MicroRNA

Research ProductsmicroRNA Research Products
2

Contents
microRNA and Ago-Subfamily Proteins ……………………………… 3,4
1. microRNA Experiment Flow ………………………………………………… 5
2. Summary of Ago Immunoprecipitation
Benefit 1 Concentration of Ago-bound microRNA …………………………… 6
Benefit 2 Concentration of RISC-binding target mRNA ……………………… 7
Benefit 3 Concentration of free Ago-bound microRNA
from blood samples …………………………………………………… 8

3. Anti-Ago Monoclonal Antibody Series ………………………………… 9
Immunoprecipitation of recombinant Ago proteins …………………………… 10

4. microRNA Isolation Kit Series …………………………………………… 11
4-1. MagCaptureTM microRNA Isolation Kit (magnetic beads) ……………… 11
MagCaptureTM microRNA Isolation Kit, Human Ago2 ………………… 12
MagCaptureTM microRNA Isolation Kit, Mouse Ago2 …………………… 13
MagCaptureTM microRNA Isolation Kit, Ago1-4 ………………………… 13
MagCaptureTM microRNA Isolation Kit, Protein G ……………………… 15
4-2. microRNA Isolation Kit (silica-based beads) …………………………… 16
microRNA Isolation Kit, Human / Mouse Ago1 ………………………… 16
microRNA Isolation Kit, Human Ago2 …………………………………… 16
microRNA Isolation Kit, Mouse Ago2 …………………………………… 16
microRNA Isolation Kit, Human Ago3 …………………………………… 16

5. Total RNA Purification
5-1. microRNA Extractor® SP Kit ……………………………………………… 17
5-2. ISOGEN® II …………………………………………………………………… 18
5-3. ISOGEN® …………………………………………………………………… 18
5-4. ISOGEN®-LS ………………………………………………………………… 18

6. microRNA Analysis Tools
Electrophoresis
6-1. SuperSepTM RNA, 15%, 17-Well ………………………………………… 19
6-2. R-MarkTM Small RNA Marker (20-50nt) …………………………………… 19
6-3. CLEAR STAIN Ag ………………………………………………………… 20
6-4. 5×TBE ………………………………………………………………………… 20
cDNA Cloning
6-5. microRNA Cloning Kit Wako ……………………………………………… 21
6-6. Target mRNA Cloning Kit Wako …………………………………………… 21
Microarray Analysis
6-7. Toray 3D-Gene ® …………………………………………………………… 22

7. Wako Pure Chemical Industries, Ltd. Publication list ……… 23
8. Products List
Other Reagents for microRNA Research ……………………………………… 23
Monoclonal Antibodies for microRNA Research ……………………………… 24
Reagent Kits for microRNA Research ………………………………………… 24

Front cover: Tomari Lab., The University of Tokyo, JapanmicroRNA and Ago-Subfamily Proteins
microRNA (miRNA) are small functional RNA molecules (about 22 nucleotides) which act as posttranscriptional regulators of gene expression. They serve many functions within biological organisms (Ref.
1), and more than 1,000 types are known to exist in humans and mice. Worldwide there are many efforts
to identify new types of microRNA with unknown functions and elucidate functions of those involved
in disease. Recently it has been reported that microRNA exist not only within cells, but also blood andother bodily fluids. They are now garnering attention as possible clinical markers for cancer and otherdiseases (Ref. 2).
In cells, microRNA pass through many steps before being incorporated into a multiprotein complex
called a RISC (RNA-induced silencing complex). After binding with the main component of Agosubfamily proteins, they bind with mRNA targets and either cleave them or inhibit translation, thus
regulating gene expression (Fig. 1) (Ref. 3, 4, 5). The Ago-subfamily of proteins are characterized bycontaining both a PAZ domain and a PIWI domain (Fig. 2). There are four types in humans (hAgo1 tohAgo4), and although each expression levels differ from each them in each cell lines (Fig. 3) (Ref. 6).
The most commonly expressed Ago-subfamily protein is Ago2, which has unique "Slicer" activity which
directly cleaves target RNA. Thus, Ago2 is considered to play an important role in microRNA pathways(Ref. 7, 8, 9, 10, 11, 12, 13). Anti-Ago antibodies are used for immunoprecipitation of RISC, allowing
recovery of both microRNA and their mRNA targets. This immunoprecipitation method is vital for
elucidating the various functions of microRNA (Ref.14, 15, 16, 17, 18, 19, 20, 21).
We offer a full selection of antibodies for various human and mouse Ago-subfamily proteins, as well as
immunoprecipitation kits for them. These tools allow comprehensive analysis of microRNA and mRNA
contained in RISC in cells and tissue, as well as Ago-bound microRNA in blood.

Figure 1 Molecular mechanism of RNA silencing

3Ago-subfamily

Figure 2 PAZ domain and PIWI domain

Data: Haruhiko Siomi, Ph. D., Keio University, Japan

Figure 3 Expression
comparison ofhAgo1–hAgo4

Figure 4 Localization of Ago2

References
1.Bartel DP: MicroRNA:genomics, biogenesis, mechanism, and function. Cell 2004, 116:281-297.
2.Cortez MA, Calin GA : MicroRNA identification in plasma and serum: a new tool to diagnose and monitor diseases.
Expert Opin Biol Ther 2009, 9:703-711
3.Hammond SM, Bernstein E, Beach D, Hannon GJ: An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.
Nature 2000, 404:293-296.
4.Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ: Argonaute2, a link between genetic and biochemical analyses of RNAi.
Science 2001, 293:1146-1150.
5.Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R: Human RISC couples microRNA biogenesis and posttranscriptional gene silencing.
Cell 2005, 123:631-640.
6.Sasaki T, Shiohama A, Minoshima S, Shimizu N: Identification of eight members of the Argonaute family in the human genome.
Genomics 2003, 82:323-330.
7.Pillai RS, Artus CG., Filipowicz W: Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis.
RNA 2004, 10:1518-1525.
8.Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T: Human Argonaute2 mediates RNA cleavage targeted by miRNAs andsiRNAs. Mol Cell 2004, 15:185-197.
9.Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ: Argonaute2 is the catalytic
engine of mammalian RNAi. Science 2004, 305:1437-1441.
10.Song JJ, Smith SK, Hannon GJ, Joshua-Tor L: Crystal structure of Argonaute and its implications for RISC slicer activity. Science 2004,
305:1434-1437.
11.Meister G, Tuschl T: Mechanisms of gene silencing by double-stranded RNA. Nature 2004, 431:343-349.
12.Hutvagner G, Zamore PD: A microRNA in a multiple-turnover RNAi enzyme complex. Science 2002, 297:2056-2060.
13.Yekta S, Shih IH, Bartel DP: MicroRNA-directed cleavage of HOXB8 mRNA. Science 2004, 304:594-596.
14.Beitzinger M, Peters L, Zhu JY, Kremmer E, Meister G: Identification of human microRNA targets from isolated Argonaute protein
complexes.
RNA Biol 2007, 4:76-84.
15.Karginov FV, Conaco C, Xuan Z, Schmidt BH, Parker JS, Mandel G, Hannon GJ: A biochemical approach to identifying microRNA targets.
Proc Natl Acad Sci USA 2007, 104:19291-19296.
16.Easow G, Teleman AA, Cohen SM: Isolation of microRNA targets by miRNP immunopurification. RNA 2007, 13:1198-1204.
17.Hendrickson DG, Hogan DJ, Herschlag D, Ferrell JE, Brown PO: Systematic identification of mRNAs recruited to Argonaute2 by specificmicroRNAs and corresponding changes in transcript abundance. PloS ONE 2008, 3:e2126.
18.Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L, Ojo T, Hafner M, Zavolan M, Tuschl T: Molecular characterization ofhuman Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA 2008, 14:2580-2596.
19.Chi SW, Zang JB, Mele A, Darnell RB: Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 2009, 460:479-486.
20.Wang WX, Wilfred BR, Hu Y, Stromberg AJ, Nelson PT: Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns ofmRNA recruitment to microribonucleoprotein complexes. RNA 2009 Dec.30.
21.Hayashida Y, Nishibu T, Inoue K, Kurokawa T: A useful approach to total analysis of RISC associated RNA . BMC Res Notes 2009, 2:169.

