I am doing an experiment with micro RNA mimic and inhibitor. I am using HEK293T cells for micro RNA mimic and inhibitor transfection. I am using hsa-miR-185-5p mimic and inhibitor for the experiment. I have question regarding experiment design. I have checked expression of this micro RNA in HEK293T cells. So my question is:
How can I transfect the inhibitor to HEK cells? Should I transfect inhibitor alone or with mimicto HEK cells?
I am also using negative control for inhibitor. I think here negative control will work as a scramble sequence. After transfection how can I analyze the data?
So I am taking 3 wells,
1) control well (no inhibitor): only HEK cells
2) Well with inhibitor: HEK cells transfected with inhibitor
3) with negative control: HEK cells transfected with negative control.
Then how should I analyze the data? How can I find the relative expression or fold change for my candidate genes targeted by this microRNA?
Now suppose I want to check inhibitor effect on gene X by qPCR. Then I was thinking to first find our delta Ct value for control by subtracting ct value of control from ct value of negative control.
and then find delta ct value of target gene by subtracting inhibitor ct value from negative control. Then find out delta delta ct value from above two delta Ct value and then calculate the fold change.
So is it a right method to find out relative expression in presence of inhibitor or there is another method?
Hi,
You need to design it as:
1) HEK cells + Negative control
2) HEK cells + Inhibitor
3) HEK cells + mimic
Then you have to extract the total RNA from the cells.
You should make 2 different sets of cDNA. One for miRNA studies, and one for mRNA studies.
For qPCR step and for miRNA evaluation, you need to use U6 as a control gene, and then measure the miR-185-5p using 2^-ddCt method. This step should be done to show that mimic and inhibitor worked properly.
For qPCR step and for mRNA evaluation (target mRNA), you need to use GAPDH or Actin as a control gene, and then calculate the changes in target mRNA(s).
For qPCR analyses, always subtract Ct value of target mRNA(or miRNA) from control gene, which is known as delta Ct. Then calculate delta delta Ct, which is the subtracting each delta Ct value from the average Ct value of Negative Contol group.
Then you can calculate the final fold change.
Hi,
You need to design it as:
1) HEK cells + Negative control
2) HEK cells + Inhibitor
3) HEK cells + mimic
Then you have to extract the total RNA from the cells.
You should make 2 different sets of cDNA. One for miRNA studies, and one for mRNA studies.
For qPCR step and for miRNA evaluation, you need to use U6 as a control gene, and then measure the miR-185-5p using 2^-ddCt method. This step should be done to show that mimic and inhibitor worked properly.
For qPCR step and for mRNA evaluation (target mRNA), you need to use GAPDH or Actin as a control gene, and then calculate the changes in target mRNA(s).
For qPCR analyses, always subtract Ct value of target mRNA(or miRNA) from control gene, which is known as delta Ct. Then calculate delta delta Ct, which is the subtracting each delta Ct value from the average Ct value of Negative Contol group.
Then you can calculate the final fold change.
use mirVana miRNA mimics and inhibitors, and the best delivery reagent for short RNA is RNAiMax (optimize the dose). cells, cells + mimic, cells + inhibitor, plus negative and positive controls. can try 2 concentrations of mimic and inhibitor, eg 5 nM and 30 nM. measure miRNA levels by qRT-PCR with miRNA TaqMan assays. for sample prep can use Cells to Ct kit - which is the fastest and easiest.
Dear
For transfection of Reposter plasmid with mimic and inhibitor what is the ratio of these three?
Thanks
I dont have a scramble inhibitor... I inhibit one micro RNA with miR inhibitor compared to my negative control (juste a transfection with lipofectamin)... The scramble have an effect to cell proliferation?
Mohamed Abdelkarim the sample with the lipofectamin only is called a "Mock" sample not negative or scrambled control. This mock sample can only control for the toxicity that may be exerted by lipofectamin. You need to have a negative control (something like Allstar negative control, qiagen) that has siRNA that not known to have any homology with any mammalian genes. You should also have the untreated control.
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