7. microRNA Research

 

Lane M : Molecular weight makar
Lane 1 : Single strand RNA (22nt) 1ng
Lane 2 : HeLa
Lane 3 : HepG2 (Human)
Lane 4 : HEK293
Lane 5 : P388D1 (Mouse)
[ Figure. 1 ]
Purification of microRNA fractions by using microRNA Isolation Kit, Human Ago2. The purified microRNA fractions from HeLa cells were specifically detected by Urea-PAGE. Cell number is approximately 5 x 106.
microRNA Isolation Kit,Human Ago2
10 Reactions (Wako Catalog No. 292-66701)

 

M: RNA Molecular weight makar
Std: Single strand RNA(22nt) 1ng
Lane 1: HeLa (Human) (Negative control)
Lane 2: P388D1 (Mouse)
Lane 3: CHO-K1 (Chinese Hamster)
Lane 4: PC-12 (Rat)
[ Figure. 2 ]
Urea-PAGE pattern of purifi ed RNA by using microRNA Isolation Kit, Mouse Ago2 (Wako catalog #292-67301). The purifi ed 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 5 x 106. The applied volume per lane is half of isolated sample by this kit.
microRNA Isolation Kit, Mouse Ago2
Wako Catalog No. 292-67301 (10 Reactions)
[Purifi cation of microRNA fractions from several rodent cell lines]


[Cloning of purified microRNA from P388D1 cells]
High efficiency of microRNA cloning by the combination use of microRNA Cloning Kit Wako
( Wako Cat. No. 290-66501 )


[ Figure 3 : ]
Cloning effi ciency of microRNA from P388D1 cell lysate. The presence ratio of microRNA was more than 90%. Others indicated cDNAs which were listed in miRBase of other organism species. Unknowns indicated cDNAs which were found in genome sequence, but not listed in miRBase. The contents of cloned microRNA are indicated on Table 1.
[Procedure of microRNA cloning]
1) The microRNA fraction was prepared by microRNA Isolation Kit, Mouse Ago2.
2) The cDNA encoding microRNA was synthesized by microRNA Cloning Kit Wako and inserted it into T-vector.
3) The 96 transformants of E. coli were randomly selected from selection LB agar medium.
4) Inserted cDNA sequences were determined by DNA sequencer and colleted sequences by using data base of Sanger miRBase.
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