4

1.

microRNA Experiment Flow

We offer a microRNA Isolation Kit series containing monoclonal antibodies for various Ago-subfamily
proteins and immunoprecipitation methods (Ago IP) for each of these. We also offer tools such as
the ISOGEN® series for purification of total RNA from samples or the microRNA Extractor® SP Kit, allallowing purification of fractions containing microRNA from samples depending on research needs.
Finally, we offer additional powerful tools to support microRNA research, such as tools to clone cDNAfrom purified microRNA or target mRNA, electrophoresis tools, and microarray analysis services.

Cell or tissue sample

Conditioned culture medium sample

Plasma or serum sample

Purification for microRNA/target mRNA

【Related products for Ago-immunoprecipitation】

Anti Ago Monoclonal Antibody series【Related products for total RNA purification】

microRNA Extractor® SP kit

microRNA Isolation Kit series

ISOGEN® II
ISOGEN®
ISOGEN®-LS

Analysis for purified microRNA/target mRNA

【Related products for electrophoresis p.19】 〈Related products for cDNA cloning p.21〉

SuperSep

TM

Urea

Small RNA Marker

microRNA Cloning Kit〈Microarray Analysis p.22〉

3D-Gene ® (Toray) analysis service

Target mRNA Cloning Kit

CLEAR STAIN Ag

5

2.

Summary of Ago Immunoprecipitation

We offer a full selection of human and mouse antibodies for Ago-subfamily proteins as well as the
microRNA Isolation Kit series based on immunoprecipitation (Ago IP) using these antibodies. These tools
offer the following benefits over traditional total RNA purification.

Benefit 1➡

Concentration of Ago-bound microRNA

Improvement of detection sensitivity of microRNA incorporated in RISC andsuppress detection of non-specific detection of microRNA not incorporated in RISC.
ExampleComparing populations of Ago IP RNA fractions and total RNA fractions from HeLa cells

Starting with HeLa cells, we used a micro-array (3D-Gene ®, Toray) to compare populations of RNA
obtained with immunoprecipitation from monoclonal antibodies specifically recognizing each Ago
subfamily protein (Ago1 to Ago4) (Ago IP RNA fraction) with microRNA contained in a total RNA fraction
prepared using ISOGEN (AGPC) (Fig. 1). Results showed that more microRNA was concentrated in each
Ago IP RNA fraction than the total RNA fraction (excluding the Ago4 IP RNA fraction), yielding a stronger
signal (Fig. 2). One signal was strongly detected in the total RNA fraction while remaining largely absent
in the Ago IP RNA fraction, suggesting that microRNA not incorporated into RISCs were non-specificallydetected in the total RNA fraction.

Ago IP RNA fraction

Total RNA fraction

Other non-coding RNA

Figure 1 Experiment flow

Figure 2 microRNA signal strength comparison
between each Ago IP RNA fraction and total RNA fraction6➡

Benefit 2

Concentration of RISC-binding target mRNARNA fractions obtained by Ago IP method are applicable to analysis of target mRNA
by cDNA cloning, qPCR, microarray, Next-generation DNA sequencer because
target mRNA is concentrated.
ExampleConcentration of target mRNA of liver-specific microRNA(miR-122)

Either miR-122 or the control of luciferace siRNA (Luc siRNA) were delivered to HepG2, a liver cancer
cell line with low expression of miR-122, then Ago2 IP RNA (using anti-Ago2 monoclonal antibody 4G8)
and total RNA (using ISOGEN) purified from both samples. Quantitative PCR was then used to compare(Fig. 1) quantities of Aldo A mRNA as a miR-122 target (normalized with GAPDH mRNA) in both cell
types. Results show that the quantity of Aldo A mRNA decreased by RISC cleavage, in the total RNA
fraction while Aldo A mRNA in the Ago2 IP RNA fraction was concentrated (Fig. 2).
miR-122 transfected cells

Luc siRNA transfected cells

Anti Ago2 MAb(4G8)Anti Ago2 MAb(4G8)Quantitative PCR
Aldo A mRNA relative quantity (Luc siRNA transfected cells:1.0)

Figure 1 Experiment flow

Luc siRNA transfected cells
miR-122 transfected cells

Figure 2 Quantities of Aldo A mRNA (quantitative PCR)

7

Benefit 3➡ Concentration of free Ago-bound microRNA from blood samples

Blood is known to include microRNA contained in exosomes, HDL-bound microRNA,
and Ago-bound microRNA. Ago immunoprecipitation method allows for specificisolation of Ago-bound microRNA, and it can be used for biomarker searches andother uses.
Example 1

Isolation of Ago2-bound microRNA from human plasma

Quantitative PCR (Ct value) was used to measure 12 types of arbitrarily-selected microRNA in pooled
plasma of healthy individuals from immunoprecipitated Ago2 IP RNA (using anti-Ago2 monoclonal
antibody 4G8) and total RNA (using microRNA Extractor® SP Kit) (Fig. 1). Comparison reveals thatmost microRNA detected in the total RNA sample was also detected in the Ago2 IP RNA fraction, while
types such as miR-22 and miR-92a were highly concentrated in the Ago2 IP RNA fraction (low Ct value
compared to total RNA) (Fig. 2).
Human plasma

Human plasma

Anti Ago2 MAb(4G8)Quantitative PCR
Figure 1 Experiment flow

Total RNA
Ago2 IP RNA

Figure 2 Comparison of microRNA quantities (Ct value)(quantitative PCR)

8

Example 2

Isolation of each Ago-bound microRNA from human plasma

Quantitative PCR was used to compare
microRNA (miR-92a and miR-122) contained
in immunoprecipitated RNA (Ago 1, 2, 3,
4 IP RNA) using anti-Ago (Ago1 to Ago4)
monoclonal antibodies from pooled plasmaof healthy individuals (Fig. 1). Results show
microRNA present in other Ago IP RNA
fractions in addition to the Ago2 IP RNA
fraction, with liver-specific miR-122 highly
concentrated in the Ago3 IP RNA fraction(Fig. 2).

Human plasma

Anti Ago1,2,3,4 MAb

Figure 1 Experiment flow

500,000
450,000
400,000
350,000
300,000
250,000
200,000
150,000
100,000
50,0000

20,00018,00016,00014,00012,00010,000
8,000
6,000
4,000
2,000
0

Figure 2 Comparison of microRNA quantities from each Ago IP RNA fractions (quantitative PCR)

3.

Anti-Ago Monoclonal Antibody Series

We offer a full line of mouse monoclonal antibodies both for Western blot detection andimmunoprecipitation recognizing various human and mouse Ago-subfamily proteins. These tools allow
selective immunoprecipitation of various Ago-subfamily proteins from human and mouse samples inorder to obtain microRNA bonded to each. Proteins obtained can also be confirmed with Western blot.
Antigen

Cross-reactivity

Ago1

Human,
Mouse

Description
Anti Ago1, Monoclonal antibody
Anti Ago1, Monoclonal antibody

Clone No.
2A7
1F2

Subclass
IgG2a･k
IgG2a･kApplication
IP, ICC, RIP
WB

Human

Anti Human Ago2, Monoclonal antibody

4G8

IgG1

IP, WB
ICC, RIP

Mouse

Anti Mouse Ago2, Monoclonal antibody

2D4

IgG1

IP, WB
ICC, RIP

Anti Human Ago3, Monoclonal antibody
Anti Ago3, Monoclonal antibody
Anti Mouse Ago3, Monoclonal antibody
Anti Mouse Ago3, Monoclonal antibody
Anti Ago4, Monoclonal antibody
Anti Ago4, Monoclonal antibody

1C12
6-107
S09
S011
2G7
2B2

IgG1
IgG1･k
IgG1･k
IgG1･kIgG2b
IgG1

IP, RIPWB
IP, RIPWB
IP, RIP
WB

Anti Ago1–4, Monoclonal antibody

1G3

IgG1

IP, WB

Ago2

Human
Ago3
Mouse
Ago4

Human,
Mouse

Ago1–4

Human,
Mouse

Concentration

1.0mg/mL

9

Immunoprecipitation of recombinant Ago proteins
We evaluated immunoprecipitation performance of each antibody using human and mouse recombinantAgo proteins (DYKDDDDK tag was fused to C-terminal). Each anti-Ago1 to anti-Ago4 antibody
specifically immunoprecipitated
Recombinant human Ago proteins
Recombinant mouse Ago proteinseach recombinant Ago protein,demonstrating that anti-Ago1
to anti-Ago4 antibodies arecapable of immunoprecipitationof all Ago-subfamily proteins.

