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TRANSCRIPT
Kit for the extraction of total RNA from wide range of tissue using MagListo™
Version No.: 2.0 (2017-03)
Please read all the information in booklet before using the unit
Bioneer Corporation
8-11, Munpyeongseo-ro, Daedeok-gu, Daejeon
34302, Republic of Korea
Tel: +82-42-930-8777
Fax: +82-42-930-8688
Email: [email protected]
www.bioneer.com
MagListo™ is a trademark of Bioneer Corporation.
Copyright 2017. Bioneer Corporation. All Rights Reserved.
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. Kit Specifications 4
Extraction of tissue total RNA from small amount of sample 4
Recommended amounts of starting sample 4
X. Sample Preparation 5
XI. Principle 5
XII. Magnetic Nano Bead Information 6
XIII. Guidelines for MagListo™ Magnetic Separation Rack 6
XIV. Materials and Equipment Needed But Not Provided 7
Types of the Magnetic Separation Rack 7
XV. Procedure 8
XVI. Protocols 9
Before you begin 9
A. RNA Extraction from Animal Tissue 9
B. RNA Extraction from Cultured Cells 14
C. ONE-Step RNA Cleanup (DNase Treatment) 18
D. RNA Cleanup (RNA Purification) 20
XVII. Appendix 21
Troubleshooting Guide 21
Experimental Data 23
XVIII. Ordering Information 25
XIX. Explanation of Symbols 26
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I. Overview
Description
MagListo™ 5M Tissue Total RNA Extraction Kit utilizes Magnetic Nano Beads to extract total RNA from various
of sources, such as animal tissue or cultured cells, with the aid of the MagListo™ Magnetic Separation Rack.
The use of MagListo™ Magnetic Separation Rack along with the kit greatly increases user convenience by
shortening the extraction time without centrifugation.
Features and Benefits
- Magnetic Nano Beads enable the rapid nucleic acid extraction
- No requirement of expensive instruments
- A single kit serves mini or midi scale experiment
Applications
Applicable to RNA extraction step for assays requiring RNA, including, but not limited, RT-PCR, cDNA
synthesis, Northern, dot, and slot blot analyses, Microarrays, and RNAseq
II. Kit Components
MagListo™ 5M Tissue Total RNA Extraction Kit *K-3613
Buffer ① (Binding) 25 ml x 2 ea
Buffer ② (1st Washing) 100 ml x 1 ea
Buffer ③ (2nd
Washing) 100 ml x 1 ea
Buffer ④ (3rd Washing) 120 ml x 1 ea
Buffer ⑤ (Elution) 20 ml x 1 ea
Magnetic Nano Beads - RNA 1.8 ml x 6 ea
*Mini – 100 rxn, Midi 10 rxn
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III. Storage
MagListo™ 5M Tissue Total RNA Extraction Kit should be stored dry at room temperature. It can be
stored for up to 2 years if it remains sealed.
IV. Intended Use
MagListo™ 5M Tissue Total RNA Extraction Kit is intended for research use only. This kit is not intended for
human or veterinary diagnostics.
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 using this kit, all potentially hazardous materials (i.e. materials that may have
come in contacted with genetically recombinant samples) including tubes, tips and other kit contents
should be processed and discarded in accordance with 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 extraction experiments described in this
User’s Guide.
Some inventions applied in this kit may infringe existing patents in certain countries. The purchase of this
kit does not include or provide a license to perform such patented inventions. Users may be required to
obtain a license depending on the patent law of the country where this product is being used. We do not
condone nor recommend the unlicensed use of patented inventions.
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 you find any issues regarding the product quality, please immediately contact
BIONEER’s Customer Service Center ([email protected]).
BIONEER does not assume any liability for the misuse of the product, i.e. using the product for any
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purposes other than its intended purpose as described in the User’s Guide. BIONEER will only assume
liability under the condition when the users disclose all related information regarding the issue to BIONEER
in written form within 30 days after occurrence of the issue.
