4. Refer to the manufacturer's documentation for instructions for installing the program on the instrument. Before first use: prepare Wash Solutions Prepare the Wash Solutions from the concentrates: • Add 10 mL of isopropanol to Wash Solution 1 Concentrate, mix, and store at room temperature.
USER GUIDE MagMAXTM mirVanaTM Total RNA Isolation Kit High-throughput isolation of RNA (including small RNA) from cells Catalog Number A27828 Pub. No. MAN0011138 Rev. B.0 WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support. Materials required but not supplied Unless otherwise indicated, all materials are available from Life Technologies (thermofisher.com). MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier. Product description The MagMAXTM mirVanaTM Total RNA Isolation Kit is designed for isolation of total RNA, including microRNA, from a wide variety of sample matrices. The kit uses MagMAXTM magnetic-bead technology, ensuring reproducible recovery of high-quality RNA that is suitable for a broad range of applications, including TaqMan® miRNA Detection Assays. This protocol describes isolation of RNA from cells, optimized for use with the MagMAXTM Express-96 Deep Well Magnetic Particle Processor, the KingFisherTM Flex Magnetic Particle Processor 96DW (96well deep well setting), or the KingFisherTM Duo Prime Magnetic Particle Processor (12-well deep well setting). Kit contents and storage Table 1 MagMAXTM mirVanaTM Total RNA Isolation Kit (Cat. no. A27828, 96 reactions) Contents Box 1 of 2 Proteinase K[1], 50 mg/mL Lysis/Binding Enhancer TURBO DNaseTM, 20 U/µL Box 2 of 2 Lysis Buffer PK Digestion Buffer[1] RNA Binding Beads[2] Wash Solution 1 Concentrate[3] Wash Solution 2 Concentrate[3] Rebinding Buffer MagMAXTM TURBO DNaseTM Buffer Elution Buffer Processing Plate[1] Elution Plates Plate Covers Amount 0.48 mL 0.96 mL 0.2 mL 115 mL 4.4 mL 2 mL 20 mL 60 mL 4.8 mL 4.6 mL 9.6 mL 1 2 4 Storage 25°C to 15°C 15°C to 30°C [1] Not used for RNA isolation from cells. [2] Do not freeze the RNA Binding Beads. [3] Final volume; see "Before first use: prepare Wash Solutions" on page 2. Item Source Magnetic particle processor, one of the following: MagMAXTM Express96 Deep Well Magnetic Particle Processor Cat. no. 4400079 KingFisherTM Flex Magnetic Particle Processor 96DW[1] Thermo Scientific Cat. no. 5400630 KingFisherTM Duo Prime Magnetic Particle Processor[1] Thermo Scientific Cat. no. 5400110 Other equipment Thermo ScientificTM Compact Digital Microplate Shaker Fisher Scientific Cat. no. 11-676-337 Fisher ScientificTM Analog Vortex Mixer Fisher Scientific Cat. no. 02-215-365 Economy Lab Incubator Fisher Scientific Cat. no. S50441A VWRTM Digital Mini Incubator VWR Cat. no. 10055-006 Adjustable micropipettors MLS Multi-channel micropipettors MLS Ice bucket MLS Plates and combs[2] Deep Well Plates, one of the following: MagMAXTM Express-96 Deep Well Plates Cat. no. 4388476 KingFisherTM Flex Microtiter Deepwell 96 Plate, Sterile Thermo Scientific Cat. no. 95040460 Standard Well Plates, one of the following: MagMAXTM Express-96 Standard Plates Cat. no. 4388475 KingFisherTM 96 KF Microplate Thermo Scientific Cat. no. 97002540 One of the following tip combs, depending on the magnetic particle processor used: MagMAXTM Express-96 Deep Well Tip Combs Cat. no. 4388487 KingFisherTM 96 Tip Comb for DW Magnets Thermo Scientific Cat. no. 97002534 KingFisherTM Duo 12-Tip Comb, for Microtiter 96 Deepwell plate Cat. no. 97003500 Other consumables MicroAmpTM Clear Adhesive Film Cat. no. 4306311 Nonstick, RNase-free Microfuge Tubes (1.5 mL) Cat. no. AM12450 Nonstick, RNase-free Microfuge Tubes (2.0 mL) Cat. no. AM12475 Conical tubes (15 mL) Cat. no. AM12500 Aerosol-resistant pipette tips MLS Reagent reservoirs MLS For Research Use Only. Not for use in diagnostic procedures. Item Reagents Isopropanol, 100% (molecular grade or higher) Ethanol, 200 proof (absolute) 2-Mercaptoethanol Source MLS