[Detection] Primary antibody: Anti-DYKDDDDK Antibody(1E6)Secondary antibody: Anti-mouse IgG, Peroxidase ConjugatedWestern blot analysis of recombinant Ago proteins
We evaluated Western blot performance of each antibody using human and mouse recombinant Ago
proteins (DYKDDDDK tag was fused to C-terminal). Each anti-Ago1 to anti-Ago4 antibody specificallydetected each recombinant Ago
Recombinant human Ago proteins
Recombinant mouse Ago proteinsprotein, demonstrating that antiAgo1 to anti-Ago4 antibodies
are capable of detection of all
Ago-subfamily proteins.

[Detection] Primary antibody: Each Anti-Ago Monoclonal Antibody
Secondary antibody: Anti-mouse IgG, Peroxidase ConjugatedEndogenous Ago protein immunoprecipitation and Western blot analysis
We immunoprecipitated and then used Western blot analysis for detection of endogenous Ago proteinsusing anti-human Ago antibodies from human K562 cells and anti-mouse Ago antibodies from mouse
P388D1 cells or mouse lung tissue. Each anti-Ago1 to anti-Ago4 antibody specifically immunoprecipitatedeach endogenous Ago protein and
Human endogenous Ago
Mouse endogenous Agoused for Western blot analysis,
indicating that anti-Ago1 to antiAgo4 antibodies are capable ofseimmunoprecipitation of endogenous
se
ou
ou
lm
m
a
l
a
rm
Ago1 to Ago3.
rm
no
no

[Detection] Primary antibody: Each Anti-Ago Monoclonal Antibody
Secondary antibody: Anti-mouse IgG, Peroxidase Conjugated

10

4.

microRNA Isolation Kit Series

We offer a microRNA Isolation Kit series to simplify an entire range of procedures from Ago
immunoprecipitation to RNA purification. This kit series allows recovery of Ago-bound microRNA andtarget mRNA from both human and mouse cells and tissues, and free Ago-bound microRNA from culture
supernatant, serum, and plasma. Obtained RNA can then be analyzed by quantitative PCR, microarrays,
and next-generation sequencing. This series includes MagCaptureTM microRNA Isolation Kits, which
adopt magnetic beads, and microRNA Isolation Kits, which adopt silica-based beads. Features andbenefits of each product are described below.
Anti Ago MAb
conjugated
Beads

Cell/tissue
Culture supernatant/serum/plasma<Applications>RNApurification

Ago IP◦ Quantitative PCR◦ Cloning◦ Microarrays◦ Next-generation

sequencing

4-1. MagCaptureTM microRNA Isolation Kit (magnetic beads)
We offer four types of MagCaptureTM microRNA Isolation Kits: three types contain magnetic beads
immobilized to anti human Ago2 antibody (4G8), anti mouse Ago2 antibody (2D4), or anti-Ago1-4
antibody (1G3), and one type contain with Protein G magnetic beads which can immobilize an optional
anti Ago subfamily antibodies. All of these kits adopt magnetic beads for easy immunoprecipitation.
After immunoprecipitation, two protocols for the RNA purification step are selectable: One-step method
for obtaining high-yield microRNAs■ Outline
by a simple procedure or Spincolumn method for obtaining highSample
preparation
purity microRNAs.
Cells/tissue

Culture supernatant/serum/plasma■ Kit contents(Example: Human Ago2, 10 reactions)

1) Anti-Human Ago2 Magnetic Beads Solution 600μL×1 tube
2) Cell Lysis Solution
20mL×1 tube
3) Washing Solution for IP
40mL×1 tube4) Elution Solution I for IP
500μL×1 tube5) Elution Solution II for IP
500μL×1 tube
6) Binding Solution for Spin Column
2mL×1 tube
7) Binding Enhancer for Spin Column
100μL×1 tube
8) Washing Solution I for Spin Column
3mL×1 tube
9) Washing Solution II for Spin Column
4mL×1 tube
10) Elution Solution for Spin Column
1mL×1 tube
11) Spin Column/Collection Tube
10 tubes

Immunoprecipitation
reactionRNA
purification

Cell solution

One-step method

Spin-column method

Heated elution

Purification
completemicroRNA
Target mRNA
purification
Compatible
samples

Description

Antibody
clone no.

Animal

MagCapture™ microRNA Isolation Kit, Human Ago2

4G8

Human, monkey, dog

MagCapture™ microRNA Isolation Kit, Mouse Ago2

2D4

MagCapture™ microRNA Isolation Kit, Ago 1-4

1G3

Cell, tissue,
Mouse, rat, dog
serum, plasma
Human, monkey, dog,mouse, rat

MagCapture™ microRNA Isolation Kit, Protein G

Any

Depends on antibody

Cell, tissue

RecoveredRNA

IP B/F separation RNA purification
methodmethodmicroRNA andtarget mRNA
microRNA
Depends on
antibody

Magnetic
separation

Spin column or
one-stepSpin column

11MagCaptureTM microRNA Isolation Kit, Human Ago2
Recovering microRNA from human cell strains
We used MagCapture TM microRNA Isolation Kit, Human Ago2 and a previous product to obtainmicroRNA from human K562 cells (1×107 cells), then compared quantities of microRNA obtained withdenaturing polyacrylamide gel electrophoresis (silver staining) (Fig. 1) and quantitative PCR (miR-92a
quantity) (Fig. 2). Results showed that the two products yielded equivalent quantities of microRNA.
M

1

2

3

3.5×1010
3.0×1010
2.5×1010

LaneM:Marker
Lane1:synthesized RNA oligo (23 base)1ng
Lane2:MagCaptureTM (Spin Column Method)
Lane3:MagCaptureTM (One Step Method)

2.0×1010
1.5×1010
1.0×1010
5.0×109
0.0

MagCaptureTM(Spin Column
Method)MagCaptureTM(One Step
Method)silica-based
beads**4-2 microRNA Isolation Kit (292-66701)

Figure 1 Detection of purified microRNAs from human K562
cells by silver staining

Figure 2 Comparison of recovery quantities ofpurified microRNAs from human K562 cells

Recovering free Ago2-bound microRNA from human plasma
We used MagCaptureTM microRNA Isolation Kit, Human Ago2
and a previous product to obtain microRNA from pooled plasmaof healthy individuals, then compared quantities obtained withquantitative PCR (miR-92a quantity). As shown below, using
the spin column method with the MagCapture TM resulted inapproximately equivalent yield to our previous product, while
the one-step method demonstrated greater quantities of
microRNA recovered compared to our previous product.

