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A help for siRNA experiment - (Feb/04/2014 )

Dear All,

at first, I am still a beginner in siRNA experiments, this is my first one

I want to silence specific two genes

I bought siRNA from sante cruz company.

http://www.scbt.com/

with all kit, like transfecting agent, medium and control siRNA.

and I cultured my cells and followed the protocol as written exactly,

after finishing the experiment and waiting 24 hrs, I wanted to test the presence of this protein in the cell i silenced, so I stained my cells by immunofluorescnece.

However, I could not see any difference, between, control siRNA and gene silenced and even normal cells.

so I tried again, with 2 experiment, 1 changing the transfection reagent into RNAi fect, and 2- repeat the same old experiment but waited for both exp longer time, perhaps my protein half life time is long (65 hrs)

however, today, I can see the same under microscope.

for both genes I used.

 I know that I need rt-PCR or western blot to prove silencing of specific gene.

 

my questions?

The use of immunofluorescence staining is good way to prove silence gene?

Do u have any suggestion for my to think about since I am beginner?

Do u have any experience regarding this company and siRNA products

 

at the end, I am very grateful for those who is welling to share their experience with me

Thank u

 

 

-madelingirly-

I have two comments - does this antibody work for you with IF in other experiments?

 

and

 

I would start with looking at KD using western blot - that is the functional end of things.  Yes, IF is similar, but the major difference is that IF i mostly not a very quantitative technique, whereas westerns can be.  So, if you KD and only see a 50% reduction in the WB, you might not be able to visualize this on IF.

-bob1-

Hi madelingirly,

 

here is a list of things you need to think about when trouble shooting:

1) Transfection efficiency: how well have the siRNAs been transfected into your cells? In general, siRNA transfection efficiency is not a big concern, but may vary in different cells. How about you purchase a fluorescence labeled siRNA as a control for transfection efficiency?

2) I don't think IF is a good way of accessing gene knockdown. IF itself can give you false positive signal. Try RT-PCR and western.

3) Do you know the basal expression of the target genes in your cells? Are they already expressed low?

-pcrman-