MLS MLS [1] See "If needed, download the KingFisherTM Flex or Duo program" on page 2 [2] KingFisherTM Duo Combi Pack (Cat. no. 97003530) includes plates and combs for the KingFisherTM Duo Prime Magnetic Particle Processor. Sample collection and storage We recommend using up to 1 × 106 cells grown in 96-well or 24-well cell culture plates. Cells can be adherent or in suspension. We recommend the methods described in this User Guide to prepare cells from adherent and liquid cultures. · See "Lyse the cells and bind the RNA to the RNA Binding Beads" on page 3 if you are using the MagMAXTM Express-96 Deep Well Magnetic Particle Processor or the KingFisherTM Flex Magnetic Particle Processor 96DW. · See "Lyse the cells and bind the RNA to the RNA Binding Beads" on page 5 if you are using the KingFisherTM Duo Prime Magnetic Particle Processor. Important procedural guidelines · Perform all steps at room temperature (2025°C) unless otherwise noted. · When mixing samples by pipetting up and down, avoid creating bubbles. · Cover the plate during the incubation and shaking steps to prevent spill-over and cross-contamination. The same Plate Cover can be used throughout the procedure, unless it becomes contaminated. · If you use a titer plate shaker other than the Thermo ScientificTM Compact Digital Microplate Shaker, verify that: The plate fits securely on your titer plate shaker. The recommended speeds are compatible with your titer plate shaker. Ideal speeds should allow for thorough mixing without splashing. · Volumes for reagent mixes are given per well. We recommend that you prepare master mixes for larger sample numbers. To calculate volumes for master mixes, refer to the per-well volume and add 5% overage. · Lysed samples can be stored in Lysis Binding Mix at 20°C for up to 4 days before adding the Binding Beads Mix. Thaw frozen samples to room temperatures before use. If needed, download the KingFisherTM Flex or Duo program The program required for this protocol is not pre-installed on the KingFisherTM Flex Magnetic Particle Processor 96DW or on the KingFisherTM Duo Prime Magnetic Particle Processor. 1. On the MagMAXTM mirVanaTM Total RNA Isolation Kit web page, scroll down to the Product Literature section. 2. Right-click on the appropriate program for your instrument: · A27828_FLEX_Tissue_Cells for KingFisherTM Flex Magnetic Particle Processor 96DW. · A27828_DUO_Tissue_cells for KingFisherTM Duo Prime Magnetic Particle Processor. 3. select Save as Target to download to your computer. 4. Refer to the manufacturer's documentation for instructions for installing the program on the instrument. Before first use: prepare Wash Solutions Prepare the Wash Solutions from the concentrates: · Add 10 mL of isopropanol to Wash Solution 1 Concentrate, mix, and store at room temperature. · Add 48 mL of ethanol to Wash Solution 2 Concentrate, mix, and store at room temperature. Before each use: prepare TURBO DNaseTM Solution and Binding Beads Mix · Prepare the TURBO DNaseTM Solution as indicated in the following table, mix, and store on ice until use. Component Volume per well MagMAXTM TURBO DNaseTM Buffer 48 µL TURBO DNaseTM 2 µL Total TURBO DNaseTM Solution 50 µL · Prepare the Binding Beads Mix as indicated in the following table, mix, and store on ice until use. Component RNA Binding Beads Lysis/Binding Enhancer Total Binding Beads Mix Volume per well 10 µL 10 µL 20 µL 2 MagMAXTM mirVanaTM Total RNA Isolation Kit (cells) User Guide Perform RNA extraction from cells Isolate RNA using the MagMAXTM Express96 Deep Well Magnetic Particle Processor or the KingFisherTM Flex Magnetic Particle Processor 96DW 1 Lyse the cells and bind the a. Prepare sufficient Lysis Binding Mix, according to the following table. RNA to the RNA Binding Component Beads Lysis Buffer Volume per well 99 µL Isopropanol 100 µL 2-Mercaptoethanol 1 µL Total Lysis Binding Mix 200 µL b. Collect the cells according