(Spin Column) For purification of DNA and RNA from formalin-fixed, paraffin-embedded. tissue sections. Instruction for Use. For Research Use Only

Transcription

1 AmoyDx FFPE DNA/RNA Kit (Spin Column) For purification of DNA and RNA from formalin-fixed, paraffin-embedded tissue sections Instruction for Use For Research Use Only Instruction Version: B1.0 Revision Date: May 2013 Store kit dry at room temperature (15~25 ) Store DNase I at -20±5

2 Introduction The AmoyDx FFPE DNA/RNA Kit provides silica-based membrane and special lysis buffer system for FFPE tissue DNA/RNA extraction effectively. This kit is specially designed for simultaneously isolation and purification of DNA and RNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections. The purified DNA/RNA is suitable for downstream applications such as reverse transcription, RT-PCR, and real-time quantitative RT-PCR (qrt-pcr). Principle FFPE specimen tissue sections are first deparaffinized with xylene/ethanol method, then incubated in Buffer RTL and Proteinase K solution, to release DNA and RNA from the sections. After centrifugation RNA is in the supernatant, and DNA is in underlying the precipitates. DNA Extraction: The precipitates incubated in Buffer DTL and Proteinase K solution to completely release DNA. A short incubation in Buffer DES at a higher temperature partially reverses formalin crosslinking of the released nucleic acids, improving DNA yield and quality as well as DNA performance in downstream assays. The lysate is mixed with Buffer DTB and ethanol to provide appropriate binding conditions for DNA, then the mixture is applied to a DNA spin column, where the DNA binds to the membrane and impurities are removed with wash buffer. The DNA is eluted in Buffer DTE. RNA Extraction: The supernatant incubated at 80 to partially reverse formalin crosslinking of the released nucleic acids, improving RNA yield and quality as well as RNA performance in downstream enzymatic assays. Next, genomic DNA in the solution is removed with the DNase I. The lysate is mixed with Buffer RTB and ethanol to provide appropriate binding conditions for RNA, and the sample is then applied to a RNA spin column, where the total RNA binds to the membrane and impurities are removed with wash buffer. The total RNA is eluted in buffer RTE. Kit Contents This kit contains sufficient reagents to perform 36 tests (Table 1). Table 1 Kit Contents Item Amount Tube No. DNA Spin Columns 36 RNA Spin Columns 36 Collection Tubes (2 ml) 2 72 Centrifugal Tubes (1.5 ml) 2 72 Buffer RTL 10 ml 1 Proteinase K Solution 1.8 ml 2 DNase I Magic Buffer 1.5 ml 3 DNase I (30 U/µL) 40 µl 4 Buffer RTB 15 ml 5 Buffer RTW (concentrate) 2 7 ml 6 Buffer RTE 8 ml 7 RNase-free Water 1.5 ml 8 1 / 6

3 Buffer DTL 10 ml 1 Buffer DES 900 µl 2 Buffer DTB 10 ml 3 Buffer DW1 (concentrate) 13 ml 4 Buffer DW2 (concentrate) 6 ml 5 Buffer DTE 8 ml 6 Instruction for Use 1 Note: 1. Buffer RTB, Buffer DTB and Buffer DW1 contain guanidine salt, not compatible with disinfectants containing bleach or acidic solutions. 2. For the first time use, add 21 ml ethanol (96~100%) into Buffer RTW (concentrate), add 17 ml ethanol (96~100%) into Buffer DW1 (concentrate) and mix thoroughly; add 24 ml ethanol (96~100%) into Buffer DW2 (concentrate ) and mix thoroughly. Tick the check box on the bottle label. 3. DNase I (30 U/µL) should be stored at -20±5 upon receipt. For the first time use, add 360 µl RNase-free Water into DNase I (30 U/µL) to obtain 3 U/µL solution, mix well by pipetting gently up and down, then subpackage into small volume and store at -20±5. Equipment and Reagents Not Supplied With Kits 1. Sterile, RNase-free pipet tips 2. Ethanol (96~100%) 3. Xylene 4. RNaseA (optional) 5. Water bath or heated orbital incubator (37~90 adjustable) 6. Microcentrifuge (8000~14000 rpm adjustable) 7. Vortexer 8. Palm centrifuge 9. Recommend: microtome suitable for sectioning paraffin-embedded tissue that is capable of producing 5~10 μm sections. Storage and Expiry Date The shelf life of the kit is 12 months. DNase I (30 U/µL) should be stored at -20±5. After DNase I (30 U/µL) is diluted to 3 U/µL, please avoid unnecessary freezing and thawing. Do not use the DNase I after eight freeze-thaw cycles. The other reagents should be stored dry at room temperature (15~25 ). Starting material Standard formalin-fixation and paraffin-embedding procedures always result in significant fragmentation of nucleic acids. To limit the extent of DNA/RNA fragmentation, be sure to: 1. Fix tissue samples in 4~10% neutral formalin solution as quickly as possible after surgical removal. 2. Use a fixation time of 14~24 hours (longer fixation time leads to more severe DNA/RNA fragmentation, resulting in poor performance in downstream assays). 3. Thoroughly dehydrate samples prior to embedding (residual formalin maybe inhibit the digestion of the Proteinase K). 2 / 6

