microRNA Research
using Anti Ago2 Antibody
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The Pathway of Translational Silencing
by microRNA
The microRNA matured in multiple steps
in the cells is taken into a protein
compound called an RNA-induced
silencing complex (RISC), and it is known
that it bonds with Argonaute (Ago)
subfamily protein as its main component.
Specific purification of RNA bound to
protein of the Ago subfamily is
considered to be an important
technology for microRNA function
determination, discrimination of new
microRNA, and analysis of interaction
between microRNA and mRNA.
Wako has developed and launched
high-quality anti-Ago2 monoclonal
antibodies, microRNA Isolation kits, and
high-efficiency microRNA Cloning kits, and
the line-up includes research reagents with
powerful support for microRNA research.
for Highly Efficient Adapter Ligation
microRNA Cloning Kit Wako
for Effective 65°C
Ligation of ssDNA, ssRNA and ssRNA-ssDNA
ssDNA Ligase, thermostable
Immunoprecipitation method with anti-Ago2 mAb
microRNA Isolation Kit, Human / Mouse Ago2
Anti Human / Mouse Ago2, Monoclonal Antibody
Wako Cat. No. 290-66501
Wako Cat. No. 298-65103; 292-65101
microRNA Isolation Kit, Human Ago2
(Wako Cat. No. 292-66701)
microRNA Isolation Kit, Mouse Ago2
(Wako Cat. No. 292-67301)
Anti Mouse Ago2, Monoclonal Antibody
(Clone No. 2D4), immunostained image
microRNA ‘‘Specific’’ Purification
microRNA Isolation Kit
, Human Ago2(
, Mouse Ago2(
Wako Cat. No.
292-66701
Wako Cat. No.
292-67301
)
)
microRNA Isolation Kit, Human/Mouse Ago2 can prepare high purified fractions of microRNA, which binds
with Argonaute2 (Ago2) protein, based on immunoprecipitation method by using a high affinity monoclonal
antibody against Ago2.
The purified microRNA fraction will contain very little contaminated degradation fragments of rRNA and tRNA.
These kits will highly improve the microRNA cloning efficiency compared with that by using conventional
microRNA purification method.
Features
Achieve Immunoprecipitation of internal Ago2 from
human, mouse, rat, and hamster. Note)
microRNA bound to Ago2 protein can be isolated with
high purity.
Few rRNA or tRNA decomposition products or
impurities like smallRNA etc.
The isolated microRNA fraction can be used for
cloning and micro arrays.
Note)
Wako Cat. #292-66701 is for human cells and tissues and #292-67301
is for cells and tissues of mice and rats.
Simple procedure
Outline of procedure
Comparison with conventional method
Kit contents (10 reactions)
Conventional
method
Wako's
Latest Method
Total RNA extraction Necessary Unnecessary
small RNA(≦200nt) purification
Denaturing PAGE
Gel extraction from denaturing PAGE
Gel purification from denaturing PAGE
Contamination ratio of rRNA fragments High Low
Contamination ratio of tRNA fragments High Low
Handling time ≦12hr ≦4hr
Anti Human Ago2 Antibody Beads Solution
・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 500 μL×1 vial
Cell Lysis Solution・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 50 mL×1 vial
Elution Solution・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 500 μL×1 vial
Ethachinmate・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 30 μL×1 vial
3mol/L Sodium Acetate・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 400 μL×1 vial
Specific Purification of microRNA fraction from several cell lines
Figure.1   Purification of microRNA fraction by using microRNA
Isolation Kit, Human Ago2
(Wako catalog #292-66701).
The purified microRNA fraction from human cultured cell
lines(HeLa, HepG2, HEK293) were specifically detected by
Urea-PAGE. Cell number of each cell line is approximately
5×106;. The applied volume per lane is half of 10μL of final
solution prepared with an IP by this kit.
Figure.2   Urea-PAGE pattern of purified RNA by using microRNA
Isolation Kit, Mouse Ago2
(Wako catalog #292-67301).
The purified microRNA fractions from cultured rodent cell
lines(P388D1, CHO-K1, PC-12) were detected by silver
stain. Cell number of each cell line is approximately 1×107.
The applied volume per lane is half of isolated sample by this
kit.
Cloning of purified microRNA
High efficiency of microRNA cloning by using these kits followed by using microRNA Cloning Kit Wako.
