Bioneer Corporation 8-11, Munpyeongseo-ro, Daedeok-gu, Daejeon 34302, Republic of Korea Tel: +82-42-930-8777 Fax: +82-42-930-8688 Email: sales@bioneer.co.kr
Contents I. Overview 1 II. Kit Components 1 III. Storage 2 IV. Intended Use 2 V. Safety Warnings and Precautions 2 VI. Warranty and Liability 2 VII. Technical Assistance 3 VIII. Quality Management 3 IX. Product Specifications 4 X. Principle 4 XI. Materials and Equipment Needed But Not Provided 5 Magnetic Separation Rack Choice 5 XII. Protocols 6 Before you begin 6 Protocol for Micro/Mini/Midi Extraction 7 Summary of reagent volumes required in each step 10 RNA Clean up Protocol 10 ONE Step RNA Cleanup 11 Experimental data 12 XIII. Troubleshooting Guide 14 XIV. Ordering Information 15 XV. Explanation Symbols 16
I. Overview Description MagListo TM 5M Cell Total RNA Extraction Kit is an innovative product to extract total RNA from a wide range of cell types. This kit is designed to be applicable to variable scales of total RNA extraction (micro/mini/midi extraction) by using a proper volume of each solution suggested in this User s Guide. MagListo TM 5M Cell Total RNA Extraction Kit guarantees RNA of high purity in unprecedented short period of time. MagListo TM Magnetic Separation Rack, which is also available from Bioneer (TM- 1000~1020), will greatly enhance user s convenience and save processing time by removing the necessity of the centrifugation. Features and Benefits - Magnetic Nano Beads enable the rapid extraction of RNA : 8 min - No need of expensive instruments. - One kit serves both mini and midi scale extraction. Applications Applicable to assays requiring RNA, including, but not limited, RT-PCR, cdna synthesis, Northern, dot, and slot blot analyses, Primer extension, Poly A + RNA selection, Microarrays II. Kit Components MagListo TM 5M Cell Total RNA Extraction Kit *K-3610 ** K-3611 Buffer 1 (Binding) 4 ml x 1 ea 25 ml x 2 ea Buffer 2 (1 st Washing) 4 ml x 1 ea 40 ml x 1 ea Buffer 3 (2 nd Washing) 1.6 ml x 1 ea 16 ml x 1 ea Buffer 4 (Elution) 1 ml x 1 ea 25 ml x 1 ea Magnetic Nano Bead - RNA 1 ml x 1 ea 1.8 ml x 6 ea *mini- 8 reactions **mini 100 RXNs, midi 20 reactions 1
III. Storage MagListo TM 5M Cell Total RNA Extraction kit is stored dry at room temperature and It can be stored for up to one year if it remains sealed. Buffers may form precipitates during storage. If this occurs, please warm the buffer to 37 until the precipitates are completely dissolved. IV. Intended Use MagListo TM 5M Cell Total RNA Extraction kit is intended for research use only. This kit is not intended for human or veterinary diagnostics use. V. Safety Warnings and Precautions Please inquire BIONEER s Customer Service Center to obtain a copy of the Material Safety Data Sheet (MSDS) for this product. Before, during and after use of this kit as described in this User s Guide, all potentially hazardous materials (i.e. materials that may have come in contact with genetically recombinant samples) including tubes, tips and materials should be processed and disposed of according to applicable and appropriate regulations of the municipality/government in which this product is being used. A user must also be equipped with basic experimental techniques required for correct execution of the experiments described in this User s Guide. Some applications that may be performed with this kit may infringe upon existing patents in certain countries. The purchase of this kit does not include or provide a license to perform patented applications. Users may be required to obtain a license depending on country and application. We do not condone nor recommend the unlicensed use of a patented application. VI. Warranty and Liability All BIONEER products are manufactured and tested under strict quality control protocols. BIONEER guarantees the quality of all directly manufactured products during the warranty period of one (1) year from the date of purchase. If any issues are discovered relating to compromise in product quality, immediately contact BIONEER s Customer Service Center (sales@bioneer.com). 2