VII. Technical Assistance
At Bioneer, we pride ourselves on being responsive to your needs. Our Technical Service Departments are
staffed by experienced scientists with extensive practical and theoretical expertise in Molecular Biology and
the use of Bioneer products. If you have any questions or would like to find out more information about
MagListo™ 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: [email protected]
- In North America
Tel: +1-877-264-4300
Email:[email protected]
VIII. Quality Management
Every aspect of our quality management system from product development to supplier qualification
ensures that our products meet the world-class standards. Each lot of MagListo™ 5M Tissue Total RNA
Extraction Kit is carefully tested by the quality control team.
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IX. Kit Specifications
Mini scale Midi scale
Starting sample ≤ 20 mg tissue or 3 x 106 cells ≤ 200 mg tissue or 2 x 10
7 cells
Extraction time < 10 min < 15 min
Minimum elution volume 50 µl 500 µl
Expected purity A260/280 > 1.9
*RNA content can vary greatly according to tissue type.
Extraction of tissue total RNA from small amount of sample
MagListo™ 5M Tissue Total RNA Extraction Kit is also able to extract total RNA from a small quantity of
sample. Refer to “RNA Extraction from Cultured Cell for Mini” in page 14 for more details about RNA
extraction from samples with a low number of cells (≤ 3x106).
Recommended amount of starting sample
It is recommended to use the amounts in Table 1 as starting sample amount.
Table1. Growth area and Average cell yield in various culture dishes.
Cell culture dishes Growth area (cm2) Average cell yield
Multi well plate
6 well 9.6 1.2 x 106
12 well 4 4 x 105
24 well 2 2 x 105
48 well 1 1 x 105
96 well 0.35-0.6 4 x 104
Dishes
35 mm 8 1.2 x 106
60 mm 21 3 x 106
100 mm 55 8 x 106
150 mm 148 2 x 107
Flasks
50 ml 25 2.5 x 106
300 ml 75 1 x 107
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X. Sample Preparation
Several factors, such as harvesting method and storage of starting samples can influence the yield and
purity of RNA. All specimens must be stored in a -70℃ freezer or used immediately after collection. It is
recommended to put the sample as soon as possible on ice, and to avoid repeated freezing and thawing.
Repeating freeze-thawing of the sample will result in the degradation of RNA.
Tissue
Tissue samples should immediately be used or stored at -70℃ for optimal results. To disrupt tissue
sample, grind it in a pestle and a mortar with liquid nitrogen. Alternatively, a homogenizer or a bead-beater
can be used.
Cultured cells
Cultured cells can easily be harvested using a centrifuge. However, it might be difficult to extract total
RNA if cultured cells are too clustered. In this case, trypsin can be used to scatter cells from the cluster.
For optimal extraction, the number of cells should be less than 2 x 107, which is calculated with a cell
counter. It is recommended to keep samples on ice before use.
XI. Principle
The MagListo™ 5M Tissue Total RNA Extraction Kit is designed for the extraction of high purified total
RNA from tissue and cultured cells. The overall principle is based on adsorption of RNA onto the
Magnetic Nano Bead by chaotropic salt. For example, chaotropic agents in Buffer ① (Binding)
contains guanidine hydrochloride and guanidine thiocyanate, as which remove water molecules around
RNA and silica coated magnetic beads surface resulting in RNA then being captured by magnetic
beads. The Magnetic Nano Beads and RNA complexes are pulled and fixed on the tube wall using a
magnetic force, followed by washing with ethanol to remove debris and excessive salts. Finally, the
captured RNAs are then eluted by Buffer ⑤ (Elution), an aqueous solution with optimal pH.
Sample Lysis Binding Washing Elution
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XII. Magnetic Nano Bead Information
Description
Magnetic Nano Beads have been developed to overcome shortcomings of existing resin and to automate
purification process. The principle of extraction using the Magnetic Nano Beads is that coated functional
group on the surface of the Magnetic Nano Beads bind with DNA and the Magnetic Nano Beads are then
isolated using external magnetic field.