4.0×105
3.5×105
3.0×105
2.5×105
2.0×105
1.5×105
1.0×105
5.0×104
0.0

MagCaptureTM(Spin Column
Method)MagCaptureTM(One Step
Method)silica-based
beads**4-2 microRNA Isolation Kit (292-66701)

Figure 3 Comparison of recovery quantities ofpurified microRNAs from human plasma

Recovering target mRNA of microRNA
Either miR-122 or the control of luciferase siRNA (Luc siRNA) were delivered to HepG2, a liver cancer
cell line with low expression of miR-122, then immunoprecipitated Ago2 IP RNA obtained using either
MagCaptureTM microRNA Isolation Kit, Human Ago2 or our previous product. Total RNA was preparedwith ISOGEN. Quantitative PCR was then used to compare quantities of miR-122 (Fig. 4) and its target
mRNA (Aldo A corrected with GAPDH mRNA) (Fig. 5). Results showed that miR-122 and Aldo A mRNA
were concentrated in both MagCaptureTM and our previous kit, while the former also allowed recovery of
microRNA's target mRNA. In contrast, total RNA obtained with ISOGEN showed decreased quantities of
Aldo A mRNA due to cleavage accompanying increase of miR-122.
5.0×105
4.5×105
4.0×105
3.5×105
3.0×105
2.5×105
2.0×105
1.5×105
1.0×105
5.0×104
0.0
TM
TM
A)
re
re
RN
ptu hod)
ptu hod)
tal
a
a
t
t
gC Me
gC Me
(To
a
a
N
M mn
M tep
GE
S
lu
ISO
ne
Co
(O
in
p
(S
*4-2 microRNA Isolation Kit (292-66701)

ds

ea

s

-ba

ca

sili

b
ed

*

Figure 4 Comparison of recovery quantities of miR-122

12

TM
TM
A)
re
re
RN
ptu hod)
ptu hod)
tal
a
a
t
t
gC Me
gC Me
(To
a
a
N
M mn
M tep
GE
S
lu
ISO
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Co
(O
in
p
(S
*4-2 microRNA Isolation Kit (292-66701)

ds

ea

s

-ba

ca

sili

b
ed

*

Figure 5 Comparison of recovery quantities of target mRNA

MagCaptureTM microRNA Isolation Kit, Mouse Ago2
Recovering microRNA from mouse cell strains
We used MagCaptureTM microRNA Isolation
M 1 2 3
Kit, Human Ago2 and a previous productto obtain microRNA from mouse P388D1
cells (2×107 cells), then compared quantities
of microRNA obtained with denaturing
polyacrylamide gel electrophoresis (silver
staining) (Fig. 1) and quantitative PCR (miR92a quantity) (Fig. 2).
LaneM:Marker
Lane1:synthesized RNA oligo (23 base)1ng
Results showed that theLane2:MagCapture (Spin Column Method)
Lane3:MagCapture (One Step Method)
two products yielded
equivalent quantities of Figure 1 Detection of purified microRNAs from mouse
P388D1 cells by silver staining
microRNA.

7.0×109
6.0×109
5.0×109
4.0×109
3.0×109
2.0×109
1.04×109
0.0

TM

MagCaptureTM(Spin Column
Method)TM

MagCaptureTM(One Step
Method)silica-based
beads**4-2 microRNA Isolation Kit (292-67301)

Figure 2 Comparison of recovery quantities of purified
microRNAs from mouse P388D1 cells

Recovering free Ago2-bound microRNA from rat plasma
We used MagCaptureTM microRNA Isolation Kit, Mouse Ago2 anda previous product to obtain microRNA from 200 μL rat plasma,then compared quantities obtained with Quantitative PCR (miR92a quantity). As shown below, using the spin column methodwith the MagCaptureTM resulted in approximately equivalent yieldto our previous product, while the one-step method demonstrated
greater quantities of microRNA recovered compared to ourprevious product.

1.8×106
1.6×106
1.4×106
1.2×106
1.0×106
8.0×105
6.0×105
4.0×105
2.0×105
0.0

MagCaptureTM(Spin Column
Method)MagCaptureTM(One Step
Method)silica-based
beads**4-2 microRNA Isolation Kit (292-67301)

Figure 2 Comparison of recovery quantities of purified
microRNAs from mouse plasma

MagCaptureTM microRNA Isolation Kit, Ago 1-4
Recovering Ago1-4-bound microRNA

Signal intensity

Signal ratio

Ago1 IP

Ago2 IP

Ago3 IP

Ago4 IPhsa-miR-22

3521.421696.5

3771.2hsa-miR-16

6886.328527.2

hsa-miR-1260b

2060.3hsa-miR-4286

678.8

Name

Ago2/Ago1

Ago2/Ago3

Ago2/Ago4

292.4

6.16

5.75

74.20

5013.4

582.5

4.14

5.69

48.97

540.9

1270.6

1977.9

0.26

0.43

0.27

202.4

2019.2

471.2

0.30

0.10

0.43

Table 1 Result of microarray analysis

microRNA level (previous product*=1)

We used a microarray (3D-Gene ®, Toray) to compare microRNA expression profile of RNA fractions
obtained from HeLa cells by immunoprecipitation of anti-Ago1 to anti-Ago4 antibodies. While large
quantities of miR-22 and miR-16 were incorporated into Ago2, large quantities of miR-1260b and miR4286 were incorporated into Ago1, Ago3, and Ago4 (see table below).
Based on these results, we decided to study whether MagCaptureTM could recover microRNA binding
Ago1, Ago3, or Ago4 at high levels, which was difficult with our previous product (microRNA Isolation
Kit, Human Ago2 ). Both products were used to recover microRNA from HeLa cells (1×107 cells), andquantitative PCR used to measure quantities of miR-22, miR-16, miR-1260b, and miR-4286 recovered,using miR-92a as an internal standard. Results showed equivalent recovery between the two productsof miR-22 and miR-16, which are shown present in Ago2 in high levels, while MagCaptureTM microRNA
Isolation Kit, Ago1-4 recovered miR-1260b and miR-4286 at higher levels than the previous product(see below). These results show that the new MagCaptureTM
microRNA Isolation Kit, Ago1-4 is capable of the detection
previous product*(Human Ago2)of not only microRNA binding Ago2, but also in Ago1, Ago3,
MagCapture (Ago1-4)
and Ago4.
TM*4-2 microRNA Isolation Kit (292-66701)

Figure 1 Comparison of quantities of each microRNAs
from HeLa cells

13

Recovering microRNA from human and mouse cell lines

MagCaptureTM microRNA Isolation Kit, Ago1-4 and our previous product (microRNA Isolation Kit, Human
Ago2 or Mouse Ago2) were used to recover microRNA from human K562 cells (1×107 cells) and mouse
P388D1 cells (2×107 cells). Quantitative PCR (miR-92a quantity) comparisons shown below indicate that
MagCaptureTM recovered more microRNA more than the previous product.
4.0×1010

3.0×109

3.5×1010

2.5×109

3.0×1010
2.0×109

2.5×1010
10

1.5×109

2.0×10

1.5×1010

1.0×109

1.0×1010

5.0×108

5.0×109
0.0

previous product(Human Ago2)

0.0

MagCaptureTM

previous product(Mouse Ago2)

MagCaptureTM

Figure 3 Comparison of recovery quantities of purified
microRNAs from mouse P388D1 cells

Figure 2 Comparison of recovery quantities ofpurified microRNAs from human K562 cells

Recovering free Ago-binding microRNA from human and rat plasma

MagCaptureTM microRNA Isolation Kit, Ago1-4 and our previous product (microRNA Isolation Kit, Human
Ago2 or Mouse Ago2) were used to recover microRNA from 200 μL human and rat plasma. Quantitative
PCR (miR-92a quantity) comparisons shown below indicate that MagCaptureTM recovered microRNA
more than the previous product.
3.5×105

2.0×106

3.0×105

1.8×1065

1.6×106

2.5×10

1.4×106

2.0×105

1.2×106

5

1.0×106

1.5×108.0×105

1.0×105

6.0×105

5.0×104

4.0×105
2.0×105

0.0
previous product(Human Ago2)

MagCapture

0.0

TM

Figure 4 Comparison of recovery quantities of purified
microRNAs from human plasma

previous product(Mouse Ago2)

MagCaptureTM

Figure 5 Comparison of recovery quantities ofpurified microRNAs from rat plasma

Recovering microRNA from various animal cell lines

MagCapture TM microRNA Isolation Kit, Human Ago2, Mouse Ago2, and Ago1–4 were used toimmunoprecipitate microRNA from K562 cells, monkey COS-7 cells, dog MDCK cells, mouse P388D1
cells, and rat SCC-131 cells (each: 2×106 cells). On the other hand, ISOGEN was used to obtain total
microRNA fractions from the same cell samples.
As a result of comparison of relative value of miR-92a quantities between each Ago IP fractions and total
RNA fractions, MagCaptureTM recovered microRNAs from all cell types.

