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problem with siRNA transfection of HEK cells - (Jul/27/2011 )

Hello there,

I purchased 5 siRNA sequences from sigma and tried to transfect HEK cells (using Lipofectamine). However, when the times come to verify the efficiency of the silencing, those cells didnt show any decrease of their RNA expression. I tried those sequences on other cell line and they dont have any affect on the expression level of the targeted gene. I used different concentration of the sequences (20 pmol-160 pmol) without any effect.

Can anyone please help to increase the efficiency of my transfection and wither I should use something other lipofectamine? Does it make a difference if I dont change the media 6hrs after transfection (leaving them O/N)? I am concern that the sequences are not good and I want to troubleshoot them before I get another set of sequences?

-HELP !!!-

1. Are those siRNA validated (pre-design...)by Sigma? how many siRNA did you get for one gene? I didn't try sigma's siRNA, but for Dharmacon' s pre-design series, they will let you select 2 or 3 siRNA for one gene, and they guarantee at least one siRNA can knockdown your target by >80%. in case you failed in all 2/3 siRNA, they will re-send you another siRNA (with different sequence of course) until you say OK.
2. you may change your lipofectamine to Lipofectamine™ RNAiMAX.
3. is the lipofectaine toxic to your HEK cells? if not, i think it is ok not to change medium after 6 hours and it won't lead to low transfection efficiency or change RNAi efficiency.

-woraw-

woraw on Fri Jul 29 03:45:36 2011 said:


1. Are those siRNA validated (pre-design...)by Sigma? how many siRNA did you get for one gene? I didn't try sigma's siRNA, but for Dharmacon' s pre-design series, they will let you select 2 or 3 siRNA for one gene, and they guarantee at least one siRNA can knockdown your target by >80%. in case you failed in all 2/3 siRNA, they will re-send you another siRNA (with different sequence of course) until you say OK.
2. you may change your lipofectamine to Lipofectamine™ RNAiMAX.
3. is the lipofectaine toxic to your HEK cells? if not, i think it is ok not to change medium after 6 hours and it won't lead to low transfection efficiency or change RNAi efficiency.


1- I got 5 sequences from sigma, and unfortunately non of the sequences they have for my gene were validated, so I picked the top five they recommended. I may try to call sigma and see if they can send me different ones.

2- I will check if we can try that one

3-It is not toxic and actually I just did this today and decided to leave the lipofectamin O/N for my cells to see if this will increase the efficiency

Thank you

-HELP !!!-

HELP !!! on Fri Jul 29 04:57:18 2011 said:


woraw on Fri Jul 29 03:45:36 2011 said:


1. Are those siRNA validated (pre-design...)by Sigma? how many siRNA did you get for one gene? I didn't try sigma's siRNA, but for Dharmacon' s pre-design series, they will let you select 2 or 3 siRNA for one gene, and they guarantee at least one siRNA can knockdown your target by >80%. in case you failed in all 2/3 siRNA, they will re-send you another siRNA (with different sequence of course) until you say OK.
2. you may change your lipofectamine to Lipofectamine™ RNAiMAX.
3. is the lipofectaine toxic to your HEK cells? if not, i think it is ok not to change medium after 6 hours and it won't lead to low transfection efficiency or change RNAi efficiency.


1- I got 5 sequences from sigma, and unfortunately non of the sequences they have for my gene were validated, so I picked the top five they recommended. I may try to call sigma and see if they can send me different ones.

2- I will check if we can try that one

3-It is not toxic and actually I just did this today and decided to leave the lipofectamin O/N for my cells to see if this will increase the efficiency

Thank you



Did you try siGlow (or whatever it's called -flourescently labeled siRNA to check for the efficiency). This would rule out problems with the transfection.

-Andras-

Some target expression/gene... are really hard to control/decrease using siRNA. You might think about switching to shRNA,viral particle instead for a stably target knockdown expression.

If you have RT-PCR work to show that SIgma produc doesn't work, get your refund quite as possible. I have tried 4 different cells lines, 5 different siRNA sequences and n=6 for total of 5 times with western blot for verification of its knockdown expression, I had no luck. Of course I varied with cell density curve (5 different ones), lipofectamine concentration curve, positive control, and different cell lines on same day. Positive control and negative control work, except the TARGET. Because we couldn't provide PR-PCR work since we only did WB, Sigma didn't credit our purchase. I wasted total of more than 4 months or work because PI didn't want to purchase or save money. It never worked, and did not work. --> Other lab also told us that they also were having problem. They also switched. Then PI wanted to move on from siRNA to shRNA.

Get your credit and purchase shRNA lentiviral particle transduction. It worked like a charm in 1st time, and these results were consistent.

-powertoeki-

powertoeki on Sat Aug 13 07:54:53 2011 said:


Some target expression/gene... are really hard to control/decrease using siRNA. You might think about switching to shRNA,viral particle instead for a stably target knockdown expression.

If you have RT-PCR work to show that SIgma produc doesn't work, get your refund quite as possible. I have tried 4 different cells lines, 5 different siRNA sequences and n=6 for total of 5 times with western blot for verification of its knockdown expression, I had no luck. Of course I varied with cell density curve (5 different ones), lipofectamine concentration curve, positive control, and different cell lines on same day. Positive control and negative control work, except the TARGET. Because we couldn't provide PR-PCR work since we only did WB, Sigma didn't credit our purchase. I wasted total of more than 4 months or work because PI didn't want to purchase or save money. It never worked, and did not work. --> Other lab also told us that they also were having problem. They also switched. Then PI wanted to move on from siRNA to shRNA.

Get your credit and purchase shRNA lentiviral particle transduction. It worked like a charm in 1st time, and these results were consistent.


Thank you guys,
I have contacted Sigma and they are looking into the issue!, and most likely to get a refund. I have been trying this for 3 months with no luck. We choose siRNA because our cells are resistant to many of the antibiotics used on the shRNAs available.

thank you all for your help

-HELP !!!-