4 4. The starting material for DNA/RNA purification should be freshly cut sections of FFPE tissue, each with a thickness of up to 5~10 μm. (Thicker sections may result in lower DNA/RNA yields, even after prolonged incubation with proteinase K). 5. The FFPE tissue area should be 0.5~1 cm 2. If the FFPE tissue surface area is less than 0.5 cm 2, please use more sections. 6. FFPE sample s storage time should be less than three years. Guidelines for Sectioning Paraffin Blocks To use this kit, it needs 5~10 μm sections of the tissue in paraffin block. You may use any method for sectioning the paraffin blocks. General guidelines for sectioning paraffin blocks are outlined below: 1. Avoid nuclease contamination by using a clean, sharp microtome blade and tweezers. 2. When multiple samples are processed, clean the microtome blade and tweezers with RNase-inactivating agents to avoid cross-contamination of nucleic acids and RNases. UV irradiation for 10 minutes is recommended after cleaning. 3. Always wear latex or nitrile gloves. 4. Cut 5~10 μm thick sections from trimmed paraffin blocks with a tissue surface area about 0.5~1 cm 2. General Handling of RNA Observe the following guidelines to prevent RNase contamination and maximize the RNA yield: 1. Use disposable, sterile plastic ware. 2. Use sterile, new pipette tips and microcentrifuge tubes. 3. Wear latex or nitrile gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin. 4. Use proper microbiological aseptic technique when working with RNA. 5. Use proper method to remove RNase contamination from surfaces. Safety Information Follow the safety guidelines below when using the kit. 1. Treat all reagents supplied in the kit as potential irritants. 2. Wear a lab coat, disposable gloves, and protective goggles. 3. If a buffer spill occurs, clean with a suitable laboratory detergent and water. 4. Dispose any tissue in designated biohazard containers. 5. If a spill contains potentially infectious reagents, clean the affected area first with laboratory detergent and water, then with 1% (v/v) sodium hypochlorite or a suitable laboratory disinfectant. Protocol 1. Deparaffinization 1.1 Using a scalpel, trim excess paraffin off the sample block. 1.2 Cut sections with a thickness of 5~10 μm and a surface area between 0.5~1 cm Immediately place 2~5 sections in a 1.5 ml centrifugal tube. 1.4 Add 1 ml xylene, close the lid and vortex vigorously for 10 seconds. 1.5 Centrifuge at rpm for 2 min at room temperature. 1.6 Remove the supernatant by pipetting (do not remove any of the pellet). 1.7 Add 1mL ethanol (96~100%) to the pellet, and mix by vortexing for 10 seconds. 1.8 Centrifuge at rpm for 2 min at room temperature. 3 / 6

5 1.9 Remove the supernatant by pipetting (do not remove any of the pellet) Open the tube and incubate at room temperature for 10 min, or at 37 for 5 min. Make sure all residual ethanol has evaporated before proceeding. 2. Tissue Lysis 2.1 Add 200 µl Buffer RTL and 25 µl Proteinase K Solution into the tube above, mix gently by pipetting up and down. 2.2 Incubate at 56 for 15 min. 2.3 Centrifuge at rpm for 2 min. 2.4 Transfer 180 µl supernatant to a clean 1.5 ml centrifugal tube for RNA extraction. 2.5 The remaining solution and precipitates are used for DNA extraction. 3. DNA Extraction For the first time use, please add 17 ml ethanol (96~100%) into Buffer DW1 (concentrate), add 24 ml ethanol (96~100%) into Buffer DW2 (concentrate), and mark it clearly. Before the DNA extraction, please check the reagents without leakage. Shake the reagents gently to mix the solutions. If the reagents contain precipitates, dissolved by heating at Add 140 µl Buffer DTL and 15 µl Proteinase K Solution into the underlying precipitates, mix gently by pipetting up and down. 3.2 Incubate at 56 for 1 hour for lyse the sample tissue. If the tissue has not been completely lysed, or need higher concentration of DNA, incubate for further time or overnight. 3.3 Add 10 µl Buffer DES, mix gently by vortexing and briefly centrifuge for 5~10 seconds. Transfer the centrifugal tube to heated orbital incubator and incubate at 90 for 2 hour. 3.4 Briefly centrifuge for 5~10 seconds. If RNA-free genomic DNA is required, allow the sample to cool to room temperature, add 2 µl RNase A (100 mg/ml) and incubate for 5 min at room temperature. 3.5 Add 200 µl Buffer DTB and 200 µl ethanol (96~100%), mix by vortexing. 3.6 Briefly centrifuge for 5~10 seconds. 3.7 Transfer the entire lysate to the DNA Spin Column (in a 2 ml collection tube) without wetting the rim, close the lid, and centrifuge at 8000 rpm for 1 min. 3.8 Discard the flow-through in collection tube. 3.9 Add 600 µl Buffer DW1 to DNA Spin Column, centrifuge at 8000 rpm for 1 min Discard the flow-through in collection tube Add 600 µl Buffer DW2 to DNA Spin Column, centrifuge at 8000 rpm for 1 min Discard the collection tube with flow-through Place the DNA Spin Column in a clean 2 ml collection tube, centrifuge at rpm for 3 min Discard the collection tube with flow-through Place the DNA Spin Column in a clean 1.5 ml centrifugal tube Apply 30~100 µl Buffer DTE to the center of the membrane. Do not touch the membrane Incubate at room temperature (15~25 ) for 1~5 min Centrifuge at rpm for 1 min The eluted DNA is immediately ready for use or for storage under -20. Note: Decrease the volume of Buffer DTE properly to obtain a higher concentration of DNA. 4. RNA Extraction 4 / 6