Anti Human Ago2,MAb
Anti Mouse Ago2,MAb
Anti Ago1-4,MAb(Human)(Monkey)(Dog)(Mouse)(Rat)

Figure 6 Comparison of recovery quantities of purified microRNAs from each cells

14

MagCaptureTM microRNA Isolation Kit, Protein G

5.0×108

6.0×106

4.0×108
4.0×106

3.0×1082.0×1082.0×106
0.0

1.0×108
MagCaptureTM

previous product

0.0

MagCaptureTM

previous product

MagCaptureTM: Ago1 antibody (2A7)Previous product: microRNA Isolation Kit, Human/Mouse
Ago1 (291-70201)

MagCaptureTM: Anti human Ago2 antibody (4G8)Previous product: microRNA Isolation Kit, Human Ago2(292-66701)

Figure 1 Comparison of recovery quantities of purified microRNAs
Immobilized antibody: Anti-Human Ago1(2A7)

Figure 2 Comparison of recovery quantities of purified microRNAs
Immobilized antibody: Anti-Human Ago2(4G8)

1.2×108

1.0×108

1.0×1088.0×1077

8.0×10

6.0×107

6.0×107
4.0×107

4.0×1072.0×1072.0×107
0.0

0.0
MagCaptureTM

MagCaptureTM

previous product

MagCaptureTM: Anti human Ago3 antibody (1C12)Previous product: microRNA Isolation Kit, Human Ago3(297-70301)

Figure 4 Comparison of recovery
quantities of purified
microRNAs
Immobilized antibody: AntiMouse Ago2(2D4)

l-m

iR

-2

38

1n

g

Figure 3 Comparison of recovery
quantities of purified
microRNAs
Immobilized antibody: AntiHuman Ago3(1C12)

previous product

MagCaptureTM: Anti mouse Ago2 antibody (2D4)
Previous product: microRNA Isolation Kit, Mouse Ago2(292-67301)

hA 2
go
3
hA
go
4

go

1

go

hA

（base）

hA

ke

r
he
siz

ed

ce

K562 cells(Human)
1.0×109
1.0×108
1.0×107
1.0×106
1.0×105
1.0×104
1.0×103
1.0×102
1.0×101.0

nt

MagCapture TM microRNA Isolation
Kit, ProteinG (using anti-Ago1 (2A7),
anti-Ago2 (4G8), anti-Ago3 (1C12),and anti-Ago4 (2G7) antibodies) wereused to obtain microRNA from human
K562 cells (1×10 7 cells). Quantities
of microRNA were measured withdenaturing polyacrylamide gelelectrophoresis (silver staining) andquantitative PCR (miR-92a quantity)(Fig. 5). Next, MagCapture TM (using
anti-Ago1 (2A7), anti-Ago2 (2D4),
anti-Ago3 (S09), and anti-Ago4(2G7) antibodies) was used to obtainmicroRNA from mouse P388D1 cells(2×107 cells). Quantities of microRNA
were measured with denaturing
polyacrylamide gel electrophoresis(silver staining) and quantitative PCR(miR-92a quantity) (Fig. 6). Results
show that MagCapture TM with an
optional anti-Ago antibody enables
recovery of each Ago-binding
microRNA.

6.0×108ar

Recovering different types of Agobinding microRNA from human andmouse cell lines

7.0×1088.0×106

M

MagCapture microRNA Isolation
Kit, ProteinG (using anti-Ago1(2A7), anti-Ago2 (4G8), and antiAgo3 (1C12) antibodies) and ourprevious products were used toobtain microRNA from human K562
cells (1×10 7 cells). Figure 1, 2, 3
shows quantitative PCR (miR-92a
quantity) comparisons of quantities
of microRNA recovered. Next,
MagCapture TM (using mouse antiAgo2 antibodies (2D4) and ourprevious product were used to obtainmicroRNA from mouse P388D1 cells(2×10 7 cells) (Fig. 4). Results showthat MagCaptureTM with an optionalanti-Ago antibody enables microRNA
recovery superior to that of ourprevious product.

8.0×108sy

TM

1.0×107

hAgo1: Anti Ago1 monoclonal antibody (2A7)
hAgo2: Anti human Ago2 monoclonal antibody (4G8)hAgo3: Anti human Ago3 monoclonal antibody (1C12)
hAgo4: Anti Ago4 monoclonal antibody (2G7)

Figure 5 Comparison of recovery quantities of purified microRNAs from human
K562 cells and detection by silver staining
P388D1(Mouse)
1.0×108
1.0×107
1.0×106
1.0×105

M
ar
ke
sy r
nt
he
siz
m
ed
Ag
ce
o1
l-m
m
Ag
iR
-2
o2
38
m
Ag
1n
o3
g
m
Ag
o4

Recovering microRNA from humanand mouse cell lines (productcomparison)（base）

1.0×104
1.0×103
1.0×102
1.0×10
1.0
mAgo1: Anti Ago1 monoclonal antibody (2A7)
mAgo2: Anti mouse Ago2 monoclonal antibody (2D4)
mAgo3: Anti mouse Ago3 monoclonal antibody (S09)
mAgo4: Anti Ago4 monoclonal antibody (2G7)

Figure 6 Comparison of recovery quantities of purified microRNAs from mouse
P388D1 cells and detection by silver staining15

4-2. microRNA Isolation Kit (silica-based beads)■ Outline

Our microRNA Isolation Kit uses anti-Ago antibodies
conjugated to silica-based polymer beads with
low non-specific absorbency for centrifuge
immunoprecipitation. Post-immunoprecipitation
RNA purification steps are performed by phenol/
chloroform and ethanol precipitation. We offer alineup of four types: anti-Ago1 antibody (2A7), anti
human Ago2 antibody (4G8), anti mouse Ago2
antibody (2D4), and anti human Ago3 antibody(1C12).
■ Kit contents (Example: Human Ago2, 10 reactions)
1) Anti-Human Ago2 Antibody Beads Solution
2) Cell Lysis Solution3) Elution Solution
4) Ethachinmate5) 3 mol/L Sodium Acetate

500μL×1 tube
50mL×1 tube
500μL×1 tube
30μL×1 tube
400μL×1 tube

Antibody
clone no.

Description

AnimalmicroRNA Isolation Kit, Human/Mouse Ago1

2A7

Human, mousemicroRNA Isolation Kit, Human Ago2

4G8

Human, monkey, dog

microRNA Isolation Kit, Mouse Ago2

2D4

Mouse, rat

microRNA Isolation Kit, Human Ago3

1C12

Human

Compatible
samples

Recovered RNA

Cells, tissue

microRNA andtarget mRNA

IP B/F separation RNA purification
methodmethodCentrifuge

Phenol/chloroform

Application Data
The microRNA Isolation Kit, Human Ago2 was used to purify microRNAs from human HeLa, HepG2, andHEK293 cell lines (5 × 106 cells) and mouse P388D1 cell lines (5 × 106 cells). The purified microRNAs
were detected by silver staining (311-03961, CLEAR STAIN Ag, 20stains) after Urea-PAGE. As a result,the microRNA Isolation Kit, Human Ago2 purified microRNAs specifically from human HeLa, HepG2, andHEK293 cell lines. In addition, the microRNA Isolation Kit, Mouse Ago2 was used to purify microRNAs
from human HeLa cell lines (5 × 106 cells) and rodent P388D1, CHO, PC-12 cell lines (5 × 106 cells).
The purified microRNAs were detected by the previous described method. As a result, the microRNA
Isolation Kit, Mouse Ago2 purified microRNAs specifically from rodent P388D1, CHO, PC-12 cell lines.
（base）（base）

LaneM:Marker
Lane1:synthesized ss
RNA(22nt)1ng
Lane2:HeLa
Lane3:HepG2
Human
Lane4:HEK293
Lane5:P388D1
Mouse

Figure 1 Detection of microRNA purified with Human Ago2 kit
by silver staining.

16

LaneM:Marker
Lane1:synthesized ss
RNA(22nt)1ng
Lane2:HeLa—Human
Lane3:P388D1—Mouse
Lane4:CHO-K1—Chinese hamster
Lane5:PC-12-Rat

Figure 2 Detection of microRNA purified with Mouse Ago2 kit bysilver staining.

5.

