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.
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
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
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
Comparison with conventional method
Purification of microRNA fraction from several human 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
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
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.