BIONEER does not assume liability for misuse of the product, i.e. usage of the product for any purposes other than its intended purpose as described in the User s Guide. BIONEER assumes liability under the condition that users disclose all information related to the problem to BIONEER in written form within 30 days of occurrence. VII. Technical Assistance At Bioneer, we pride ourselves on being responsive to your needs. If you have any questions or would like to find out more information about MagListo TM products, please contact us. We look forward to hearing from you! Technical Support For all technical questions and troubleshooting on Bioneer products and applications. Tel: +82-42-930-8777 Email: sales@bioneer.com - In North America Tel: +1-877-264-4300 Email:support@bioneer.us.com VIII. Quality Management Every aspect of our quality management system from product development, production to quality assurance and supplier qualification meets the world-class standards. Each lot of MagListo TM 5M Cell Total RNA Extraction kit is carefully tested by the quality control team. 3
IX. Product Specifications Mini scale Midi scale Starting Cell Number 10 5 ~ 5 x 10 6 Cells 5 x 10 6 ~ 2 x 10 7 Cells Extraction time < 8 min < 13 min Elution volume 80 µl ~ 100 µl 100 µl ~ 500 µl Expected RNA yield Up to 100 µg Up to 400 µg Expected purity A 260/280 > 1.9 *RNA content can vary greatly between cell types. X. Principle MagListo TM 5M Cell Total RNA Extraction kit is designed for the extraction of highly purified total RNA from cultured cells. For instance, chaotropic agents in Binding Buffer contains guanidine hydrochloride or guanidine thiocyanate, as which removes water molecules around RNA and silica coated magnetic bead surface resulting in RNA captured by silica coated magnetic beads. The magnetic bead and nucleic acid complexes are pulled and fixed on the tube wall using a magnetic force, followed by washing with Washing Buffers to remove debris and excessive salts. The captured nucleic acids are then eluted by Elution Buffer, an aqueous solution with optimal ph. Lysis Binding Washing Elution 4
XI. Materials and Equipment Needed But Not Provided 1. Table-top microcentrifuge, 16,000 x g (>13,000 rpm) (mini scale) 2. Centrifuge with rotor capable of 3,000 x g (midi) 3. 1.5 ml or 2 ml tube (micro/mini scale) 4. 15 ml tube (midi scale) 5. Vortex mixer 6. Absolute ethanol 7. Blow dryer or heat gun or dry oven 8. MagListo TM Magnetic Separation Rack Magnetic Separation Rack Choice Tube MagListo TM Magnetic Separation Rack Cat.No 1 ml tube with 8-cap strip MagListo TM -8Ch Magnetic Separation Rack TM-1000 1.5 ml or 2 ml microcentrifuge tube MagListo TM -2 Magnetic Separation Rack TM-1010 15 ml tube MagListo TM -15 Magnetic Separation Rack TM-1020 (Note) Please refer to the ordering information table on the latter part of the manual which contains the appropriate catalog number for specific size of tubes. 5