Total RNA Purification

5-1. microRNA Extractor® SP Kit
This kit is for extraction of microRNA from serum or plasma of humans and animals. It enables microRNA
extraction without use of phenol, chloroform, and other hazardous reagents required in previous methods.
Protocol flow

Kit contents
Washing Solution 1(including 2-propanol)

Plasma/serum
sample (200 μL)

SupernatantProteose Solution
Lysis solution(including reducing agent)
37℃ incubation

Dispose offiltrate

Dispose ofFiltrate

Lysis Solution
Reducing reagent
Protease Solution
Enhancer
Washing Solution 1
Washing Solution 2
Elution Solution
Spin column/2 mL tube

Washing Solution 2(including ethanol)

Elution Solution

1- Butanol

The following necessary reagents are
not included.
1-Butanol (code No.022-16035)
2-Propanol (code No.168-21675)
Ethanol (code No.054-07225)

Supernatant

2- Propanol
Load (×2)

Sample (microRNA)

Extraction from normal human plasma

microRNA fractions
from can be usedwith qPCR.

Endogenous hsa-miR-16

Extraction from rat plasma and serum

Dispose offiltrate

20 mL x 1
60 μL x 1
600 μL x 1
600 μL x 1
12 mL x 1
22.5 mL x 1
4 mL x 1
50 sets

Endogenous rno-miR-16 extraction

For plasma coagulation, please use EDTA or citric acid. Using heparin inhibit
PCR detection after RNA extraction.

Endogenous hsa-miR-451

6.0×106
5.0×106

2.5×107

Sample 1
Sample 2
Sample 3

4.0×106

Sample 1
Sample 2
Sample 3

2.0×107

1.5×1076

3.0×10

1.0×1072.0×106
5.0×106

1.0×106

2.0×108

1.5×108

1.0×108

0.0

Extraction
from animal
experiments
possible

Extraction from normal human plasma with added cel-miR-238
Sample 1: 104copy/test (=5×104copy/mL plasma)
Sample 2: 105copy/test (=5×105copy/mL plasma)
Sample 3: 106copy/test (=5×106copy/mL plasma)
Sample 4: 107copy/test (=5×107copy/mL plasma)
Sample 5: 108copy/test (=5×108copy/mL plasma)

Recovery rate of addition

2.5×108

Recovery rate and reproducibility

0.0

0

5.0×107

Higher extraction
rates observed
than fromprevious methods

Sample1 Sample2

Sample3 Sample4

Sample5High
reproducibility
exhibited

Code No.

Description

Package size

295-71701

microRNA Extractor® SP Kit

50 Extractions17

5-2. ISOGEN® II
ISOGEN® II is a reagent for extracting total RNA or small RNA from animal tissue or cultured cells. It is a
uniform fluid containing phenol and guanidine that can isolate RNA in a single step through interactions
with cellular components. There is not require to separate the liquid phase with chloroform as previous
products (ISOGEN® or ISOGEN®-LS).
ISOGEN®

ISOGEN® II

Total RNA was extracted with
ISOGEN ® and ISOGEN ® II, thenanalyzed with a 3D-Gene ® Human
microRNA Oligo chip (Toray).
Results show ISOGEN® II efficacy
in small RNA extraction.

Effective spots: 638 microRNA

supernatantprecipitation

Effective spots: 688 microRNA
Code No.
317-07363
311-07361

Description®

ISOGEN II

Package size
10ml
100ml

5-3. ISOGEN® 5-4. ISOGEN®-LS
ISOGEN® and ISOGEN®-LS enable the isolation of undamaged RNA in high yield about 1 hour without
contaminating DNA or protein. Therefore, Northern analysis or dot blot hybridization can be performed
without DNase or other processing (Fig. 1).
Figure 1
ISOGEN® Flow

IPA

®

upper
chloroform

RNA

RNA

middleunderDNA
Protein

EtOH

wash

precipitation

Homogenize

SampleISOGENor ®
ISOGEN -LS

DNA
Protein

IPA

SampleYield
Liver
SpleenKidneySkeletal
Brain
Placenta

Mouse
TissueRat, pituitary gland, or human liver
needle biopsy sample

6–10 µg RNA/mg tissue
6–10 µg RNA/mg tissue
3–4 µg RNA/mg tissue
1–1.5 µg RNA/mg tissue
1–1.5 µg RNA/mg tissue
1–4 µg RNA/mg tissue
4–8 µg RNA/mg tissue

Plant

Arabidopsis or tobacco

Cultured cells

Epithelial
Fibroblasts

150–200 µg RNA/0.5–1 g weight tissue
8–15 µg RNA/106 cell
5–7 µg RNA/106 cell

Blood

Human or animal

7–15 µg RNA/mL blood
Code No.

Description

315-02504
317-02503

10ml
ISOGEN®

311-02504
Code No.
317-02623
311-02621

18

Package size
50ml
100ml

Description®

ISOGEN -LS

Package size
10ml
100ml

6.

microRNA Analysis Tools

Electrophoresis
6-1. SuperSep RNA, 15%, 17-Well
This product is an polyacrylamide gel with 4M urea. It is a ready-touse precast gel, so it does not require steps such as dissolving urea or
preparing gels. Acrylamide concentration is 15%.

■(base)(base)(base)(base)

1 Mouse brain
2 Mouse liver
3 Mouse kidney
4 HeLa cells
5 Jurkat cells
Each sample: 500 ng run
EtBr stain

1: Total RNA 2.5μg
2: High-molecule RNA 2.5μg
3: Small RNA 250ng
EtBr staining

Sample: RNA Prestaine Marker
Running buffer: TBE Buffer
Current: 10 mA constant

Sample: RNA Marker
Running buffer: 0.5×TBE Buffer
Current: 10 mA constant
Stain: Silver
Exposure time: 5 min

RNA extracted from HeLa cells
according to ISOGEN II protocol
before performing polyacrylamide
gel electrophoresis.

Small RNA extracted from various samples
using ISOGEN II (with optional operations)
before performing polyacrylamide gelelectrophoresis.

Code No.

Description

Package size

194-15881

SuperSepTM RNA, 15%, 17well

5 sheets6-2. R-MarkTM Small RNA Marker (20–50 nt)
This product is consisted of four types of single-chain RNA (20, 30, 40, 50 nt). Use after mixing with RNA
loading buffer.
Recommended quantity: 1 μL/lane (50 times)
Component: Water (RNase free)
Total RNA quantity: 112.5 μg/mL
RNA quantity in each band/μL: 20 nt (37.5 ng), 30 nt, 40 nt, 50 nt (25 ng each)

Code No.

Description

Package size

182-02711

R-MarkTM small RNA Marker

50 μl

19

6-3. CLEAR STAIN Ag
CLEAR STAIN Ag is a silver staining kit developed especially for nucleic acids (DNA/RNA), enabling highsensitivity detection after polyacrylamide gel electrophoresis with low background noise. This method
offers 50 to 100 higher sensitivity than ethidium bromide staining (measured at 302 nm), allowing
detection of 20 to 50 pg DNA per band.
■ Kit contents (140×140×1mm 6% polyacrylamide gel, 20 stains)
1) Fixing solution (×20): CTAB, ethanol, 200 mL2) Ammonia solution (×20): ammonia, 200 mL3) Staining solution A (×20): silver nitrate, 200 mL4) Staining solution A (×20): sodium hydroxide, ammonia, 200 mL5) Developing solution A(×10): sodium carbonate, 200 ml×2 (10× concentrate)6) Developing solution B(×20): formaldehyde, sodium thiosulfate, 200 mL
7) Preserving solution (×20): glycerol, acetic acid, 200 mL8) Product manualSample: Marker 9 ( φ X174/Hinf I digest)
Sample quantity: From left: 500, 250, 125, 63, 32, 16, 8, 4, 2, 1,
0.5, 0.25 ng/lane
Buffer: TBE
Gel size: 140×140×1 mm (6% polyacrylamide gel)

Code No.

Description

Package size

311-03961

Clear Stain Ag

20 stains

6-4. 5×TBE
Components

0.445 mol/l Tris-borate, 10 mmol/l EDTA

Storage

Room temp.

Comments

Dilution amounts
Agarose gel electrophoresis: About 10×
Polyacrylamide gel electrophoresis: About 5×

Code No.

