DLK knock down - (Aug/24/2005 )

Hi,

I ordered the 2 pre-designed human DLK siRNA oligos from Ambion. I transfected the 50pmol and 150 pmol oligo each by using lipofectamine 2000 to DLK overexpressed hMSC. One day before transfection, I plate 750,000cell per well of 6-well plate. RNAs were collected 1 day, 3 days and 6 days after transfection. But suprisingly, for the day 1 and day 6 samples, the expression level of DLK in transfected cell are higher than controls. The knock down effect only observed in day 3 samples.

Could you give me some reasonable explanation for this?

Thanks

Weimin

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Hi Weimin,

First of all, is the result reproducible and what method do you use to assess the expression of your gene, RT-PCR, or western? To confirm such unusual finding, you have to repeat the transfection and assess expression by Western or Northern. Although siRNA can sometimes activate expression, in your case, it is unlikely.

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Thank you for your answer.

In my case, I use real-time PCR to dectect the dlk expression. The result is not repeated. Also, I collected the cells for immunostaining and the medium for ELISA. But I still wait for the result.

Why in my case, it is unlike that siRNA activate expression?

Thanks

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siRNA can induce gene expression in a sequence independent (interferon response) or dependent way. We have observed the latter but I will not reveal the detail because the data is unpublished. In your case I doubt that the finding is reproducible. If it is, I may help you check if the induction is sequence specific if you give me your siRNA sequence.

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Hi,

the sequences are: gcuuacuauacgcggucugtt sense
cagaccgcguauaguaagctc antisense

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Hi Weimin,

I checked your sequence and found a partial match between your siRNA and a particular DNA sequence. It is too early to say this match has anything to do with the observed induction. Only if the induction is reproducible, this "off-target" effect may provide some explanation.

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