XII. Protocols Before you begin 1. Buffer 1 (Binding) and Buffer 2 (1 st Washing) contains chaotropic salt. You should take the appropriate laboratory safety precautions and wear gloves and lab goggle when handling. 2. The relative centrifugal force (RCF) is calculated in g as follows: RCF = 1.12 x r x (rpm/1,000) 2 Where r is the radius of a rotor in cm, and rpm is the speed of the rotor in revolutions per minute. 3. Buffer 2 (1 st Washing) and Buffer 3 (2 nd Washing) are supplied as concentrated solutions. Before using for the first time, add absolute ethanol as indicated on the bottles. 4. To inhibit RNase activity, we recommend adding β-mercaptoethanol to Buffer 1 (Binding) before use. Add 10 µl β-mercaptoehanol(>99%) per 1 ml Buffer 1 (Binding). Typical Yields of Total RNA Cell type (1x 10 6 ) Yield Hela 10 ~ 15 µg 293T 20 ~ 30 µg Balb/c 3T3 20 ~ 30 µg Huh7 10 ~ 15 µg (Note) We recommend using our MagListo TM Magnetic Separation Rack for guaranteed results. 6
Protocol for Micro(~10 4 )/Mini(1 x 10 5 ~ 5 x 10 6 )/Midi (5 x 10 6 ~ 2 x 10 7 ) Extraction Harvest of Cells 1. Cells grown in suspension : Count the cell number and centrifuge the proper number of cells at 300 x g for 5 min. Discard supernatant carefully and go to lysis & homogenization (go to step 3). 2. Cells grown in a monolayer: There are 2 different ways to collect cells grown in a monolayer. A. Direct cell lysis on the culture dish: Completely remove cell culture medium and go to lysis & homogenization (go to step 3). (Remaining medium may inhibit the RNA extraction) B. Harvesting cells with trypsin: Remove cell culture medium and wash the monolayer with DPBS. Add 0.1%-0.25% typsin to the washed cell monolayer. When the cells are detached, add Cell Culture Medium to inactivate the typsin. Transfer the cells into a RNase-free tube and centrifuge at 300 x g for 5 min. Discard supernatant carefully and go to lysis & homogenization (go to step 3). Lysis & homogenization 3. Add 50 µl (micro) / 400 µl (mini) / 2 ml (midi) of Buffer 1 (Binding) to the cell culture dish or the collected cell pellet and completely homogenize the cells by pipetting or vortexing for at least 1 min. (Note) Insufficient homogenization can decrease the RNA purification yield, and also cause clogging of Magnetic Nano beads in the following steps. For the sufficient homogenization of the lysate, make the lysate passed through blunt 20-gauge needle (0.9 mm diameter) 5 to 10 times. A. For (micro/mini) scale extraction, please transfer the lysate to a 2 ml (or 1.5 ml) tube. B. For (midi) scale extraction, please transfer the lysate to a 15 ml tube. RNA binding 4. Add 50 µl (micro) / 400 µl (mini) / 2 ml (midi) of absolute ethanol to the tube and mix by vortexing or shaking. 5. Add 50 µl (micro) / 100 µl (mini) / 400 µl (midi) of Magnetic Nano Bead solution to the tube and mix by vortexing or shaking. (Note) Magnetic Nano Bead solution contains magnetic nano beads. Please Vortex well before use. 6. Place the tube in MagListo TM -2 (micro/mini) / MagListo TM -15 (midi) Magnetic Separation Rack with the magnet plate attached and invert the tube 3 ~ 4 times gently until the beads tightly bind to magnet. 7
* How to Use - Attachment Combine the magnet plate to the stand. 1 - Discard solution Discard solution by inverting the MagListo rack. The silicone immobilizer inside the stand holds the tubes from falling in an upside down position. When discard solution, invert the rack completely for the solution not to smear on the rack. 2 - Detachment Push up the magnet plate gently. 3 7. Keeping the tubes in MagListo TM rack, discard the supernatant and remove the remaining supernatant using a paper towel by blotting. (Optional : If performing optional ONE Step RNA Cleanup, follow steps (page 11) after performing this step.) Washing 8. Detach the magnet plate from MagListo TM rack. Add 350 µl (micro) / 700 µl (mini) / 3.5 ml (midi) of Buffer 2 (1 st Washing) to the tube. Close the cap and mix by vortexing or shaking until the bead are fully resuspended. 9. Place the tubes in MagListo TM rack with the magnet plate attached and invert the tube 3 ~ 4 times gently until the beads tightly bind to the magnet. 8