Features
Fast binding guarantees higher throughput automation
Large surface area enables more sensitive assay
Globular structure increases specificity by decreasing non-specific binding
Specification
AccuNanoBeadTM Silica Magnetic Nano Beads
Matrix Silica-coated Fe3O4
Average size 400nm
Ligand -OH
Working Temp. 0~100℃
Storage Store at room temperature upon receipt
XIII. Guidelines for MagListoTM Magnetic Separation Rack
Description
MagListo™ Magnetic Separation Rack is designed for a fast, easy separation of the Magnetic Nano
Beads. These racks of different sizes allow users to choose the product according to their needs.
The following are recommended when handling the MagListo™ Magnetic Separation Rack
The product is made of acryl and plastic. Be careful not to drop the product as the dropping may
break the product.
When moving the product, take extra care not to drop the product as it may cause injury.
If the product is broken, do not discard it with bare hands as the sharp edges may cause injury.
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When an extracted or purified nucleic acid is spilled on the product, immediately rinse it with running
water and clean it with 70% ethanol.
Acetone, Toluene, or organic solvent may damage the acrylic and plastic part of the product, which
may lead to malfunction of the product. Rinse the product immediately when spillage of any above
mentioned solvents occurs as the expected DNA yield may not be obtained if the product is
damaged.
Make sure that a corrosive liquid on the magnet plate part of the product. If spillage occurs,
immediately rinse it off with running water as it may corrode the magnet during storage and may
degrade its performance.
XIV. 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 (mini scale)
4. 15 ml tube, 50 ml tube (midi scale)
5. Vortex mixer
6. Absolute ethanol
7. Thermal block or dry oven
8. MagListo™ Magnetic Separation Rack
Types of the Magnetic Separation Rack
Tube MagListo™ Magnetic Separation Rack Cat.no
1.5 ml or 2 ml
microcentrifuge tube MagListo™-2 Magnetic Separation Rack TM-1010
15 ml tube MagListo™-15 Magnetic Separation Rack TM-1020
50 ml centrifuge tube MagListo™-50 Magnetic Separation Rack TM-1030
(Note) Please refer to the ordering information in this User’s Guide for more information regarding
catalog number of racks designed for specific size of tubes.
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XIV. Procedure-Tissue Total RNA Extraction
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XVI. Protocols
Before you begin
1. Buffer ① (Binding) contains chaotropic salt. You should take appropriate laboratory safety
precautions and wear gloves and lab goggles 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. 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).
A. RNA Extraction from Animal Tissue for Mini/Midi Scale
1. (Tissue lysis & homogenization) Disruption and homogenization using a rotor-stator homogenizer :
Place the weighed (fresh, frozen, or RNAlater™-stabilized) tissue (~20 mg (mini) / ~200 mg (midi))
in a suitable-sized vessel.
Add 400 µl (mini) / 4 ml (midi) of Buffer ① (Binding) to the tissue sample. Immediately disrupt
and homogenize the tissue using a conventional rotor-stator homogenizer until it becomes
uniformly homogeneous and transfer it to a new 2 or 1.5 ml tube (go to step 4).
2. Disruption using a mortar and pestle followed by homogenization using a needle and syringe:
Immediately place the weighed (fresh, frozen, or RNAlater™-stabilized) tissue in liquid nitrogen,
and grind the sample thoroughly with a mortar and pestle. Decant tissue powder and liquid
nitrogen into an RNase-free, liquid-nitrogen-cooled 2 ml tube. Allow the liquid nitrogen to
evaporate, but do not allow the tissue to be thawed (go to step 3).
3. Add 400 µl (mini) / 4 ml (midi) of Buffer ① (Binding) to each tube and mix thoroughly using a
vortex mixer. Make sure that the sample is completely resuspended the sample to achieve
maximum lysis efficiency.
(Note) Insufficient homogenization can decrease the yield of total RNA purified, and also may
cause clogging of Magnetic Nano Beads in the following steps. For a sufficient
homogenization of the lysate, make the lysate, pass through a blunt 20-gauge needle (0.9
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mm diameter) 5 to 10 times.
