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: order@bioneer.co.kr

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 (sales@bioneer.com).

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: 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 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