10. Maintaining the tubes in the MagListo TM rack, discard the supernatant and completely remove the remaining supernatant using a paper towel by blotting. 11. Detach the magnet plate from MagListo TM rack. Add 350 µl (micro) / 700 µl (mini) / 3.5 ml (midi) of Buffer 3 (2 nd Washing) to the tube. Close the cap and mix by vortexing or vigorous shaking until the bead are fully resuspended. 12. Repeat the above step 9 ~ 10 for additional washing. 13. Detach the magnet plate from MagListo TM rack. Add 350 µl (micro) / 700 µl (mini) / 3.5 ml (midi) of absolute ethanol to the tube. Close the cap and mix by vortexing or shaking until the bead are fully resuspended. 14. Repeat the above step 9 ~ 10 for additional washing. Drying 15. With a heat gun or a blow dryer, completely dry the beads with the tube open 3 cm away from the top of the tube. (micro/mini : >1 min, midi : >3 min) (Note) Alternatively, the beads can be dried with a dry oven at 65 for current times. (micro/mini : >5 min, midi : >25 min) Please use the clean bench during the drying procedure to prevent RNase or other aerosol contamination. Elution 16. Add for 80 µl (micro/mini) / 250 µl (midi) of Buffer 4 (Elution) to the tube with the magnet plate detached and resuspend by vortexing or pipetting 17. Incubate the tube at 50 ~ 65 for 2 min. (Note) Warm up the tube with a heat block, a heat gun or a blow dryer. 18. Vortex the tube for 15 sec. 19. Attach the magnet plate to MagListo TM rack and invert the tube 3 ~ 4 times gently until the beads tightly bind to the magnet. 20. Keeping the tube in MagListo TM rack, carefully take the supernatant containing RNA to a sterile microcentrifuge tube. 21. Discard the used Magnetic Nano Beads. Do not reuse the beads. 9
Summary of reagent volumes required in each step of Cell Total RNA Extraction Step Buffer Micro Mini Midi Page Cell Lysis Buffer 1 (Binding) 50 μl 400 μl 2 ml P. 7 Add EtOH Absolute Ethanol 50 μl 400 μl 2 ml P. 7 RNA Binding Magnetic Nano Bead - RNA 50 μl 100 μl 400 μl P. 7 1 st Washing Buffer 2 (1 st Washing) 350 μl 700 μl 3.5 ml P. 8 2 nd Washing Buffer 3 (2 nd Washing) 350 μl 700 μl 3.5 ml P. 9 3 rd Washing Absolute Ethanol 350 μl 700 μl 3.5 ml P. 9 Elution Buffer 4 (Elution) 80 μl 80 μl 250 ul P. 9 RNA Clean up Protocol This protocol is to remove enzymes, buffers, or chemical inhibitors and concentrate RNA for use in certain applications. 1. Transfer RNase-free water into RNA sample to make a total volume of 100 µl. 2. Add 50 µl of Buffer 1 (Binding) and mix thoroughly. 3. Add 200 µl of absolute ethanol and mix well. 4. Add 100 µl of Magnetic Nano Bead solution to the tube and mix by vortexing or shaking. (Note) Magnetic Nano Bead solution contains magnetic nano beads. Please Vortex well before use. 5. Place the tube in MagListo TM -2 with the magnet plate attached and invert the tube 3~4 times gently until the beads tightly bind to the magnet. 6. Keeping the tube in MagListo TM rack, discard the supernatant and remove the remaining supernatant using a paper towel by blotting. 7. Go to Washing step 11 (page number 9) and follow the instruction described above for the remaining steps 10
ONE Step RNA Clean Up Protocol This protocol is to remove DNA for use in certain applications. 1. Detach the magnet plate from MagListo TM rack. Add 400 µl (micro, mini) / 3.5 ml (midi) of absolute ethanol to the tube. Close the cap and mix by vortexing or shaking until the bead are fully resuspended 2. Place the tube in MagListo TM -2 with the magnet plate attached and invert the tube 3 ~ 4 times gently until the beads tightly bind to the magnet. 3. Without removing the tube from MagListo TM rack, discard the supernatant and remove the remaining supernatant using a paper towel by blotting. 