A. (Mini) please transfer the lysate to a 1.5 ml or 2 ml tube.
B. (Midi) please transfer the lysate to a 50 ml tube.
4. (RNA precipitation) Add 200 µl (mini) / 2 ml (midi) of absolute ethanol to each tube and mix well
using a vortex mixer or by pipetting.
5. (RNA binding with Magnetic Nano Bead: 5-7) Add 100 µl (mini) / 1 ml (midi) of Magnetic Nano
Beads solution to the each tube and mix thoroughly using a vortex mixer until the beads are fully
resuspend.
(Note) Please shake the Magnetic Nano Bead solution well or mix completely with a vortex mixer before
use.
6. Place the tube in MagListo™-2 (mini) / MagListo™-50 (midi) Magnetic Separation Rack with the
magnet plate attached and invert the rack gently 3 to 4 times until the beads bind tightly to magnet.
- Attachment of the magnet plate
Combine the magnet plate to the stand.
7. Without removing the tube from MagListo™ Magnetic Separation Rack, discard the supernatant
carefully and completely remove the remaining supernatant on a paper towel by blotting action.
During this process, the magnet crude pellet remains attached to the side of tube.
(Optional) If performing optional ONE Step RNA Clean up, follow steps (page 12) after performing
this step.
- How to discard the supernatant
Discard the supernatant by inverting the MagListo™ Magnetic Separation Rack. The silicone
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immobilizer inside the stand prevents the tubes from falling. When discarding the supernatant,
invert the rack completely so that the solution does not spill on the rack.
8. (1st Washing: 8-10) Detach the magnet plate from MagListo™ Magnetic Separation Rack. Add
1ml (mini) / 10 ml (midi) of Buffer ② (1st Washing) to the each tube and close the cap. Mix with a
vortex mixer until the beads are fully resuspended.
- Detachment of the magnet plate
Detach the magnet plate gently by pulling it upwards.
9. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the
magnet.
10. Without removing the tube MagListo™ Magnetic Separation Rack, discard the supernatant and
remove the remaining supernatant on a paper towel by blotting action.
11. Repeat the steps 8 through 10 by adding 1 ml (mini) / 10 ml (midi) of Buffer ③ (2nd
Washing)
instead of Buffer ② for additional washing.
12. (3rd Washing) Without removing the tube from MagListo™ Magnetic Separation Rack, add 1 ml
(mini) / 10 ml (midi) of Buffer ④ (3rd Washing) to “the opposite side of bead pellet”. Close the cap
and invert the rack twice in order to remove ethanol from the sample.
(Note) Direct pipetting of Buffer ④ onto the bead pellet, vortexing and/or vigorous shaking of the
tubes may release nucleic acid from the beads, which may result in lower RNA yield than
expected.
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13. Discard the supernatant and completely remove the remaining supernatant by blotting action.
- Add Buffer ④ and discard the supernatant
14. (Elution: 14-18) Detach the magnet plate from the MagListoTM Magnetic Separation Rack. Add 50-
100 μl (mini) / 500 μl-1 ml (midi) of Buffer ⑤ (Elution) to the tube with the magnet plate detached
and resuspend RNA by vortexing or pipetting.
15. Incubate the tube at 55 - 65℃ for 1 min.
16. Attach the magnet plate to MagListo™ Magnetic Separation Rack and invert the rack gently 3 to 4
times until the beads bind tightly to the magnet.
17. Without removing the tube from MagListo™ Magnetic Separation Rack, transfer supernatant
containing RNA carefully to a new sterile microcentrifuge tube.