6 For the first time use, please add 21 ml ethanol (96~100%) into Buffer RTW (concentrate), and mark it clearly. For the first time use, please add 360 µl RNase-free Water into DNase I (30 U/µL) to obtain 3 U/µL solution, and mix well by pipetting gently up and down. DNase I working solution should be stored at -20±5. Before the RNA extraction, please check the reagents without leakage; shake the reagents gently to mix the solution. If the reagents contain a precipitate, dissolved by heating at Transfer the centrifugal tube which contains 180 µl supernatant to heated orbital incubator and incubate at 80 for 15 min. 4.2 Allow the sample to cool to room temperature, then briefly centrifuge for 5~10 seconds. 4.3 According to the ratio of 20 µl DNase I Magic Buffer and 10 µl DNase I (3 U/µL) per sample, mix DNase I Magic Buffer and DNase I (3 U/µL) by pipetting up and down to get DNase I working solution. 4.4 Add 30 µl DNase I working solution to the sample, mix gently by pipetting up and down. 4.5 Stand at room temperature for 15 min. 4.6 Add 340 µl Buffer RTB and 750 µl ethanol (96~100%), mix thoroughly by pipetting gently up and down. 4.7 Briefly centrifuge for 5~10 seconds. 4.8 Transfer 650 µl lysate to the RNA Spin Column (in a 2 ml collection tube) without wetting the rim, close the lid, and centrifuge at rpm for 30 seconds. 4.9 Discard the flow-through in collection tube Transfer the remaining solution to the RNA Spin Column, without wetting the rim, close the lid Centrifuge at rpm for 30 second Discard the flow-through in collection tube Add 600 µl Buffer RTW to RNA Spin Column, centrifuge at rpm for 30 seconds Discard the flow-through in collection tube Add 600µL Buffer RTW to RNA Spin Column, centrifuge at rpm for 30 seconds Discard collection tube with flow-through Place the RNA Spin Column in a clean 2 ml collection tube, centrifuge at rpm for 5 min Discard the collection tube with flow-through Place the RNA Spin Column in a clean 1.5 ml centrifugal tube Apply 30~80 µl Buffer RTE to the center of the membrane. Do not touch the membrane Stand at room temperature (15~25 ) for 1~3 min Centrifuge at rpm for 1 min The eluted RNA is ready for use. If it s not used immediately, it should be stored under -70. Note: Decrease the volume of Buffer RTE properly to obtain a higher concentration of RNA. Warnings and Precautions 1. Please read the instruction carefully and become familiar with all components of the kit prior to use. 2. The Kit is intended for research use only. Prior to using it for other purpose, the user must validate the system in compliance with the applicable law, directives, and regulations. 3. The extracted DNA and RNA quality is subject to the influence of such factors as sample source, sampling 5 / 6

7 process, formalin fixation, paraffin embedding and storage conditions. 4. Sample quality has a high impact on quality and amount of the purified DNA and RNA. 5. Due to fixation and embedding conditions, nucleic acids in FFPE samples are usually heavily fragmented and chemically modified by formaldehyde. DNA/RNA purified from FFPE samples should not be used in downstream applications that require full-length DNA/RNA. 6. Buffer RTB, Buffer DTB and Buffer DW1 contain guanidine salt, which can form highly reactive compounds when combined with bleach. Do not add bleach or acidic solutions directly to the sample-preparation waste. If the liquid containing this buffer is spilt, clean with suitable laboratory detergent and water. 7. Unless otherwise indicated, perform all steps of the procedure at room temperature (15~25 C). 8. For the first time use, make sure that Buffer RTW (concentrate), Buffer DW1 (concentrate) and Buffer DW2 (concentrate) are diluted with the ethanol (96~100%) as described and mixed well. 9. Do not exchange and mix up the kit contents with different batches. 10. We recommend the use of aerosol-barrier filter pipet tips; avoid touching the membrane with the pipet tips. Open only one tube at a time, and take care to avoid generating aerosols. 11. Only trained professionals should use this kit. Please wear suitable lab coat and disposable gloves. 12. The used kit and flow-through fractions may contain hazardous waste and should be disposed of properly. 6 / 6