Hi all,

I'll be explaining the situation briefly. I'm working with two cancer cell lines: T24 and UMUC-3 cells.

I'm trying to do a knockdown for the SLC38A2 gene using siRNA and Hiperfect Transfection reagent from QIAGEN.

I have followed the protocol from Qiagen: transfecetion using a 6-well plate.

1st day: seeding of 10^5 cells.

2nd day: Transfection with 10nM-20nM-30nM of siNeg Cntrl and siSLC38A2, and for each cc, I tried 3 different vls of Hiperfect suggested in the protocol: 6.5 µl, 12 µl, and 13.5 µl.

I tried incubation for 48 hours and 72 hours.

The knockdown is done in most cases going to 80%KD but the problem is that in the same conditions, the siNC control is causing also a KD for the target gene :SLC38A2.

I assessed the knockdown using qRT-PCR.

It is not a problem of RNA or cDNA concentration. as they are all normalized before using them. the primers also I'm sure about that I used before in other experiments.

I tried all conditions for the transfection, trying to change the incubation time, the cc of siRNA, and the vl of the transfection reagent, but the problem still persists.

Can anyone please suggest a solution?

I'm attaching the gene expression bars for the UMUC-3 cells after 48 and 72 hours of incubation.

N: NC

S: SLC38A2

1/2/3: 10nM

4/5/6: 20nM

7/8/9: 30nM

1-4-7: adding 6.5 µl of Hiperfect transfection reagent

2-5-8: adding 12µl of Hiperfect transfection reagent

3-6-9: adding 13.5 µl of Hiperfect transfection reagent

Unt: Untreated cells