transfection with negtive control siRNA-cy3 and HiPerFect Transfection Reag

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guanduo8308
guanduo8308's picture
transfection with negtive control siRNA-cy3 and HiPerFect Transfection Reag

Hello,my friends!
     I am using negtive control siRNA-cy3 and HiPerFect Transfection Reagent from Qiagen to transfect negtive control siRNA-cy3 into bone marrow stromal cells.I followed traditional protocol to carry on my experiment,but I failed to transfect it into bone marrow stromal cells.I observed fluorescence at the time of 16h—24h after transfection,and take some photos,but I think that I failed to transfcet into the cells. I used DMEM(Hyclone) with 15%FBS(Hyclone) and antibiotics.
My protocol is as follow:
The first time:
1. The day before transfection, seed 2–8 x 104 cells per well of a 24-well plate in 0.5 ml of an appropriate culture medium containing serum and antibiotics.
2. Incubate the cells under normal growth conditions (typically 37°C and 5% CO2).
3. On the day of transfection, dilute 1.5ul siRNA (2um)in 100 μl culture medium without serum. Add 3 μl of HiPerFect Transfection Reagent to the diluted siRNA and mix by vortexing.
4. Incubate the samples for 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.
  I observed fluorescence at the time of 24h to 48h after transfection .but I failed to .
  The second time:
   1.I dilute 1.5ul siRNA (2um)in 100 μl culture medium without serum,and add 4.5ul of HiPerFect Transfection Reagent to the diluted siRNA and mix by vortexing..
   2. Incubate the samples for 20 min at room temperature (15–25°C) to allow the formation of transfection complexes.
   3.Other procedure is the same as last time .
   I observed fluorescence at the time of 24h to 48h after transfection .but I failed to .see too.
I am evry worrying about my experiment and I don’t know how to carry on my experiment .I need some help from you.Thank you very much.I am waiting for your answers! Good luck!

Ivan Delgado
Ivan Delgado's picture
 

 
Hi guanduo8308,
Since you are using a commercial transfection reagents (Qiagen), I do not think the problem comes from that. Yet, since I do not have experience using Qiagen for this application one suggestion I can give you is to try Lipefectamine from Invitrogen. I've used it extensively and it always works. 
From your description my best guess, assuming that you have the correct construct (i.e. it is not degraded, etc), is that you may be having issues with your cells. Since you can see fluorescence that to me means that the experiment is working (the siRNA-cy3 is getting into the cells in an active form). When you start the transfection, do the cells look overly confluent? is there any major difference in the way the cells look like 1 to 2 days after transfection? If the cells are too dilute or too confluent this could lead to very low transfection rates.

guanduo8308
guanduo8308's picture
Hi Ivan,thank you evry much!

Hi Ivan,thank you evry much!
       The transfected cells are not highly confluent,It is about 60 percent.I observed the transfected cells 24h-48h after transfection,and the cells growed evry well.I have some photos about it .Can you help me to find some questions about it .
        Good luck!

Ivan Delgado
Ivan Delgado's picture
 

 
What you say sounds good (60% confluency at transfection, very good growth 24-48 hours after transfection), which suggests that the issue may not be the cells either. I wished I could help you more, but it sounds like you are doing everything right. The only suggestion I can give when everything is going as it should is to try something else (Invitrogen's lipofectamine or another siRNA control). Alternatively call Qiagen and tell them what you are experiencing. Customer service at Qiagen is one of the best and they should be able to give you some guidance I may be missing. 
Good luck
 

ly54436731
ly54436731's picture
Hi, I have the same problem

Hi, I have the same problem with you. first time. I transfected negative control siRNA to RAW cell with Hiperfect. but cannot observed fluorescence. and second time, I mix 0.1ug GFP DNA with negative control siRNA and Hiperfect. and after transfection, I can see the fluorescence of GFP, but cannot see the siRNA......I am getting crazy

rp0497
rp0497's picture
Hi there

Hi there

It sounds weird indeed. Are you sure of the GFP fluorescence ? Can it be autofluorescence of the cells instead ? The only reason why you would see the GFP and not the siRNA is if you made 2 different mixes, and only one is taken up by the cell.

 I've never worked with RAW cells, but I'm doing a lot of transfection on primary and hard-to-transfect cell lines. My advise is to use SilenceMag or PolyMag (OZ Biosciences).

Let me know if you need more advice.