Wako Pure Chemical
 
작성일 : 15-07-28
[Wako Pure Chemical] microRNA Isolation Kit, Human Ago2

 

microRNA Isolation Kit, Human Ago2

Argonaute2 단백질에 결합하고 있는 저분자 RNA의 정제 키트로 mouse, rat, hamster 등의 microRNA 정제에

사용 실적이 있습니다.

 

 

microRNA Isolation Kit, Mouse Ago2는 고품질의 anti-mouse Ago2 단클론 항체를 이용한 면역침강법에 의해

microRNA-Ago2 복합체를 회수하여 microRNA를 특이적으로 정제하는 키트입니다. 기존의 microRNA의 정제법

(변성 polyacrylamide 겔 추출)에서는 rRNA 및 tRNA의 분해산물이 다량 함유되어, microRNA의 복제효율을

현저히 저하시키고 있었습니다. 본 키트는 Ago2에 포함된 microRNA를 고순도로 포함한 RNA fraction을 정제 할

수 있습니다. 기존 정제법(5 steps)에 비해 간편하고(1 step) 특이적으로 microRNA의 정제가 가능합니다.

 

 

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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 name microRNA (miRNA) is given to functional, low molecular weight RNA comprising approximately 22

ribonucleotides. Such microRNA is known to regulate gene expression and play a role in the transcriptional

mechanisms of eukaryotic cells. Mature microRNA in the cytoplasm and nucleus will be introduced through

several processing pathways to RNA -induced silencing complex (RISC) and transferred to target mRNAs to

suppress the translation. Argonaute 2 (Ago2) is one of the main components of RISC.

This microRNA isolation kit can be used to prepared high purity fractions of microRNA based on

immunoprecipitation methods using a high affinity anti-Human/Mouse Ago2 monoclonal antibody. The

purified microRNA fraction contains very little contamination from rRNA and tRNA degradation fragments.

This kit will highly improve the microRNA cloning effi ciency compared with conventional microRNA

purification method.

 

   

Features

 

 Kit contents (10 reactions)

 

• High purification performance of microRNA

• Human Ago2 Specific

• Little contamination of other RNAs

• High effciency of microRNA cloning

 

  ① Anti Human Ago2 Antibody Beads Solution

  ② Cell Lysis Solution

  ③ Elution Solution

  ④ Ethachinmate

  ⑤ 3 mol/L Sodium Acetate

  500 μL x 1 vial

  50 mL  x 1 vial

  500 μL x 1 vial

  30 μL x  1 vial

  400 μL x 1 vial

 

 

                                   Outline of procedure

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                                   Simple procedure

  

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                         Comparison with conventional method

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               Purification of microRNA fraction from several human cell lines

   

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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

 specifi cally 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.

 

 

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

    5 x 106 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.

 

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Figure. 2 Cloning efficiency of microRNA from

HeLa cell lysate.

The presence ratio of microRNA was more than 90%.

Others indicated that isolated cDNAsequences were

not matched in a miRBase. Unknowns indicated that

isolated cDNA sequenceswere not matched human

genome sequence.The contents of cloned microRNA

species were indicated on Table 1.

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Brochure (PDF)  

 

 

Ordering informations

   

Catalog No.

Product Name

Size

292-66701

microRNA Isolation Kit, Human Ago2

10 reactions

016-20861

Anti Human AGO2, Monoclonal Antibody

50 μL

290-66501

microRNA Cloning kit wako

8 reactions

298-65103

single Standard DNA Ligase, thermostable, recombinant, Solution

200 units

292-65101

500 units

315-02504

ISOGEN

10 mL

317-02503

50 mL

311-02501

100 mL

194-13061

SuperSep15%, 12 wells

10 gels

304-30761

BioMashar1.5ml microtube (with O-ring)

50 tubes

058-07681

EasySeparator

1 set

312-01791

Ethachinmate (Nucleic acid carrier)

0.2 mL

 

 

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