www.toyobo.co.jp/e/bio

F0982K

Instruction manual MagExtractor-RNA-2004

MagExtractor -RNANPK-201F 100 preparations

Store at 4°C

Contents[1]

Introduction[2]

Components[3]

Materials required[4]

Protocol1. Preparation of reagents required
2. Pretreatment of specimens
3. Purification[5]

Troubleshooting[6]

References[7]

Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnostic or clinical purposes. Please observe
general laboratory precaution and utilize safety while using this kit.
JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description

MagExtractor -RNA- provides a simple and reliable method for the rapid purification oftotal RNA from various specimens (e.g. cultured cells or animal tissues) using magnetic
silica beads. This kit is based on binding properties of RNA onto a silica surface in thepresence of chaotropic agents1)2) and an RNA-binding accelerator. Purified total RNA
contains primarily rRNA and mRNA. The purified total RNA can be used for RT-PCRexperiments.

LysisBinding

Washing

ElutionFig. 1 Principle of purification

Features-Purified total RNA contains primarily rRNA and mRNA.
-This kit does not contain hazardous substances, such as phenol or chloroform.
-No ethanol is used in the washing steps.
-This kit is suitable for high-throughput extraction of total RNA from various specimens.
The following table shows the typical yield and purity in each case.
SampleAmount ofspecimens

Yield

Cultured cells~5x106 cells~10 µg/106 cells

Tissue~30 mg~15 µg/30mg

Yeast~5 O.D.
(660nm)/ mL~20 µg/5 O.D.

Remarks
Total RNA yields depend on
the number of cells.
Total RNA yields depend on
tissues or storage conditions.
Pretreatment by zymolyase isnecessary.

Notes-This kit is not applicable for extraction from whole blood or serum. In the case ofwhole blood, white blood cells should be separated by a centrifugation using Ficoll.

[ 2 ] Components

This kit contains the following components for 100 preparations. Lysis & Binding Solution
and 2-mercaptethanol should be stored at 4 °C. The remaining components should be storedat 4°C or room temperature.
Lysis & Binding Solution*
Washing Solution I
Washing Solution IIElution Solution
Magnetic Beads

77 mL
66 mL
176 mL
10 mL
6 mL* Lysis & Binding Solution and 2-mercaptethanol (2-ME) should be mixed at a ratio of
100:1 prior to use. 2-ME is not supplied with this kit.

Caution:-The “Lysis & Binding Solution” and “Washing Solution I” contain chaotropic salt, which
is an irritant. Take appropriate laboratory safety measures and wear gloves when handling.
If contact with skin occurs, wash thoroughly with water. If the eyes get affected, flush
thoroughly for 15 min. with cool water, and consult a physician.
JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[3] Materials required

The following materials are required.
-2-Mercaptoethanol (2-ME)- Magnetic stand- Heating block can be set to 65°C.

[4]Protocol

1.

Fig.2 Magnetic standMagical Trapper (Code No.MGS-101)

Preparation of reagents required(1) Lysis & Binding solution (2-ME)
Lysis & Binding Solution and 2-Mercaptethanol (2-ME) should be mixed at a ratioof 100:1 prior to use. The mixed solution can be stored at 4 °C for 3 months. 2-ME
is not supplied with this kit.
(2) Washing Solution I and II should be used at room temperature.

2.

Pretreatment of specimens
Cultured cells, tissue, or yeast specimens should be treated with either of the following
methods.
(1)

Cultured cells

Pellet cultured cells by centrifugation in PBS(-). After resuspension with a small
amount of PBS(-), dispense 1x105-106 cells/100 μL PBS(-) into 1.5-mL microtubes.
Cultured cells (≤ 5x106 cells) 700 μL Lysis & Binding Buffer (containing 2-ME)
Pipette until viscosity is decreased

Vortex for 30 seconds

Incubate at room temperature for 10-15 minutes(2)

TissueFreezing tissue (≤ 30mg)

 750-900 μL Lysis & Binding Buffer (containing 2-ME)
Homogenization on iceVortex for 30-60 seconds until viscosity is decreasedCentrifuge at 3,000-5,000 rpm for 10 seconds

Supernatant 700 μL

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Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
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Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio(3)

Yeast cells
Pellet of yeast cells from 1 mL culture medium (≤ 5 O.D. [660 nm] /mL) 50 μL 30 mg/mL Zymolyase (20,000 U/g)*

Incubate at 37°C for 5-20 minutes after vortexing 700 μL Lysis & Binding Buffer (containing 2-ME)

Pipette until viscosity is decreased

Vortex for 30 seconds

Incubate at room temperature for 10-15 minutes*Zymolyase buffer : 0.9M Sorbitol, 0.1 M EDTA, 50 mM DTT (pH 7.5)

3.

