[討論] luciferase assay相關問題
麻煩大家
解決我面臨到的問題(如果做出來，我就可以順利畢業了>"<)
以下是我的問題:
我找到兩個miRNA，然後用線上預測網站去預測他的TARGET GENE
找到之後，在細胞內大量表現我的miRNA
用WESTERN BLOT 看到我要看的PROTEIN 表現量有下降(先偷看有沒有作用)
之後就要用LUCIFERASE ASSAY去證明，我的miRNA會不會直接跟我的TARGET GENE BINDING
但是我的Luciferase 沒有被壓下來或是有時候有壓下來。我調過target gene與miRNA之間的比例(1:1,1:3,1:4)
也看過Transfection 24hr,48hr
這樣的結果是在說明，我的miRNA跟我的TARGET GENE沒有直接Binding的關係嗎???
這樣子，我又要如何走我的下一步呢????
麻煩你們給我一點方向
以下是我transfection 的condition
transfection reagent:lipofetamine LTX
Seeding in 24 well plate
每個well的plasmid量
A
miRNA construct 250ng
target gene construct 250ng
renilla 13ng
opti-mem 50ul
B
lipo 2ul
opti-mem 50ul
A+B MIX
在加入WELL中並補400ul medium
過3hr換medium
放24hr
麻煩各位了(鞠躬~~)
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