18. Discard the tubes with the remaining Magnetic Nano Bead pellet. Do not reuse the beads.
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Summary of reagent volumes required in each step of Tissue Total RNA Extraction
Step Buffer Mini scale Midi scale
Tissue Lysis Buffer ① (Binding) 400 μl 4 ml
RNA precipitation Absolute ethanol 200 μl 2 ml
RNA Binding Magnetic Nano Beads - RNA 100 μl 1 ml
1st
Washing Buffer ② (1st Washing) 1 ml 10 ml
2nd
Washing Buffer ③ (2nd
Washing) 1 ml 10 ml
3rd Washing Buffer ④ (3
rd Washing) 1 ml 10 ml
Elution Buffer ⑤ (Elution) 50 - 100 μl 500 μl - 1 ml
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B. RNA Extraction from Cultured Cells in Mini/Midi Scale
1. (Harvest cell) Cells grown in suspension:
Count the cell number, then centrifuge given number of cells (~3x106 (mini) / 2x10
7 (midi)) 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 with 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).
3. (Lysis & homogenization) Add 400 µl (mini) / 4 ml (midi) of Buffer ① (Binding) to each tube and
mix thoroughly using a vortex mixer. Make sure that you must completely resuspend the sample to
achieve maximum lysis efficiency.
(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. (Mini) please transfer the lysate to a 1.5 ml or 2 ml tube.
B. (Midi) please transfer the lysate to a 50 ml tube.
4. (RNA precipitation) Add 200 µl (mini) / 2 ml (midi) of absolute ethanol to the each tube and mix
well using a vortex mixer or by pipetting.
5. (RNA binding with Magnetic Nano Bead: 5-7) Add 100 µl (mini) / 1 ml (midi) of Magnetic Nano
Beads solution to the each tube and mix thoroughly using a vortex mixer until the beads are fully
resuspended.
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(Note) Magnetic Nano Bead solution contains Magnetic Nano Beads. Please shake well or mix with
a vortex mixer before use.
6. Place the tubes on the MagListo™-2 (mini) / MagListo™-15 (midi) Magnetic Separation Rack with
the magnet plate attached and invert the rack gently 3 to 4 times until the beads bind tightly to the
magnet.
- Attachment of the magnet plate
Combine the magnet plate to the stand.
7. Without removing the tube form MagListo™ Magnetic Separation Rack, discard the supernatant
carefully and completely remove the remaining supernatant on a paper towel by blotting action.
(Optional) If performing optional ONE Step RNA Cleanup (DNase Treatment), follow steps (page
12) after performing this step.
- How to discard the supernatant
- Discard the supernatant by inverting the MagListo™ Magnetic Separation Rack. The silicone
immobilizer inside the stand prevents the tubes from falling. When discarding the supernatant,
invert the rack completely so that the solution does not to spill on the rack.
8. (1st Washing: 8-10) Detach the magnet plate from MagListo™ Magnetic Separation Rack. Add 1
ml (mini) / 10 ml (midi) of Buffer ② (1st Washing) to the each tube and close the cap. Mix by
vortexing or shaking until the beads are fully resuspended.
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- Detachment of the magnet plate
Detach the magnet plate gently by pulling it upwards.
9. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the
magnet.
10. Without removing the tubes from the MagListo™ Magnetic Separation Rack, discard the
supernatant and remove the remaining supernatant on a paper towel by blotting action.
11. (2nd
Washing) Repeat the steps 8 through 10 by adding 1 ml (mini) / 10 ml (midi) of Buffer ③ (2nd
Washing) instead Buffer ② for additional washing.
12. (3rd Washing) Without removing the tubes from the MagListo™ Magnetic Separation Rack, add 1
ml (mini) / 10 ml (midi) of Buffer ④ (3rd Washing) to “the opposite side of bead pellet”. Close the
cap and gently invert the rack twice in order to remove ethanol from the sample.
(Note) Direct pipetting of Buffer ④ onto the bead pellet, vortexing and/or vigorous shaking of the
tubes may release nucleic acid from the beads, which may result in lower RNA yield than expected.
13. Discard the supernatant and completely remove the remaining supernatant by blotting action.
- Add Buffer ④ and discard the supernatant
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14. (Elution: 14-18) Detach the magnet plate from the MagListo™ Magnetic Separation Rack. Add
50-100 μl (mini) / 500 μl-1 ml (midi) of Buffer ⑤ (Elution) to the tube with the magnet plate
detached and resuspend by vortexing or pipetting or vortex mixer for 15 sec.