Purification(1) [Binding] Add 50 μL magnetic beads to the 700 μL pretreated solution (seeabove section) and vortex for 20 seconds (maximum speed) and incubate for 4060 seconds at room temperature.
Notes
Completely resuspend the magnetic beads prior to use.
(2) Place each tube in the magnetic stand. The magnet will
attract the magnetic beads, separating from the specimen
solution.
(3) After magnetic capture, carefully remove the supernatant.

Fig. 3Magnetic separation(4) [Washing] Add 600 μL Washing Solution I to the beads and vortex for 10
seconds by maximum speed.
(5) Place each tube in the magnetic stand and collect the beads.
(6) After magnetic capture, carefully remove the supernatant.
(7) [Washing] Add 800 μL Washing Solution II and vortex for 5 seconds bymaximum speed.
(8) Place each tube in the magnetic stand and collect the beads.
(9) After magnetic capture, carefully remove the supernatant.
(10) [Washing] Repeat (7) - (9)(11) [Elution] Add 40 μL Elution Solution and mix well.
(12) Heat at 65°C for 2 minutes, and mix well.
(13) Place the tube in the magnetic stand.
(14) Collect the supernatant into a fresh tube.
JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 5 ] Related Protocol

1.

DNase I treatment of total RNA

Total RNA prepared by general methods contains genomic DNA. Genomic DNA can beeliminated by the following method.
(1) Mix the following reagents.
Nuclease-free water

X μL

Total RNA (<10 µg)

Y μL

10 x DNase I Buffer[e.g. 100 mM Tris-Cl, 20 mM MgCl2(pH 7.5)]

1 μL

RNase-free DNase I (10 U/μL)

0.5 μL

Total volume10 μL(2) Incubate on ice for 10-30 min.
(3) Purify the treated RNA according to the following step.
DNase I-treated RNA  Add nuclease-free water (adjust volume to 100 μL)
  Add 100 μL TE-saturated phenolVortex
Keep on ice for 5 min.
 Centrifuge at 12,000 rpm for 5 min.
Supernatant  Add 100 μL chloroform: isoamyl alcohol (24:1), Vortex
 Centrifuge at 12,000 rpm for 5 min. at 4 °C
Supernatant Add 100 μL 5 M ammonium acetate + 200 μL isopropanol+ [5 μL 2 mg/mL glycogen* (for coprecipitation) : optional]
Vortex
Incubate at - 20 °C for 30 min.
 Centrifuge at 12,000 rpm for 10-15 min. at 4 °C
Discard supernatant
Precipitate Add 1 mL 70% ethanol Centrifuge at 12,000 rpm for 5 min.
Discard supernatant
Precipitate Dissolve in appropriate volume of nuclease-free water
RNA solution*Molecular biology grade

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 5 ] Troubleshooting
Symptom

Low yield

Low 260/280 nm ratio

Poor amplification
with RT-PCR

Degradation of RNA[ 6 ] References

Cause

Solution

Insufficient pipetting in Lysis &Binding Solution

Pipette specimens in Lysis & Binding Solution
until viscosity is decreased.

Insufficient incubation in Lysis &Binding Solution
Excess centrifugation of treatedsolution in Lysis & Binding Solution

Increase incubation time > 15 minutes.

Excess specimen
Low RNA concentrationDecrease the amount of specimen.
Low RNA concentration tends to result in low
260/280 nm ratio. Dilute the RNA with 10 mM
Tris-HCl (pH 8.0)
Treat the RNA with DNase I. (see [5])Contamination with genomic DNA
RNA degradation
Heating of RNA
Excess specimen

In order to remove insoluble tissue, centrifuge at≤ 5,000 rpm for 10 seconds.

-Confirm storage condition of specimens forpurification.
-See “Degradation of RNA”
Heating of RNA in DNase I buffer may accelerate
RNA degradation.
RNA extracted from excess specimens might
contain residual ribonuclease.

1) B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci. USA. 76: 615-619 (1979)
2) R. Boom, C. J. A. Sol, M. M. M.Salimans, C. L. Wertheim-van Dillen, P. M. E. Dillen
and J. van der Noordaa, J. Clin. Microbiol., 28: 495-503 (1990)[ 7 ] Related products
Product name

Package1 piece

Magnetic standMagical Trapper

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

Code No.
MGS-101