Can you provide some more details to help us help you:

• What cell type are you using? How many cells per well?
• Have you tested more than 1 siRNA for each isoform?

Are you concerned because you have toxicity/low transfection, or do you think that might explain your results?

thanks for your answer

i am using primary human tubular cells, plated 36 thousand x well in 96 well plate and after 24h transfected x 24h . i am testing only 2 sirna ( one for each isoform)

IMP

i discovered that siRNA against the isoforms U is instead able to bind also the other isoform (s) so we can consider it as a siRNA against all XBP1 isoforms(XBP1t). it explain why cell treatred with siRNA called anti U show low expression of both XBP1u and S.

now my question is: i used a siRNA final concentration of 33 nM, do you think should i increse it to have better transfection efficiency? i am also worried about the amount of lipofectamine i am using that might explain the results of the scramble siRNA.

do you have any different interpretation of the results and suggestion for me?

I would definitely consider trying a higher concentration of siRNA to try and get stronger silencing. I've used 200 nM final concentration when transfecting microRNA mimics and inhibitors (same protocol as doing siRNA transfection) with no issues.

If you are unable to achieve the desired level of silencing with the two siRNAs that you have, you may need to design others. It is usually recommended that you design multiple siRNAs for each target from the start as the efficiency of knockdown can vary quite a lot.

I don't expect that it would affect your actual transfection efficiency though (this would have to be tested with a fluorescently-tagged siRNA). You can try altering the ratio of siRNA and Lipofectamine to optimize the transfection efficiency in your specific cells. You are also plating quite a lot of cells per well. I don't have any experience with tubule cells, so I'm not sure how confluent the monolayers would be or how confluent those cells like to be. However, if you're doing that many cells to get enough RNA, you might benefit from scaling up to a 48 or 24 well plate (increasing your transfections as well).

Lipofectamine can be pretty hit or miss on toxicity depending on the cell type. If you are seeing large amounts of cell death you may want to try changing the media after transfection. When working with HUVECs (primary endothelial cells) I found that changing to fresh media 4 hours after starting the transfection drastically cut down on cell death without affecting transfection efficiency.

As a last resort, there are some transfection reagents that are designed to give better transfection efficiency when using small RNAs like siRNA. I've used the Dharmafect series with good success in primary human smooth muscle cells and endothelial cells. However, Lipofectamine should perform well enough in most applications.