microRNA "Specific" Purification Kit
microRNA Isolation Kit, Human Ago2
(Wako catalog #292-66701)
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microRNA Isolation Kit, Human Ago2 can prepare high purified fractions of
microRNA, which binds with human Argonaute2 (hAgo2) protein, based on
immunoprecipitation method by using a high affinity monoclonal antibody
against hAgo2.
The purified microRNA fraction will contain very little contaminated
degradation fragments of rRNA and tRNA.
This kit will highly improve the microRNA cloning efficiency compared with
conventional microRNA purification method.
Features
High purification performance of microRNA
Human Ago2 Specific
Little contamination of other RNAs
High efficiency of microRNA cloning
Kit contents (10 reactions)
① Anti Human Ago2 Antibody Beads Solution
② Cell Lysis Solution
③ Elution Solution
④ Ethachinmate
⑤ 3 mol/L Sodium Acetate
500µL1 vial
50mL1 vial
500µL1 vial
30µL1 vial
400µL1 vial
Outline of procedure
Simple procedure
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http://www.e-reagent.com
Comparison with conventional method
Purification of microRNA fraction from several human cell lines
Specific purification of
microRNA
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
5106. The applied volume per lane is half of
10µL of final solution prepared with an IP by
this kit.
Cloning of purified microRNA from HeLa cells
High efficiency of microRNA cloning by using this kit followed by using microRNA Cloning Kit Wako.
Procedure of microRNA cloning
1) The microRNA fraction was prepared by microRNA Isolation Kit, Human Ago2 (#292-66701) from 5106 HeLa cells.
2) The cDNA encoding microRNA was synthesized by microRNA Cloning Kit Wako (#290-66501) and was inserted into
T-vector.
3) The 96 transformants of E.coli were randomly selected from LB agar medium containing antibiotics.
4) Inserted cDNA sequences were determined by DNA sequencer and were collected sequences by using Sanger
miRBase.
High cloning efficiency
Figure.2
Cloning efficiency of microRNA from HeLa cell
lysate.
The presence ratio of microRNA was more
than 90%. Others indicated that isolated cDNA
sequences were not matched in a miRBase.
Unknowns indicated that isolated cDNA sequences
were not matched human genome sequence.
The contents of cloned microRNA species were
indicated on Table 1.
"High Efficiency" microRNA Cloning Kit
microRNA Cloning Kit Wako
microRNA Cloning efficiency
≥70%
(Case of HeLa cell line)
(Wako catalog #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), thermostable
single strand DNA ligase (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
High cloning efficiency
Cloning of secondary structured microRNA
High reproducibility of microRNA cloning
Outline of procedure
Kit contents (8 reactions)
① SAP
② 5xSAP Buffer
③ 40xLigation Buffer
④ RNase Inhibitor
⑤ 10mmol/L MnCl
⑥ Reverse Transcriptase
⑦ 10xRT Buffer
⑧ dNTP Mixture
⑨ 0.5 mol/L EDTA, pH 8.0
2
16µL
64µL
16µL
16µL
16µL
8µL
16µL
112µL
16µL
⑩ 1mol/L Tris-HCI,pH 7.5
⑪ Ethachinmate
⑫ 10mol/L Ammonium Acetate
⑬ 3' Adaptor (50pmol/µL)
⑭ 5' Adaptor (50pmol/µL)
⑮ RT Primer (50pmol/µL)
⑯ 5' PCR Primer (50pmol/µL)
⑰ 3' PCR Primer (50pmol/µL)
⑱ Control RNA (30ng/µL)
160µL
24µL
960µL
8µL
8µL
8µL
16µL
16µL
8µL
Thermostable ligase and DNA polymerase are not included in this kit.
Precaution
microRNA Cloning Kit Wako must be used together with
Single Strand DNA Ligase, Thermostable (Code No.
292-65101, 298-65103), which is sold separately as a single
product.
This ligase will be used for the high efficient adaptor ligation.
Therefore, reaction buffers of this kit are optimized with this
ligase.
This ligase will be used for ligation of ssDNA-ssDNA and
ssRNA-ssRNA depending on ATP. Reaction of this ligase
at 55-65ºC, which is the optimal temperature of enzymatic
reaction, will promote the ligation efficiency of single strand
nucleic acids.
Comparison with conventional method
Please visit the Wako Online Catalog.
http://www.e-reagent.com
Cloning of microRNA from HeLa cells
Procedure of microRNA cloning
1) Preparation of total RNA from HeLa cells (1107 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.3
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 2.
Product Information
Listed products are intended for
laboratory research use only, and not
to be used for drug, food or human
use.
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