RNAi transfection

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HJGuan
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RNAi transfection

I am using the RNAi starter kit from Qiagen to transfect siRNA into HeLa cells. I follow the fastforward protocol but only got one or two in 200000 cells transfected with the Alexa Fluor labeled control siRNA. The transfected cell was round loosely attached to other heathy cells. I tried not to use serum but still the same bad but with more dead cells. I tried HeLa from other department but still doesn't work. I tried Escort V transfection reagent from Sigma with or without serum but could still only see one or two cells with fluorescence (the cell were abnormally shaped) or none at all (Escort V is more toxic to cells. I tried Escort V using the same protocol as Qiagen except for the preparation of transfection cocktail.). I observed fluorescence usually at the time of 12h or 21h post-transfection. I used RPMI1640(Hyclone) with 10%FBS(Hyclone) and antibiotics.

The protocol is as follow:
Protocol: Fast-Forward Transfection of HeLa cells with siRNA using HiPerFect Transfection Reagent
This protocol has provided successful results when tested in experiments with this cell type. It is provided as a starting point for optimization of siRNA transfection in a 6-well plate.
The amounts given are for one well of a 6-well plate. In this protocol, cell seeding and transfection are performed on the same day.
Procedure
1.Shortly before transfection, seed 2 x 105 cells per well of a 6-well plate in 2300 µl of an appropriate culture medium containing serum and antibiotics.
2.For the short time until transfection, incubate the cells under normal growth conditions (typically 37°C and 5% CO2). Cells may alternatively be seeded after step 3 of this protocol.
3. Dilute 30 ng siRNA in 100 µl culture medium without serum (this will give a final siRNA concentration of 1 nM after adding complexes to cells in step 5).
Add 12 µl of HiPerFect Transfection Reagent to the diluted siRNA and mix by vortexing.
IMPORTANT: The amount of transfection reagent and siRNA required for optimal performance may vary, depending on the cell line and gene target.
4. Incubate the samples for 5-10 min at room temperature (15-25°C) to allow the formation of transfection complexes.
5. Add the complexes drop-wise onto the cells. Gently swirl the plate to ensure uniform distribution of the transfection complexes.
6. Incubate the cells with the transfection complexes under their normal growth conditions and monitor gene silencing after an appropriate time (e.g., 6-72 h after transfection, depending on experimental setup). Change the medium as required.
Note: The optimal incubation time for gene silencing analysis depends on the cell type, the gene targeted, and the method of analysis. This can be determined by performing a time-course experiment. When using fluorescently labeled siRNA,
microscopic analysis should be performed 4-24 h after transfection.

I hope the Technician Nikky from Qiagen doesn't mind that I post the protocol here.

I don't know what is wrong. The next thing I want to try is to use DMEM as medium. Can you guys help a little? It's been months now.

HJGuan
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I forgot to say that I also

I forgot to say that I also tried HepG2 and HCT116+ch3 cells but got similar results. Thx.

R Bishop
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HJGuan,

HJGuan,

Im seeing this problem all over the place these days. Super frustrating to spend months on it Im sure.

For starters, if you bought the kit and followed all the recommended optimization techniques to transfect the control Alexa labeled siRNAs, I would start to suspect the kit first. However you changed transfection reagents and still got no results at all. That makes me suspect the control siRNAs are garbage. Have you bought some new siRNAs? Is there anyone at your facility that also does silencing? If so perhaps you could borrow a bit of the controls siRNAs they use.

I guess you already done all the classic troubleshooting methods?

There are 3 things you need to very Time, Temperature, and Concentration. These are cells so Temp is fixed. That leaves you mainly with concentration and you fill us in a bit on the different reagent concentrations you have tried on HeLas?

I still think the siRNA you bought is no good for what ever reason.

HJGuan
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Sorry. It's the Chinese New

Sorry. It's the Chinese New Year.
I tried GlycoDoc's recommendation (thanks by the way) and used the traditional protocol (transfecting cells the day after cell seeding) in HeLa and HCT116+ch3 cells using the fluorescence control siRNA but it didn't work. I'm starting to think that maybe there is a problem with the fluorescence control siRNA  (maybe it's been exposed to light?). You know, there are dead cells (?) that can cause light-refraction. And all the light that can pass through the filter of the fluorescence microscope is green, which gives people an illusion that those are transfected cells. I think that's what I observed in the past experiments? The technician from Qiagen asked me to try the positive siRNA targeting MAPK1 and validate with PCR. I'm starting to do so. And also, next time I will try not to use any filter when using the fluoescence microscope. I will keep you posted.
Many thanks and Happy Chinese New Year of the Ox to all of you!

Jen_Floyd
Jen_Floyd's picture
 I do not know how true this

 I do not know how true this is but I have heard that the fluorescent si RNA oligos are a poor measure of transfection.  I have two solutions for this:
1.  Choose a cell surface marker to knock down and check for knockdown by FACS.  That will tell you %transfected.  You could follow up with a western blot to determine the amount of knockdown.
2.  Check for any gene with an siRNA that you know works well (either confirmed by Qiagen or a collegue) and just test by QPCR or western blot.  I don't care about % transfected because I just want to know if my knockdown worked.  If it's about 90%, the transfection was, at worst, 90%, probably higher.
I have had equal luck using Invitrogen, Qiagen, and Dharmacon siRNA oligos.  I don't think it matters too much, I usually go with whomever is cheapest or fastest.

HJGuan
HJGuan's picture
Hi! Guys! I have an update.

Hi! Guys! I have an update.
I tried the transfection in HeLa using Qiagen validated siRNA and transfection reagent, and real-time PCR showed that 24 and 48 hour post-transfection, there were 39% and 40% target gene left, respectively. I couldn't believe it. And then I tried western blot. 24 hour post-transfection had about 40% left. I am like, what?! I tried the transfection again and got the same result. But when I tried 48 hour, the Western blot showed only 10% to 20% knock-down (maybe it's my technical problem). This is really ironic cause the moment when I got the products, I tried the exact same thing but I harvested cells 4 days after transfection cause I thought the protein needs time to degrade. And the Western showed no knock-down, which started a whole process of trouble shooting. It seems that the siRNA is rapidly cleared away and 24 hour is the right time to detect my protein. I don't know whether it is true. I don't know why the fluorescence labeled negative siRNA didn't show any fluorescence. But Qiagen sends me a new one. I will try to make it work some other time.
I really want to thank all of you guys who care. I am so ashamed that I never have the time or mood to do this to people. I can finally see a faint  light of my graduation out of the profound darkness.