REPORTER GENE ASSAY

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ksowmya123
ksowmya123's picture
REPORTER GENE ASSAY

I have a question regarding the luciferase reporter gene assay. I performed transient transfection of MCF-7 cells using CYP1B1 reporter plasmid and exposed the cells to 100nM TCDD and found the increase in activity of CYP1B1 promotor activity when i performed luciferase reporter gene assay,I repeated the same experimental procedure for seven times and i could't get the same results and plzz suggest what could be the posiible reason for my poor results.

Trish
Trish's picture
1. What is your transfection

1. What is your transfection efficiency? - may switch to a better reagent. In our case, we found Gencarrier-1 is the best for MCF-7 with high efficiency and extremely low cell death if any.
2. Is your CYP1B1 promotor a strong one in MCF-7 cells? - try other cell types which may also serve as your positive control if you really need MCF-7.
3. Is your CYP1B1 promoter full length? - may lack some enhancer elements.
4. Try TCDD dose respnose and time course study to determine the optimal conc. and time to harvest samples.

Rajeshwari patel
Rajeshwari patel's picture
Hi,

Hi,
You are performing the assay with transient transfected vector,I f you consider more than 90% effeciency in your transfection than also you will get assay variation on day to day, so to rule out it you have to applly some normalization factor with your assay like, total protein estimation or else you go for dual luciferase assay