15. Incubate the tube at 55 - 65℃ for 1 min.
16. Place the tubes on the MagListo™ Magnetic Separation Rack. Attach the magnet plate and invert
the rack gently 3 to 4 times until the beads bind tightly to the magnet.
17. Without removing the tubes from the MagListo™ Magnetic Separation Rack, transfer supernatant
containing DNA supernatant to a new sterile microcentrifuge tube.
18. Discard the tubes with remaining Magnetic Nano Bead pellet. Do not reuse the beads.
Summary of reagent volumes required in each step of Cell Total RNA Extraction
Step Buffer Mini scale Midi scale
Cell Lysis Buffer ① (Binding) 400 μl 4 ml
RNA precipitation Absolute ethanol 200 μl 2 ml
RNA Binding Magnetic Nano Beads - RNA 100 μl 1 ml
1st
Washing Buffer ② (1st Washing) 1 ml 10 ml
2nd
Washing Buffer ③ (2nd
Washing) 1 ml 10 ml
3rd Washing Buffer ④ (3
rd Washing) 1 ml 10 ml
Elution Buffer ⑤ (Elution) 50 - 100 μl 500 μl - 1 ml
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C. ONE Step RNA Clean Up (DNase Treatment)
1. (RNA precipitation) Detach the magnet plate from the MagListo™ Magnetic Separation Rack. Add
1 ml (mini) / 10 ml (midi) of absolute ethanol to the each tube and close the cap. Mix by vortexing
or shaking until the beads are fully resuspended.
2. Place the tubes on the MagListo™-2 (mini) / MagListo™-50 (midi) Magnetic Separation Rack with
the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet.
3. Without removing the tubes from the MagListo™ Magnetic Separation Rack, discard the
supernatant carefully and completely remove the remaining supernatant using a paper towel by
blotting action.
4. The beads can be dried with a dry oven at 65℃ for following times. (mini: > 5 min, midi: >15 min)
Please use a clean bench during the drying procedure to prevent RNase or other aerosol
contamination.
5. Add DNase Reaction Buffer and RNase-Free DNase to the each tube.
(mini: up to 70 µl, midi: up to 700 µl)
6. Detach the magnet plate from the MagListo™ Magnetic Separation Rack and close the cap. Mix
with a vortex mixer until the beads are fully resuspended.
7. Place on the benchtop (20 – 30°C) for 20 min.
8. Detach the magnet plate from MagListo™ Magnetic Separation Rack. Add 300 µl (mini) / 3 ml
(midi) scale extraction of Buffer ② (1st Washing) and 300 µl (mini) / 3 ml (midi) scale extraction of
absolute ethanol to the each tube. Close the cap and mix with a vortex mixer until the beads are
fully resuspended.
9. Place the tubes on the MagListo™ Magnetic Separation Rack with the magnet plate and invert the
rack gently 3 to 4 times until the beads bind tightly to the magnet.
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10. Without removing the tubes from the MagListo™ Magnetic Separation Rack, discard the
supernatant carefully and completely remove the remaining supernatant using a paper towel by
blotting action.
11. Go to step 11 of “A. RNA Extraction from Animal Tissue” in page 11 and follow the instructions
accordingly.
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D. RNA Clean up (RNA Purification)
1. Transfer RNase-free water into RNA sample to a volume of 100 µl.
(Optional) If you DNA-free RNA is required, add DNase Reaction Buffer and RNase-Free DNase to
each tube and make the volume up to 100 µl with RNase-free water. Incubate the tubes for 10 min
at room temperature.
2. Add 100 µl of Buffer ① (Binding) to each tube and mix completely using a vortex mixer.
3. Add 200 µl of absolute ethanol to each tube and mix completely using a vortex mixer.
4. Add 100 µl of Magnetic Nano Beads solution to the each tube and mix thoroughly using a vortex
mixer until the beads are fully resuspended.