Figure.3   Cloning efficiency of microRNA from HeLa cell lysate
The microRNA fraction was prepared by microRNA Isolation
Kit, Human Ago2 (#292-66701)
from 5×106 HeLa cells.
The cDNA encoding microRNA was synthesized by microRNA
Cloning Kit Wako (#290-66501)
and was inserted into T-vector.
The presence ratio of microRNA was more than 90%.
The contents of cloned microRNA species are indicated on
Table 1.
Figure.4   Cloning efficiency of microRNA from P388D1 cell lysate
The microRNA fraction was prepared by microRNA Isolation
Kit, Mouse Ago2 (#292-67301)
from 5×106 P388D1 cells.
The cDNA encoding microRNA was synthesized by microRNA
Cloning Kit Wako (#290-66501)
and was inserted into T-vector.
The presence ratio of microRNA was more than 90%.
The contents of cloned microRNA are indicated on Table 2.
Purification of microRNA fraction from tissues
Figure.5   microRNA fractions, which were isolated by this kit from
human liver and testis lysate, were detected by silver stain
after Urea-PAGE.


 (50mg Tissues→2mL Total tissue lysate→1mL Tissue lysate
  →Immunoprecipitation→10μL Total RNA solution→5μL
  RNA solution→Urea-PAGE.)
Figure.6   microRNA fractions, which were isolated by this kit from
mouse lung, liver and brain lysate, were detected by silver
stain after Urea-PAGE.


 (50mg Tissues→2mL Total tissue lysate→1mL Tissue lysate
  →Immunoprecipitation→10μL Total RNA solution→5μL
  RNA solution→Urea-PAGE.)
Achieve Highly Efficient, Accurate & Easy-to-Use Adapter Ligation
microRNA Cloning Kit Wako
Wako Cat. No.
290-66501
(
)
The microRNA Cloning Kit Wako can prepare the cDNA encoding microRNA. The cloning procedure will be
completed within 1.5 days after preparation of microRNA fraction.
This kit is supported by shrimp alkaline phosphatase (SAP), single strand DNA Ligase, thermostable
(Wako Cat. No. 298-65103; 292-65101) (selling separately), and original modified adaptors.
The cloning efficiency using this kit is improved higher than that of the conventional methods, which used
bacterial alkaline phosphatase and T4 RNA ligase.
Features
Outline of procedure
Highly efficient and accurate adapter ligation by
thermostable ligase.
Suitable for cloning of microRNA forming
secondary structures.
Simple operation achieves forming of cDNA
coding microRNA in 1.5 days.
①SAP 16 μL
②5×SAP Buffer 64 μL
③40×Ligation Buffer 16 μL
④RNase Inhibitor 16 μL
⑤10mmol/L MnCl2 16 μL
⑥ Reverse Transcriptase 8 μL
⑦10×RT Buffer 16 μL
⑧dNTP Mixture 112 μL
⑨0.5mol/L EDTA, pH8.0 16 μL
⑩1mol/L Tris-HCl, pH7.5 160 μL
⑪Ethachinmate 24 μL
⑫10mol/L Ammonium Acetate 960 μL
⑬3’ Adaptor (50pmol/μL) 8 μL
⑭5’ Adaptor (50pmol/μL) 8 μL
⑮RT Primer (50pmol/μL) 8 μL
⑯5’ PCR Primer (50pmol/μL) 16 μL
⑰3’ PCR Primer (50pmol/μL) 16 μL
⑱Control RNA (30ng/μL) 8 μL
Thermostable ligase and DNA polymerase are not included in this kit.
IMPORTANT
microRNA Cloning Kit Wako must be used together with Single Strand
DNA Ligase, Thermostable (Wako Cat. No. 298-65103; 292-65101)
,

which is sold separately as a single product.
This ligase will be used for the high efficient adaptor ligation and
reaction buffers of this kit are optimized with this ligase.
This ligase is ATP-dependent and used for ligation of ssDNA-ssDNA
and ssRNA-ssRNA. Reaction of this ligase at 55-65°C, which is the
optimal temperature of enzymatic reaction, will promote the ligation
efficiency of single strand nucleic acids.