4. With a heat gun or a blow dryer, completely dry the beads with the tube open 3 cm away from the top of the tube. (micro/mini : >1 min, midi : >3 min) (Note) Alternatively, the beads can be dried with a dry oven at 65 for current times. (micro/mini : >5 min, midi : >25 min) Please use the clean bench during the drying procedure to prevent RNase or other aerosol contamination. 5. Add 50 μl of DNase Reaction Buffer and 10 μl of RNase-Free DNaseⅠ to the tube. 6. Detach the magnet plate from MagListo TM rack and close the cap and mix by vortexing or shaking until the bead are fully resuspended. 7. Place on the benchtop (20 ~ 30 C) for 20 min. 8. Detach the magnet plate from MagListo TM rack. Add 350 µl (micro) / 700 µl (mini) / 3.5 ml (midi) of Buffer 2 (1 st Washing) to the tube. Close the cap and mix by vortexing or shaking until the bead are fully resuspended. 9. Go to Washing step 9(page number 8) and follow the instruction described above for the remaining steps. 11
Experimental Data Figure 1: Comparison of total RNA purified with Maglisto TM 5M Cell Total RNA Extraction Kit and competitor 1x10 6 and 5x10 6 Hela cells were used for the comparison. Total RNA purification was performed with the MagListo TM 5M Cell Total RNA Extraction Kit and the competitor s product respectively. The bands of purified total RNA were illustrated by 1% denaturing agarose gel electrophoresis. The equivalent level of purification yield of the MagListo TM 5M Cell Total RNA Extraction Kit and the competitor s product was confirmed through the bands. Also the distinctive 28S/18S rrna band patterns represent the superb quality of RNA purities having no RNA degradations. Figure 2: Extraction of RNA from various cell lines 1X10 6 of Huh7, Hela, 293T, and Balb/c 3T3 cells were used for the RNA purification performed with the MagListo TM 5M Cell Total RNA Extraction Kit. The figure shows the bands of purified RNA made by using 1% denaturing agarose gel electrophoresis. The effective RNA purifications from various cell lines could be confirmed through the bands. 12
Ct value Figure 3: RT-qPCR comparison of RNA isolated from 10 ~ 10 6 cells 36.00 Hela Cell-GAPDH 34.00 32.00 30.00 28.00 26.00 24.00 22.00 20.00 MagListo Competitor R² = 0.995 R² = 0.995 18.00 16.00 14.00 1.E+06 1.E+05 1.E+04 1.E+03 1.E+02 1.E+01 (Left) Human GAPDH extracted from 10, 10 2, 10 3, 10 4,10 5, 10 6 Hela cells with the MagListo TM 5M Cell Total RNA Extraction Kit were amplified using the AccuPower RoketScript RT-qPCR master mix (K-6700) kit. The figure represents fluorescent signals of amplified Human GAPDH. (Right) The graph shows the comparison results of amplified Hela cell-gapdh extracted with the MagListo TM 5M Cell Total RNA Extraction Kit and the competitor s product. According to the results, RNAs were successfully purified from 10~10 6 Hela cells. Also the equivalent level of purification yield of the MagListo TM kit and the competitor s product was confirmed through the GAPDH Ct value of each cell number. Cell Number 13
XIII. Troubleshooting Guide Comments and suggestions Insufficient homogenization: (Refer to page 7) Add Buffer 1 (Binding) to the cells and completely homogenize the cells by vortexing for at least 1min or increase the vortexing time for the sufficient homogenization up to 5 to 10 minutes. Use of an excess amount of sample: Use the proper amount of samples according to the instructions of this User s Guide. Low or no yield Insufficient bead dry: Beads must be completely dried. Any leftover of ethanol can decrease the RNA purification yield. Insufficient incubation during the elution step: RNA could remain attached to the beads if the incubation time is not enough. To prevent this, please repeat the Elution process with the increased incubation time. Incomplete removal