to the following methods: · Adherent cells: remove the media from the wells of a 96-well or 24-well culture plate containing up to 1 × 106 cells and add 200 µL of Lysis Binding Mix to each sample. · Suspension cells: pellet cells (up to 1 × 106 ) in a 96-well or 24-well culture plate by spinning the culture plate at 1000 × g for 4 minutes at 4°C, remove the media from the wells, and add 200 µL of Lysis Binding Mix to each sample. IMPORTANT! Add Lysis Binding Mix to the cells immediately after they have been harvested. c. Lyse the samples by pipetting up and down 5 times. d. Incubate for 5 minutes, then transfer the cell lysates from the cell culture plate to a MagMAXTM Express-96 Deep Well Plate. e. Cover and shake the plate as indicated. Time 5 minutes Speed 1050 rpm (Speed 8) [1] [1] Setting for Lab-LineTM shaker. During the incubation, set up the processing plates (next section). f. Remove the plate from the shaker and add 20 µL of Binding Beads Mix to each sample. g. Proceed directly to "Wash, rebind, and elute the RNA" on page 3. 2 Set up the processing platesWhile the samples are incubating, set up the Wash, DNase, Elution, and Tip Comb Plates outside the instrument as described in the following table. Table 2 Processing plates Plate ID Wash Plate 1 Wash Plate 2 DNase Plate[2] Wash Plate 3 Wash Plate 4 Elution Plate Tip Comb Plate position[1] 2 3 4 5 6 7 8 Plate type Standard Standard Standard Standard Standard Standard Deep Well or standard Reagent Volume per well Wash Solution 1 150 µL Wash Solution 2 150 µL TURBO DNaseTM Solution 50 µL Wash Solution 2 150 µL Wash Solution 2 150 µL Elution Buffer 50100 µL[3] Place a MagMAXTM Express-96 Deep Well Tip Comb in a MagMAXTM Express-96 Deep Well Plate or in a MagMAXTM Express-96 Standard Plate. [1] Position on the instrument [2] The instrument prompts the user to add 50 µL of Rebinding Buffer and 100 µL of isopropanol to the DNase Plate after the DNase treatment step. [3] Use 50 µL for low input or 100 µL for high input. 3 Wash, rebind, and elute the a. Ensure that the instrument is set up for processing with the deep well magnetic head and select the RNA program on the instrument. · AM1830DW on MagMAXTM Express-96 Deep Well Magnetic Particle Processor · A27828_FLEX_Tissue_Cells on KingFisherTM Flex Magnetic Particle Processor b. Start the run and load the prepared processing plates in their positions when prompted by the instrument (see Table 2). c. Load the sample plate (containing lysate, isopropanol, and Binding Beads Mix) at position 1 when prompted by the instrument. d. When prompted by the instrument (3035 minutes after the initial start): 1. Remove the DNase Plate from the instrument. MagMAXTM mirVanaTM Total RNA Isolation Kit (cells) User Guide 3 3 Wash, rebind, and elute the RNA (continued) 2. Add 50 µL of Rebinding Buffer and 100 µL of isopropanol to each sample well. Add Rebinding Buffer and isopropanol immediately after the prompt, to prevent excessive drying of any beads that are still captured on the Tip Comb. IMPORTANT! Do not pre-mix the Rebinding Buffer and isopropanol. Add them separately to the samples. 3. Load the DNase Plate back onto the instrument, and press Start. e. At the end of the run (approximately 45 minutes after the initial start), remove the Elution Plate from the instrument and seal immediately with a new MicroAmpTM Clear Adhesive Film. · (Optional) Eluates can be transferred to a storage plate after collection. · If excess bead residue is seen in the wells, place the Elution Plate on the Magnetic Stand-96 to capture any residue prior to downstream use of the RNA. IMPORTANT! Do not allow the purified samples to sit uncovered at room temperature for more than 10 minutes, to prevent evaporation and contamination. The purified samples are ready for immediate use. Alternatively, store the covered Elution Plate: · On ice for up to 8 hours. · At 20°C or 80°C for long-term storage. 