA can be precipitated in ethanol and stored at -20 C to ensure minimal degradation during long term storage. 13
Technical Guide Protocol for Formaldehyde Agarose Gel Electrophoresis Solutions needed. 10x Formaldehyde agarose gel buffer 200 mm 3-[N-morpholino] propanesulfonic acid (MOPS) (free acid) 50 mm Sodium Acetate 10 mm EDTA ph to 7.0 with Sodium Hydroxide. 1x Formaldehyde agarose gel buffer (1L) 100 ml 10x Formaldehyde agarose gel buffer 20 ml 37% (12.3 M) Formaldehyde 880 ml DEPC treated water 5x RNA Loading Dye 16 µl Saturated aqueous Bromophenol blue solution 80 µl 0.5 M EDTA, ph 8.0 720 µl 37% (12.3 M) Formaldehyde 2 ml 100% Glycerol 3084 µl Formamide 4 ml 10x Formaldehyde agarose gel buffer Formaldehyde Agarose Gel preparation 1.2% in 100 ml Mix the following: 1.2 g Agarose 10 ml 10x Formaldehyde agarose gel buffer 90 ml DEPC treated water Heat the mixture in a microwave oven to melt the agarose. Cool to 65 C in a water bath. Add 1.8 ml 37% (12.3 M) Formaldehyde and 2 µl of 5 mg/ml Ethidium Bromide. Swirl to mix and pour into a gel box. The gel must be pre-run for 30 minutes in 1x Formaldehyde agarose gel buffer before loading the samples. RNA sample preparation for formaldehyde gels The eluted RNA samples must be denatured before running on a formaldehyde agarose gel. To the sample, add 1 volume of 5x RNA loading dye for each 4 volumes of RNA sample (i.e. 2 µl of 5x RNA loading dye for each 8 µl of RNA sample). Mix the samples and briefly centrifuge to collect the sample at the bottom of the tube. Incubate at 65 C for 3-5 minutes, then chill on ice and load into the Formaldehyde agarose gel. Run the gel at 5-7 V/cm in 1x Formaldehyde agarose gel buffer. References 1. Beintema, J.J., Campagne, R.N., and Gruber, M. (1973). Biochim. Biophys. Acta 310: 148-160. 2. Kaplan, B.B., Bernstein, S.L., and Gioio, A.E. (1979). Biochem. J. 183: 181-184. 14
Contact Information Technical Support: Phone MO BIO Laboratories, Inc. Toll Free 800-606-6246, or 760-929-9911 Email: technical@mobio.com Fax: 760-929-0109 Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA 92010 Ordering Information: Direct: Phone MO BIO Laboratories, Inc. Toll Free 800-606-6246, or 760-929-9911 Email: orders@mobio.com Fax: 760-929-0109 Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA 92010 For the distributor nearest you, visit our web site at www.mobio.com/distributors 15
Products recommended for you For a complete list of products available from MO BIO Laboratories, Inc., visit www.mobio.com Description Catalog No. Quantity BiOstic Paraffin Removal Reagent 12251-50 2 x 25 ml UltraClean Tissue & Cells RNA Isolation Kit 15000-50 50 preps UltraClean Tissue & Cells DNA Isolation Kit PowerLyzer UltraClean Tissue & Cells RNA Isolation Kit PowerLyzer 24 Bench Top Bead-Based Homogenizer 12334-50 12334-250 50 preps 250 preps 15055-50 50 preps 13155 1 unit 13111-V Vortex Genie 2 13111-V-220 UltraClean Lab Cleaner 12095-250 12095-500 12095-1000 RNase-Free Gloves 1556-XS 1556-S 1556-M 1556-L 1 unit (120V) 1 unit (220V) 250 ml squeeze bottle 500 ml squeeze bottle 1 L bottle X-Small (Bag of 150) Small (Bag of 150) Medium (Bag of 150) Large (Bag of 150) 16