(Note) Magnetic Nano Bead Solution contains Magnetic Nano Beads. Please shake well or mix with
a vortex mixer before use.
5. Place the tubes on the MagListo™-2 (mini) Magnetic Separation Rack with the magnet plate
attached and invert the rack gently 3 to 4 times until the beads bind tightly to magnet.
6. Without removing the tubes from the MagListo™ Magnetic Separation Rack, discard the
supernatant carefully and completely remove the remaining supernatant on a paper towel by
blotting action.
7. Go to step 8 of “A. RNA Extraction from Animal Tissue” in page 11 and follow the instructions
accordingly.
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XV. Appendix
Troubleshooting guide
This troubleshooting guide will help you to solve problem that may arise during RNA extraction. For
other technical assistance or more information, please contact our technical assistance team.
Comments and suggestions
Low yield of RNA
Buffers or other reagents may have been exposed to external factors that may
have reduced its quality. Please make sure that reagents are stored at room
temperature at all times upon arrival and that all reagent bottles are closed
tightly, in order to preserve pH and stability, and to avoid contamination.
Excess amount of starting sample was used to extract DNA. Appropriate
amount of starting sample (see “Kit Specification” in page 4) should be used for
efficient extraction of RNA.
Elution may have been incomplete. Please extend incubation time up to 3
minutes at elution step to improve the yield. In addition, make sure that
Magnetic Nano Beads are suspended completely in the eluting solution during
incubation.
Some of Magnetic Nano Bead pellet may have been lost while discarding
solution. Check that all of the Nano Beads have bound tightly to the magnet
when you discard supernatant.
Insufficient shaking or vortexing during lysis step may lead to low DNA yield than
expected. Shake or mix with a vortex mixer sufficiently during incubation step.
Cell culture medium may have been incomplete. 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.
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Low A260/280 ratio
Beads may have been washed insufficiently. You must properly wash the beads
in the 3rd washing step. Remaining ethanol can decrease the purity of DNA.
Take enough time to properly wash the beads.
Incomplete suspension of beads during the washing step causes salts to remain
in the purified RNA. Make sure that the beads are suspended thoroughly during
the washing process.
Excessively clustered
Magnetic Nano Bead
Excess amount of starting sample is used to extract RNA. Appropriate amount
of starting material (see “Kit Specification” in page 4) should be used for
efficient extraction of RNA.
Presence of a white
precipitate in buffers
A white precipitate may form in Buffer ⓛ (Binding) due to prolonged storage at
low temperatures. Incubate Buffer ⓛ (Binding) at 60℃ to dissolve any
precipitate in the buffer.
Degraded RNA
RNase contamination can be degraded RNA. 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.
Cultured cell samples that have been stored at -80℃ or lysis the samples with
Buffer ① (Binding) and then store at -80℃.
Frequent freezing and thawing may result in lower RNA yield than expected.
Avoid repeated freezing and thawing.
Flotation of extracted
DNA when loaded on an
agarose gel
Floating of RNA on an agarose gel is caused by the remaining ethanol in the
eluted RNA. Ensure that the 3rd Washing (ethanol removing) step in the protocol
is properly performed. Remaining ethanol may also interrupt the enzymatic
reaction.
.