Description

Package size

318-90041

5×TBE

1,000 ml

Usage Example of Electrophoresis Products
MagCaptureTM microRNA Isolation Kit, Human Ago1-4 was used to obtain an RNA fraction from human
K562 cells (1×107 cells), then denaturing polyacrylamide gel electrophoresis performed with SuperSep
RNA, 15%, 17-well, with CLEAR STAIN Ag used for silver staining detection. Result show the detectionof microRNA of about 22 bp in the RNA fraction.
(base)

Lane 1: R-MarkTM Small RNA Marker 1μL 1/50 diluted fluid
Lane 2: Control siRNA duplex, Firefly Luciferases GL3(22mer) 0.5ng
Lane 3: MagCaputureTM microRNA Isolation Kit, Ago1-4 Spin column method
Lane 4: MagCaputureTM microRNA Isolation Kit, Ago1-4 One-step method
Gel: SuperSepTM RNA, 15%, 17-well
Running buffer: 0.5×TBE
Current: 10 mA constant for 75 min.
Stain: Clear Stain Ag (staining time: 60 min., developing time: 5 min.)20

cDNA Cloning
6-5. microRNA Cloning Kit Wako
microRNA Cloning Kit Wako adopt our original buffer solution which allows two reactions in the same
solution: dephosphorylation by shrimp alkaline phosphatase (SAP), which is easily inactivated with heat
treatment; and efficient adaptor ligation of single-strand DNA/RNA with a thermostable ligase (soldseparately). This allows simple and efficient adaptor ligation reactions, enabling easy cDNA synthesis
from microRNA.
■ Outline■ Features
1. Highly-efficient and accurate adaptor ligation by thermostable ligase.
2. Suitable for cloning of microRNA forming secondary structures.
3. Reduced RNA operations means cDNA coding microRNA can be synthesized within 1.5 days.

■ Kit contents (8 reactions)
1) SAP2) 5×SAP Buffer3) 40×Ligation Buffer
4) RNase Inhibitor
5) 10mmol/L MnCl26) Reverse Transcriptase
7) 10×RT Buffer8) dNTP Mixture9) 0.5 mol/L EDTA, pH 8.0
10) 1mol/L Tris-HCl, pH 7.5
11) Ethachinmate12) 10mol/L Ammonium Acetate13) 3’Adaptor(50pmol/μ L)14) 5’Adaptor(50pmol/μ L)
15) RT Primer(50pmol/μ L)16) 5’PCR Primer(50pmol/μ L)17) 3’PCR Primer(50pmol/μ L)18) Control RNA(30ng/μ L)

16μl
64μl
16μl
16μl
16μl
8μl
16μl
112μl
16μl
160μl
24μl
960μl
8μl
8μl
8μl
16μl
16μl
8μl

Please use Single Strand DNA Ligase, Thermostable Recombinant Solution (soldseparately)from EPICENTRE with microRNA Cloning Kit Wako . Buffer components
and reaction conditions are all optimized for this enzyme, which can be used in ligation
reactions for both DNA and RNA (ATP-dependent). Optimal reaction temperature is 5565℃, enabling significantly more efficient adaptor ligation of microRNA than with T4 RNA
ligase.

Code No.

Description

Package size

290-66501

microRNA Cloning Kit Wako

8 reactions

6-6. Target mRNA Cloning Kit Wako
Target mRNA Cloning Kit Wako is used for amplification of cDNA from target mRNA.
This kit is used for generating cDNA from mRNA interacting with microRNA in RNA fractions in
immunoprecipitated Ago protein. DNA sequences obtained from cDNA synthesized from this mRNAand then cloned can be used for screening target mRNA candidates of microRNA. Using this kit in
combination with Ago immunoprecipitation enables screening of target mRNA of microRNA not withprevious database-based prediction, but with molecular biology-based methods.
■ Principles■ Kit contents (10 reactions)fraction(Reverse Transcription)
synthesis)(RNA degradation)(2nd strand synthesis)(PCR Ampliﬁcation)

TA Cloning

1) dT(20)-RT Primer(20pmol/μL) 10μL×12) 10×RT Buffer 20μL×1
3) dNTP Mixture Solution (2.5mmol/l each) 20μL×1
4) RNase Inhibitor(20U/μL) 10μL×15) Reverse Transcriptase(200U/μL) 10μL×16) 0.5 mol/L EDTA Solution 20μL×17) 1mol/L Tris-HCl Solution 200μL×18) Ethachinmate 30μL×19) 10mol/L Ammonium Acetate Solution 480μL×110) 2nd Strand Synthesis Primer(20pmol/μL) 10μL×111) 5' PCR Primer(20pmol/μL) 10μL×112) 3' PCR Primer(20pmol/μL) 10μL×113) 5' Colony PCR Primer(5pmol/μL) 960μL×114) 3' Colony PCR Primer(5pmol/μL) 960μL×1
Code No.

Description

Package size

298-68001

Target mRNA Cloning Kit Wako

10 reactions

21

Microarray Analysis
6-7. Toray 3D-Gene ®
Please send us an RNA sample, and we will perform mRNA or microRNA gene expression analysis using
the 3D-Gene ® chip. We also offer consultations on statistical or biological analysis services by using
analyzed data.
■ High sensitivity, reproducibility, and accuracy compared to traditional methods■ Low noise through black resin and 3D pillar probe structure■ Surface covered on nano-level with dense layer of oligo DNA probes■ Promotes hybridization reaction with microbead agitation

Probe position

3D-Gene ® plate

3D-Gene ® characteristics make it
perfect for analysis of low-expression
genes and microRNA, as well as
analysis of RNA samples from small
specimens, blood, or FFPE (formalinfixed paraffin embedded).
*Our services begin at RNA extraction
step for blood and FFPE samples.

MethodDetection of small
amount sample

FFPESamples

same quantity
previous productsof RNA
3D-Gene ® plate

All human genes

All mouse genes

All rat genes

miRNA chip

Custom chip

DNA chipDNA chipDNA chip

Human, mouse, rat(Creation and analysis services)2-color1-color–*The microRNA chip is available only in 1-color. * Please inquire about samples FFPE (formalin fixed paraffin embedded) samples or blood samples■ Sample

SampleSample quantity
Sample concentration purity

Total RNA
2 μg or more
0.5 μg/μL or more (recommended)
OD260/OD280=1.8~2.0

Tissue/cell
Please inquire.

■ Turnaround

Turnaround is usually within 1 month, but may differ according to sample quality(In the case of Japan).
■ Price

Please inquire.
■ How to order

Please inquire through the Wako website:
http://www.wako-chem.co.jp → Reagents → Services → Microarray analysis → 3D-Gene ®
■ Notes◦ Please use recommended kits for RNA extraction.
◦ Recommended kits differ for gene expression analysis and microRNA analysis.
◦ Please send RNA samples dissolved in frozen DNase/RNase-free water, and use a frozen shipping services. Inquire first to determine adelivery time to set. We cannot be responsible for shipping-related problems.

◦ We may not be able to accept highly infectious samples.
◦ Please obtain informed consent from subject for any human samples.
◦ We can only accept P1 level samples as determined by the Ministry of Education, Culture, Sports, Science and Technology's Act on theConservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms.

◦ Samples fundamentally cannot be returned.
◦ This analysis service is intended for research use only. Please do not use for other purposes (medical diagnosis or drugs, food

manufacture, testing, etc.)

22

7.

Wako Pure Chemical Industries, Ltd. Publication list

hAgo2(4G8) MAb

hAgo2(4G8) MAb

Cell. 2014 Jan 16;156:146-157.
Inefficient SRP interaction with a nascent chain triggers a mRNA quality control pathway.

