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Anyone has a experience with Fluorescently labelled siRNA ?

siRNA transfection efficiency

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6 replies to this topic

#1 Darlidada

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Posted 30 January 2015 - 04:30 AM

Hello , 

 

I tried siRNA st and i had only 60% of down regulation. So my boss want me to check the transfecton efficiency, for this he recommanded fluorescently labelled siRNA . I never heard about it , so i checked there's a kit for this. 

But i'm not familiar with this kind of experiements.

 

Your comments are more than welcome.

 

Thanks



#2 CPRES

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Posted 30 January 2015 - 06:20 AM

There is nothing to it. You will be able to see if you are getting a good transfection efficiency with your method.

 

Basically, you transfect your experimental siRNA and control (fl labeled) siRNA to your cells (in separate wells of course). Then check the control cells under fl microscope to see what % of cells show fl. 


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#3 Darlidada

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Posted 30 January 2015 - 07:38 AM

Hello CPRES, 

 

thank you for your reply. I'm not sure to understand. 

on the protocol of the transfection lipofectamine RNAimax, it's written if you need to check the transfection efficiency, it's recommanded to use BLOCK-iT Alexa Fluor Red Fluorescent.  I checked and i didn't find any details of how i have to incorporte it. 

 

I usually dp this :  first i diluated the lipofectamine with OptiMem media then diluate siRNA with OptiMem and then mix the 2 . after 5 minutes of i have to add the lipofectamine siRNA mix to my cells.

It may be stupid for you but i didn't get at what moment and how i have to add this Alexa Fluor unsure.png unsure.png



#4 CPRES

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Posted 30 January 2015 - 07:47 AM

The BLOCK-iT Alexa Fluor Red is already labeled siRNA, so you don't have to label or incorporate anything (if that is what you are concerned about).

 

Just use this fluorescent siRNA exactly as you use your experimental siRNA in the protocol.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#5 Darlidada

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Posted 30 January 2015 - 08:09 AM

The BLOCK-iT Alexa Fluor Red is already labeled siRNA, so you don't have to label or incorporate anything (if that is what you are concerned about).

 

Just use this fluorescent siRNA exactly as you use your experimental siRNA in the protocol.

Ok,  so if i understood well  i need to purchase another sirna targeting my gene, labelled Alexa Fluor Red ? in this case it's better to purshase a kit for labelling siRNa. Actually i'm lostblink.png huh.png



#6 CPRES

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Posted 30 January 2015 - 08:13 AM

No, here is what you do:

 

1. Buy a siRNA that is targeting your gene. This is unlabeled siRNA directed against your gene.

 

2. Buy a control (BlockiT) alexa fluor red siRNA. This siRNA is carefully designed such that it does not target any mammalian gene. So, it is a negative control, but more importantly, it is a transfection efficiency control.

 

And that is all you need. Forget about a kit to label your siRNA, unless you want to see its cellular localization and dynamic. Most experiments are just about transfecting siRNA to knockdown the gene of your interest.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#7 Darlidada

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Posted 30 January 2015 - 08:23 AM

No, here is what you do:

 

1. Buy a siRNA that is targeting your gene. This is unlabeled siRNA directed against your gene.

 

2. Buy a control (BlockiT) alexa fluor red siRNA. This siRNA is carefully designed such that it does not target any mammalian gene. So, it is a negative control, but more importantly, it is a transfection efficiency control.

 

And that is all you need. Forget about a kit to label your siRNA, unless you want to see its cellular localization and dynamic. Most experiments are just about transfecting siRNA to knockdown the gene of your interest.

I get it .I'll do . Thanks a lot CPRES !!biggrin.png







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