METHOD FOR DETERMINING DESIRED CELL TYPE USING EXPRESSION OF miRNA AS INDICATOR
a technology of mirna and cell type, applied in the direction of instruments, biochemical equipment and processes, fluorescence/phosphorescence, etc., can solve the problems of difficult to carry out refined classification, difficult to apply to fractionation of living cells, and difficult to detect antibodies using antibodies, etc., to achieve simple detection method, reduce detection error in activity used as an indicator, and high resolution
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example 1
sional Separation of Existing Cell Lines, Using miRNA-Responsive Reporter mRNA
[0120]One-dimensional separation of existing cell lines was carried out using one or two types of miRNA-responsive reporter mRNAs. The results are shown in FIGS. 3A to 3H. FIG. 3A is a schematic view showing in vitro synthesized miRNA-responsive reporter mRNA used in the present Example. In the direction from the 5′-terminus on the left side to the 3′-terminus on the right side on the paper, ARCA (gap structure analog), a miRNA target sequence, a gene encoding a fluorescent protein, and poly(A) tail are positioned. The ARCA, the miRNA target sequence, and the initiation codon were designed, such that the ARCA could be separated from the miRNA target sequence by approximately 20 nucleotides, and the miRNA target sequence could also be separated from the initiation codon by approximately 20 nucleotides.
[0121]FIG. 3B is a graph showing a comparison in translational efficiency, when three types of miRNA-respon...
example 2
ration in a Higher-Dimensional Space
[0125]Cell separation was carried out using three types of or four types of miRNA-responsive reporter mRNAs. First, three types of miRNA-responsive reporter mRNAs, α(miR-24-3p)-EGFP, α(miR-127-3p)-EGFP (SEQ ID NO: 7), α(miR-17-5p)-EGFP (SEQ ID NO: 9) and α(miR-92a-3p)-EGFP (SEQ ID NO: 15), were measured in terms of translational efficiency in HeLa, 293FT, and MCF-7 cells. The results are shown in FIG. 4, panels (a) and (d). The numerical value at the top of each column indicates the translational efficiency ratio of two types out of the three types of cell lines. The error bars each indicate a mean value±standard deviation (n=3).
[0126]The results obtained by performing two-dimensional separation using three types of miRNA-responsive reporter mRNAs (α(miR-24-3p)-hmAG1 (SEQ ID NO: 4), α(miR-127-3p)-hmKO2 (SEQ ID NO: 8) and α(miR-17-5p)-tagBFP (SEQ ID NO: 13)) are shown in FIG. 4, panel (b). Three types of miRNA-responsive reporter mRNAs were each in...
example 3
of miRNA Target Sequence and Translational Efficiency
[0131]The position of a miRNA target sequence in reporter miRNA-responsive reporter mRNA was examined. That is, miRNA-responsive reporter mRNA (α(miR-21-5p)-EGFP (SEQ ID NO: 1)) in which one perfect complementary sequence of miR-21-5p that is the target site of miR-21-5p is present in the 5′UTR, and miRNA-responsive reporter mRNA (EGFP-4xα(4xmiR-21-5p) (SEQ ID NO: 2)) in which four perfect complementary sequences of miR-21-5p are present in the 3′UTR, were prepared. The thus prepared mRNAs were examined in terms of translational efficiency. Likewise, miRNA-responsive reporter mRNA (α(miR-17-5p)-EGFP (SEQ ID NO: 9)) in which one perfect complementary sequence of miR-17-5p that is the target site of miR17-5p is present in the 5′UTR, and miRNA-responsive reporter mRNA (EGFP-4xα(4xmiR-17-5p) (SEQ ID NO: 10)) in which four perfect complementary sequences of miR-17-5p are present in the 3′UTR, were prepared. The thus prepared mRNAs were...
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