of cell culture medium: The best approach is to remove the medium as much as possible. Any leftover in the medium can lead to the inhibition of RNA extraction. Aggregation of Magnetic Nano Beads RNA degradation Inhibition of downstream enzymatic reactions Low A 260/280 ratio Insufficient homogenization: (Refer to page 7) Add Buffer 1 (Binding) to the cells and completely homogenize the cells by vortexing for at least 1min or increase the vortexing time for the sufficient homogenization. Use of an excess amount of sample: Use the proper amount of samples according to the instructions of this User s Guide. RNase contamination: Use a heat gun or a blow dryer in a clean bench to prevent the contamination of RNase in the air. Use RNase-free pipette tips and change the gloves frequently. Inappropriate handling of harvested samples: If the extraction is not performed right after harvesting the samples, store the samples at -80 or lysis the samples with Buffer 1 (Binding) and then store at -80. Insufficient bead dry: Beads must be completely dried. Any leftover in the ethanol can decrease the RNA purification yield. Insufficient washing during the bead suspension process: Insufficient suspension of beads during the washing step causes salts to remain in the purified RNA. The recommendation is to suspend the beads thoroughly during the washing process. Insufficient bead dry: Beads must be completely dried. Any leftover in the ethanol can decrease the RNA purification yield. Insufficient washing during the bead suspension process: Insufficient suspension of beads during the washing step causes salts to remain in the purified RNA. The recommendation is to suspend the beads thoroughly during the washing process. 14
XIV. Ordering Information Cat no. Product Description Size K-3601SM MagListo TM 5M Plamid Extraction Kit, 8 reactions (mini) 1 kit K-3601 MagListo TM 5M Plamid Extraction Kit, 100 reactions (mini) 1 kit K-3600 MagListo TM 5M Plamid Extraction Kit, 500 reactions (mini) 1 kit K-3602 MagListo TM 5M Genomic DNA Extraction Kit, 8 reactions (mini) 1 kit K-3603 MagListo TM 5M Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit K-3604 MagListo TM 5M Plant Genomic DNA Extraction Kit, 8 reactions (mini) 1 kit K-3605 MagListo TM 5M Plant Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit K-3606 MagListo TM 5M Gel Extraction Kit, 8 reactions (mini) 1 kit K-3607 MagListo TM 5M Gel Extraction Kit, 100 reactions (mini) 1 kit K-3608 MagListo TM 5M PCR Purification Kit, 8 reactions (mini) 1 kit K-3609 MagListo TM 5M PCR Purification Kit, 100 reactions (mini) 1 kit K-3610 MagListo TM 5M Cell Total RNA Extraction Kit, 8 reactions (mini) 1 kit K-3611 MagListo TM 5M Cell Total RNA Extraction Kit, 100 reactions (mini) 1 kit K-3614 MagListo TM 5M Forensic Sample DNA Extraction Kit, 8 reactions (mini) 1 kit K-3615 MagListo TM 5M Forensic Sample DNA Extraction Kit, 100 reactions (mini) 1 kit TM-1000 MagListo TM -8Ch Magnetic Separation Rack 1 ml tube x 8 holes TM-1010 MagListo TM -2 Magnetic Separation Rack 2 ml tube x 8 holes TM-1020 MagListo TM -15 Magnetic Separation Rack 15 ml tube x 6 holes TM-1030 MagListo TM -50 Magnetic Separation Rack 50 ml tube x 3 holes TM-1040 MagListo TM -96 Magnetic Separation Rack 96-well plate 1ea TM-1100 MagListo TM Magnetic Separation Rack Bundle Set MagListo TM -2,-15,-50, and -96 (4 racks, 1 each) K-3601-A Blow Dryer 1 ea HT-15-NG 1.5 ml microcentrifuge tube 500 ea / pk HT-20-NG 2 ml microcentrifuge tube 500 ea / pk 15
XV. Explanation Symbols Catalog Number Contains sufficient for (n) tests USE BY Consult Instruction For Use Batch code Caution, consult accompanying documents Temperature Limitation Research Use Only Manufacturer Caution, Potential Biohazard DO NOT REUSE 16