Nature structural & molecular biology 2009 Dec 16;12:1259
Distinct passenger strand and mRNA cleavage activities of human Argonaute proteins.Wang B, Li S, Qi
HH, Chowdhury D, Shi Y, Novina CD

microRNA Isolation Kit human / mouse Ago1, and human Ago2

Nucleic Acids Research , 2014,1-13.
Novel functional small RNAs are selectively loaded onto mammalian Ago1.

microRNA Isolation Kit, Human Ago2

BIOLOGY OF REPRODUCTION 2009 81, 717–729
Human Villous Trophoblasts Express and Secrete Placenta-Specific MicroRNAs into Maternal Circulation
via Exosomes.
Luo SS, Ishibashi O, Ishikawa G, Ishikawa T, Katayama A, Mishima T, Takizawa T, Shigihara T, Goto T,
Izumi A, Ohkuchi A, Matsubara S, Takeshita T, Takizawa T.

microRNA Cloning Kit Wako, ssDNA Ligase, thermostable

Mol Cell. 2011 Nov 4;44(3):424-36.
MCPIP1 Ribonuclease Antagonizes Dicer and Terminates MicroRNA Biogenesis through Precursor
MicroRNA Degradation.
Suzuki HI, Arase M, Matsuyama H, Choi YL, Ueno T, Mano H, Sugimoto K, Miyazono K.

hAgo2(4G8) MAb

BMC Research Notes 2009, 2:169
A useful approach to total analysis of RISC-associated RNA.
Hayashida Y, Nishibu T, Inoue K, Kurokawa T.

Ago1(2A7), hAgo2(4G8), hAgo3(1C12) MAb

RNA Biol. 2011 Jan 1;8(1).
Deep-sequencing of human Argonaute-associated small RNAs provides insight into miRNA sorting andreveals
Argonaute association with RNA fragments of diverse origin.
Burroughs AM, Ando Y, Hoon ML, Tomaru Y, Suzuki H, Hayashizaki Y, Daub CO.,hAgo2(4G8) MAb

RNA 2009, 15:1078–1089
Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediatedtranslational repression.
Takimoto K, Wakiyama M, Yokoyama S.

hAgo2(4G8) MAb

PNAS 2011 Mar 29.
MicroRNA let-7 establishes expression of {beta}2-adrenergic receptors and dynamically down-regulates
agonist-promoted down-regulation.
Wang WC, Juan AH, Panebra A, Liggett SB.

mAgo2(2D4) microRNA Isolation Kit, Human Ago2

Reproduction 2008 Dec;136(6):811-22.
MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adultmouse testis and ovary.
Mishima T, Luo SS, Ishibashi O, Kawahigashi Y, Mizuguchi Y, Ishikawa T, Mori M, Kanda T, Goto T,
Takizawa T.

mAgo2(2D4) MAb

EMBO J . 2011 March 2; 30(5): 823–834.
Small RNA-mediated regulation of iPS cell generation
Zhonghan Li,Chao-Shun Yang, Katsuhiko Nakashima, and Tariq M RanahAgo2(4G8) MAb

Methods Mol Biol. 2008;442:29-43.
In vitro RNA cleavage assay for Argonaute-family proteins.
Miyoshi K, Uejima H, Nagami-Okada T, Siomi H, Siomi MC.

mAgo2(2D4) MAb

Molecular Pharmacology March 22, 2011
RISC bound siRNA is a determinant of RNAi mediated gene silencing in mice.
Wei J, Jones J, Kang J, Card A, Krimm M, Hancock P, Pei Y, Ason B, Payson E, Dubinina N, Cancilla M,
Stroh M, Burchard J, Sachs A, Hochman J, Flanagan WM, Kuklin N.

hAgo2(4G8) MAb

Nature , 2008 Sep 18;455(7211):421-4.
Prolyl 4-hydroxylation regulates Argonaute 2 stability.
Qi HH, Ongusaha PP, Myllyharju J, Cheng D, Pakkanen O, Shi Y, Lee SW, Peng J, Shi Y.

mAgo2(2D4) MAb

Nature structural & molecular biology 2011, 18, 2, Feb
Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without
mature microRNA.
Leung AK, Young AG, Bhutkar A, Zheng GX, Bosson AD, Nielsen CB, Sharp PA.

hAgo2(4G8) MAb

Ago1(2A7), hAgo2(4G8), hAgo3(1C12) MAb

hAgo2(4G8) MAb

PNAS 2008 June 10, vol. 105 no. 23 7964–7969
Characterization of endogenous human Argonautes and their miRNA partners in RNA silencing.
Azuma-Mukai A, Oguri H, Mituyama T, Qian ZR, Asai K, Siomi H, Siomi MC.
J Hepatol. 2009 Mar;50(3):453-60. Epub 2008 Jul 9.
Regulation of the hepatitis C virus genome replication by miR-199a.
Murakami Y, Aly HH, Tajima A, Inoue I, Shimotohno K.

microRNA Isolation Kit, Ago1, Human Ago2 and Human Ago3

Genome Res. August 18, 2010
A comprehensive survey of 39 animal miRNA modification events and a possible role for 39 adenylationin modulating miRNA targeting effectiveness.
Burroughs AM, Ando Y, de Hoon MJ, Tomaru Y, Nishibu T, Ukekawa R, Funakoshi T, Kurokawa T,
Suzuki H, Hayashizaki Y, Daub CO.

and others..........

hAgo2(4G8) MAb

Nucleic Acids Research , 2009, 1–13
Expanded RNA-binding activities of mammalian Argonaute 2.
Tan GS, Garchow BG, Liu X, Yeung J, Morris JP 4th, Cuellar TL, McManus MT, Kiriakidou M.

8.

Products List

Other Reagents for microRNA Research
Code No.
317-07363
311-07361

Description
ISOGEN IIISOGEN

311-02504
317-02623
311-02621
314-07513
318-07511

10ml
100ml
10ml

315-02504
317-02503

Package size

50ml
100ml

ISOGEN -LS
ISOGEN with Spin Column

10ml
100ml
10 extractions
60 extractions

194-15881

SuperSepTM RNA, 15%, 17well

182-02711

R-MarkTM small RNA Marker

311-03961

CLEAR STAIN Ag

20 stains

318-90041

5×TBE

1,000ml

5 gels
50μl

23

8.

Products List

Monoclonal Antibodies for microRNA Research
Code No.

018-22401
015-22411
015-22031
011-22033
018-22021
014-22023
018-23241
010-23821
013-25491
016-25501
012-24741
019-24751
012-25581
018-25583
016-25584
017-23451
018-23981
015-23991

Description

Anti Ago1, Monoclonal Antibody(1F2)
Anti Ago1, Monoclonal Antibody(2A7)
Anti Human Ago2, Monoclonal Antibody(4G8)Anti Mouse Ago2, Monoclonal Antibody(2D4)Anti Human Ago3, Monoclonal Antibody(1C12)
Anti Human Ago3, Monoclonal Antibody(6-107)
Anti Mouse Ago3, Monoclonal Antibody(mA3-S11)
Anti Mouse Ago3, Monoclonal Antibody(mA3-S9)
Anti Ago4, Monoclonal Antibody(2B2)
Anti Ago4, Monoclonal Antibody(2G7)Anti Ago1-4, Monoclonal Antibody(1G3)
Anti PIWIL1, Monoclonal Antibody(2C12)
Anti Human PIWIL2, Monoclonal Antibody(1A12)
Anti Human PIWIL2, Monoclonal Antibody(3C4)

Package size

50μl
50μl
100μl
50μl
100μl
50μl
50μl
50μl
50μl
50μl
50μl
50μl
200μl
1ml
5ml
100μl
50μl
50μl

Reagent Kits for microRNA Research
Code No.

295-71701
290-66501
298-68001
291-70201
292-66701
292-67301
297-70301
295-74001
297-74201
293-74801
299-74401

Description

microRNA Extractor SP Kit
microRNA Cloning Kit Wako
Target mRNA Cloning Kit Wako
microRNA Isolation Kit, Human/Mouse Ago1
microRNA Isolation Kit, Human Ago2
microRNA Isolation Kit, Mouse Ago2
microRNA Isolation Kit, Human Ago3
MagCapture™ microRNA Isolation Kit, Human Ago2
MagCapture™ microRNA Isolation Kit, Mouse Ago2
MagCapture™ microRNA Isolation Kit, Ago 1-4
MagCapture™ microRNA Isolation Kit, Protein G

Package size
50 extractions

8 reactions
10 reactions
10 reactions
10 reactions
10 reactions
10 reactions
10 reactions
10 reactions
10 reactions
20 reactions

Listed products are intended for laboratory research use only, and not to be used for drug, food or human use. / Please visit our
online catalog to search for other products from Wako; http://labchem-wako.fujifilm.com/ This leaflet may contain products thatcannot be exported to your country due to regulations. / Bulk quote requests for some products are welcomed. Please contact us.

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