4 MagMAXTM mirVanaTM Total RNA Isolation Kit (cells) User Guide Isolate RNA using the KingFisherTM Duo Prime Magnetic Particle Processor 1 Lyse the cells and bind the a. Prepare sufficient Lysis Binding Mix, according to the following table. RNA to the RNA Binding Component Beads Lysis Buffer Volume per well 99 µL Isopropanol 100 µL 2-Mercaptoethanol 1 µL Total Lysis Binding Mix 200 µL b. Collect the cells according to the following methods: · Adherent cells: remove the media from the wells of a 96-well or 24-well culture plate containing up to 1 × 106 cells and add 200 µL of Lysis Binding Mix to each sample. · Suspension cells: pellet cells (up to 1 × 106 ) in a 96-well or 24-well culture plate by spinning the culture plate at 1000 × g for 4 minutes at 4°C, remove the media from the wells, and add 200 µL of Lysis Binding Mix to each sample. IMPORTANT! Add Lysis Binding Mix to the cells immediately after they have been harvested. c. Lyse the samples by pipetting up and down 5 times. d. Incubate for 5 minutes, then transfer the cell lysates from the cell culture plate to Row H of a MagMAXTM Express-96 Deep Well Plate. e. Cover and shake the plate as indicated. Time 5 minutes Speed 1050 rpm (Speed 8) [1] [1] Setting for Lab-LineTM shaker. f. Remove the plate from the shaker and add 20 µL of Binding Beads Mix to each sample. 2 Set up the processing plate Add processing reagents as indicated in the following table. Table 3 Volume of processing reagents and plate location Row ID Elution Tip Comb Wash 4 Wash 3 DNase[3] Wash 2 Wash 1 Plate row[1] A B C D E F G Reagent Volume per well Elution Buffer 50100 µL[2] Place a KingFisherTM Duo 12-Tip Comb in Row B. Wash Solution 2 150 µL Wash Solution 2 150 µL TURBO DNaseTM Solution 50 µL Wash Solution 2 150 µL Wash Solution 1 150 µL [1] Row on the MagMAXTM Express-96 Deep Well Plate. [2] Use 50 µL for low input or 100 µL for high input. [3] The instrument prompts the user to add 50 µL of Rebinding Buffer and 100 µL of isopropanol to the DNase Plate after the DNase treatment step. 3 Wash, rebind, and elute the a. Ensure that the instrument is set up for processing with the deep well 96well plates and select the RNA program A27828_DUO_Tissue_cells on the instrument. b. Start the run and load the prepared processing plate when prompted by the instrument (see "Wash, rebind, and elute the RNA" on page 5). c. When prompted by the instrument (3035 minutes after the initial start): 1. Remove the plate from the instrument. 2. Add 50 µL of Rebinding Buffer and 100 µL of isopropanol to each sample well in Row E. Add Rebinding Buffer and isopropanol immediately after the prompt, to prevent excessive drying of any beads that are still captured on the Tip Comb. IMPORTANT! Do not pre-mix the Rebinding Buffer and isopropanol. Add them separately to the samples. 3. Load the plate back onto the instrument, and press Start. d. At the end of the run (approximately 45 minutes after the initial start), remove the Elution Plate from the instrument and transfer the eluted RNA (Row A) to an Elution Plate. e. Seal immediately with a new MicroAmpTM Clear Adhesive Film. IMPORTANT! Do not allow the purified samples to sit uncovered at room temperature for more than 10 minutes, to prevent evaporation and contamination. The purified samples are ready for immediate use. Alternatively, store the covered Elution Plate: · On ice for up to 8 hours. · At 20°C or 80°C for long-term storage. MagMAXTM mirVanaTM Total RNA Isolation Kit (cells) User Guide 5 Limited product warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support. The information in this guide is subject to change without notice. DISCLAIMER TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses. ©2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. thermofisher.com/support | thermofisher.com/askaquestion thermofisher.com 11 December 2018Antenna House PDF Output Library 6.5.1119 (Windows (x64))