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Experimental data
Figure 1. Comparison of liver’s Total RNA purified with MagListo™ 5M Tissue Total RNA Extraction Kit and
competitor’s kit (Single Column Type)
1 2 3 4 5 6 7 8
1-4: Extracted liver total RNA purified with Bioneer MagListoTM 5M
Tissue Total RNA Extraction Kit
5-8: Extracted liver total RNA purified with competitor Q kit
Sample Total Yield (ug) A260/A280 A260/A230
1 58.6 2.09 2.03
2 60.3 2.09 1.95
3 55.7 2.08 2.02
4 58.7 2.09 1.97
5 36.8 2.04 1.76
6 38.3 2.05 1.78
7 34.5 2.03 1.91
8 37.9 2.05 1.48
Figure 2. Comparison of kidney’s Total RNA purified with MagListo™ 5M Tissue Total RNA Extraction Kit and
competitor’s kit (Single Column Type)
1 2 3 4 5 6 7 8
1-4: Extracted kidney total RNA purified with Bioneer MagListoTM
5M Tissue Total RNA Extraction Kit
5-8: Extracted kidney total RNA purified with competitor Q kit
Sample Total Yield (ug) A260/A280 A260/A230
1 30.8 2.02 2.02
2 34.6 2.02 2.03
3 33.7 2.03 1.95
4 35.2 2.06 1.91
5 27.8 2.03 1.95
6 26.7 2.03 1.07
7 28.3 2.03 1.66
8 27.6 2.04 1.75
MagListo Q
MagListo Competitor
MagListo Competitor
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Table1.Result of capillary electrophoresis of Total RNA extraction from HeLa cell using LabChip
Sample
RNA
Quality
Score
5S Area 5S %Total 18S Area 18S %Total 28S Area 28S %Total
rRNA Area
Ratio
[28S/18S]
rRNA
Height
Ratio
[28S/18S]
MagListo
10.0 2.05 0.01 25.27 0.15 96.37 0.59 3.81 1.90
10.0 2.06 0.01 29.47 0.18 111.78 0.68 3.79 1.90
10.0 1.54 0.02 16.09 0.16 54.10 0.55 3.36 1.73
10.0 1.79 0.01 23.08 0.14 80.96 0.50 3.51 1.80
Competitor
10.0 1.96 0.01 31.72 0.15 119.04 0.57 3.75 1.90
10.0 1.77 0.01 23.25 0.16 102.63 0.70 4.41 2.06
10.0 1.44 0.01 20.33 0.15 84.37 0.61 4.15 2.06
10.0 2.01 0.01 25.22 0.14 99.41 0.54 3.94 1.93
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XIV. Ordering Information
Cat no. Product Description Size
K-3601 MagListoTM 5M Plamid Extraction Kit, 100 reactions (mini) 1 kit
K-3600 MagListoTM 5M Plamid Extraction Kit, 500 reactions (mini) 1 kit
K-3603 MagListoTM 5M Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit
K-3605 MagListoTM 5M Plant Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit
K-3607 MagListoTM 5M Gel Extraction Kit, 100 reactions (mini) 1 kit
K-3609 MagListoTM 5M PCR Purification Kit, 100 reactions (mini) 1 kit
K-3611 MagListoTM 5M Cell Total RNA Extraction Kit, 100 reactions (mini) 1 kit
K-3613 MagListoTM 5M Tissue Total RNA Extraction Kit, 100 reactions (mini) 1 kit
K-3615 MagListoTM 5M Forensic Sample DNA Extraction Kit, 100 reactions (mini) 1 kit
K-3617 MagListoTM 5M Viral DNA / RNA Extraction Kit, 100 reactions (mini) 1 kit
K-3619 MagListoTM 5M Circulating Cell Free DNA Extraction Kit, 50 reactions (mini) 1 kit
TM-1000 MagListoTM-8Ch Magnetic Separation Rack 1 ml tube x 8 holes
TM-1010 MagListoTM-2 Magnetic Separation Rack 2 ml tube x 8 holes
TM-1020 MagListoTM-15 Magnetic Separation Rack 15 ml tube x 6 holes
TM-1030 MagListoTM-50 Magnetic Separation Rack 50 ml tube x 3 holes
HT-15-NG 1.5 ml microcentrifuge tube 500 ea / pk
HT-20-NG 2 ml microcentrifuge tube 500 ea / pk
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XIX. Explanation of Symbols
Catalog
Number
Contains sufficient for
(n) tests USE BY
Batch code
Caution, consult
accompanying
documents
Temperature
Limitation
Manufacturer
Caution, Potential
Biohazard
DO NOT
REUSE
Consult Instruction
For Use