Kit contents (8 reactions)
Comparison with conventional method
  Conventional method microRNA Cloning kit Wako
Number of adaptor ligation steps 8 3
Phosphorylation of adaptors Necessary Unnecessary
Complete inactivation of Alkaline Phosphatase Impossible Possible
Apparatus for magnet beads Necessary Unnecessary
RI Necessary Unnecessary
BioAnalzer (Agilent) Necessary Unnecessary
Adaptor ligation time ≧ 8 hr 2.5 hr
Handling time 2.5 Days 1.5 Days
Reproducibility of adaptor ligation time Low High
Efficiency of microRNA cloning <10% >70%
Detection by EtBr Impossible Possible
Cloning efficiency of secondary structured microRNA Low (by T4 RNA Ligase) High (by Single Strand DNA Ligase, Thermostable)
ssDNA Ligase, thermostable (Wako Cat. No. 298-65103; 292-65101)
Cloning of microRNA from HeLa cells
High thermal stability
Optimum temperature: 55~65°C
High ligation efficiency
Ligation of ssRNA, ssDNA, and
ssRNA- ssDNA is applicable.
Source:  E. coli expressed thermophilic phage TS2126
single strand DNA ligase
Appearance:  10mmol/L Tris-HCl (pH 8.0), 50mmol/L KCl,
0.1mmol/L EDTA, 1mmol/L DTT
and 50% Glycerol
Activity:  Shown on each label. (approx. 10units/μL)
Application with the microRNA Cloning Kit Wako is described in the package insert of the kit.
Procedure
1) Preparation of total RNA from HeLa cells (1×107 cells) by ISOGEN (Nippon Gene #315-02504, 10mL).
2) Preparation of small RNA fraction, less than 200nt, from total RNA by microRNA Isolation Kit (Bio Chain Institute Inc. catalog #KS341025).
3) Separation of microRNA fraction by denaturing PAGE.
4) Collection of the gels of 20~23nt region after electrophoresis.
5) Cloning by using microRNA Cloning Kit Wako (Wako catalog #290-66501).
6) Construction of the plasmids harboring cDNA encoding microRNA and transformation of E. coli.
7) Random selection of the 96 transformed E. coli from selection LB agar medium.
8) Determination and verification of the cDNA sequences by using Sanger miRBase.
Figure.7   Cloning efficiency of microRNA from HeLa cell lysate
The cloning efficiency of microRNA was more than 70%. Others indicate that isolated cDNA sequences were not
matched miRBase. Unknowns indicate that isolated cDNA sequences were not matched human genome sequence.
The contents of cloned microRNA species are indicated on Table 3.
Anti Ago2, Monoclonal Antibody
Figure.8   Immunoprecipitation of hAgo2 protein from human cultured
cell lines (HeLa, HepG2, HEK293, THP-1) and mouse cultured
cell line (P388D1) by using 20μL 10% Protein G slurry
immobilized with 10μg of this antibody. The bands of hAgo2
protein were detected in approximately 100kDa by using
silver staining and western blot. Cell number was 5×106 cells.
Figure.9   Immunoprecipitation of Ago2 protein from NIH-3T3(Mouse),
SCC-131(Rat) and CHO(Hamster) cell line by using 20μL of
10% Protein G slurry immobilized with 5μg of this antibody
(2D4). The bands of endogenous Ago2 protein were detected
in approximately 100kDa by using silver staining and
western blot. The 1/1,000 diluted antibody was used as the
1st antibody for western blot. Cell number was 5×106 cells.
Product List
Description
Wako Cat. No. (Package Size)
microRNA Isolation Kit, Mouse Ago2
292-67301 (10 reactions)
microRNA Isolation Kit, Human Ago2
292-66701 (10 reactions)
Anti Mouse Ago2, Monoclonal Antibody
014-22023 (50 μL)
018-22021 (100 μL)
Anti Human Ago2, Monoclonal Antibody
011-22033 (50 μL)
015-22031 (100 μL)
microRNA Cloning Kit Wako
290-66501 (8 reactions)
Single Strand DNA Ligase, thermostable,
recombinant, Solution
298-65103 (200 units)
292-65101 (500 units)
・microRNA Isolation Kit, Human / Mouse Ago2 is patent pending (11, 30, 2007).
・microRNA Cloning Kit Wako is patent pending (1, 10, 2007).
・All products shown on the leaflet are for research use only.
Please visit the e-reagent.com to search products Wako can supply: http://www.e-reagent.com/