6 replies to this topic

[steffi333]

Hi all,

I'm a student just starting to use siRNA in my work (and also the first person in my lab to ever do so... the joys) and in the course of researching techniques, manufacturers etc I'm completely muddling myself up and getting tangled with even the basic principles of RNA!

I've managed to design myself some sequences using an online tool and the mRNA sequence of my gene, but when it comes to ordering I'm confusing myself - and I want to get it right, money doesn't grow on trees in my lab.

When you order your sequences, do you send the order with ATGC nucleotides, or ACGU? I know this is a basic thing, but I've been going round and round it all in my head for a few days now (along with a load of other things I'm having to do atm) and it feels like my brain is going to leak out of my ears!  

Also has anybody had any experience transfecting siRNAs with Oligofectamine? I know there are specific kits out there for siRNA transfections, but my supervisor is keen on me using Oligofectamine.

Thanks for any help anybody can give with my duncey question!

[rkay447]

When I ordered custom "stealth" siRNA from Invitrogen, I used the ACGU format.  You can always call the company that you plan on ordering the siRNA complexes from and talk with their technical support.  Just explain that you are new to this technique and want to make sure that the products you order from their company will give you the best opportunity for success.  I've found that with a little politeness and appreciation, these people will go out of their way to help you!  As for transfecting the siRNAs, I've had great success with Lipofectamine 2000 but I think it matters more on the cell line and what works best for that cell line in general.  If you can transfect your cell line with Oligofectamine and get good results with plasmid DNA, it should work very well for siRNA as well.  But, if you run into problems, there are specific transfection reagents specifically designed for siRNA transfection.   Does your supervisor have any specific reason(s) why he/she wants you to use Oligofectamine?  If it's already in the lab, I'd say try it first and see how good of a knockdown you get.

[steffi333]

Thanks I think the ACGU format is what I'll end up going for. We're going to try and get them synthesised by MWG and my institution has a fairly good record of dealing with them!

My supervisor liked the idea of oligofectamine because we've read some papers that used it successfully. But we do also have lipofectamine in our lab already. I haven't carried out much transfection with this cell line before and there isn't much written on transfection using it (it's a little known/used dog cell line) but we have plenty of plasmids available that I could try out on it.

Thanks for your help   

[rkay447]

steffi333, on 12 January 2011 - 08:52 AM, said:

Thanks I think the ACGU format is what I'll end up going for. We're going to try and get them synthesised by MWG and my institution has a fairly good record of dealing with them!

My supervisor liked the idea of oligofectamine because we've read some papers that used it successfully. But we do also have lipofectamine in our lab already. I haven't carried out much transfection with this cell line before and there isn't much written on transfection using it (it's a little known/used dog cell line) but we have plenty of plasmids available that I could try out on it.

Thanks for your help   

Just get a GFP vector and compare the transfection efficiency with both reagents.  It's easy to just check the cells the next day for GFP expression to see if one reagent works better than others.  You really should make sure that this cell line can be transfected at a high enough efficiency for your intended purpose.  Some cell lines just don't transfect well and you may find that this cell line will not be appropriate for your desired experiment!!  I would ensure that this cell line will work before ordering siRNA.

[UBClabbie]

steffi333, on 12 January 2011 - 04:23 AM, said:

Hi all,

I'm a student just starting to use siRNA in my work (and also the first person in my lab to ever do so... the joys) and in the course of researching techniques, manufacturers etc I'm completely muddling myself up and getting tangled with even the basic principles of RNA!

I've managed to design myself some sequences using an online tool and the mRNA sequence of my gene, but when it comes to ordering I'm confusing myself - and I want to get it right, money doesn't grow on trees in my lab.

When you order your sequences, do you send the order with ATGC nucleotides, or ACGU? I know this is a basic thing, but I've been going round and round it all in my head for a few days now (along with a load of other things I'm having to do atm) and it feels like my brain is going to leak out of my ears!  

Also has anybody had any experience transfecting siRNAs with Oligofectamine? I know there are specific kits out there for siRNA transfections, but my supervisor is keen on me using Oligofectamine.

Thanks for any help anybody can give with my duncey question!

I recommend using DNAfectin or RNAfectin, there are 4 types and you choose which one based on the cell type you are transfecting. It has much lower toxicity than Lipofectamine and is also much cheaper. 100% full guarantee. http://www.abmgood.c...n=3770&dsn=3876

I should add that you can request samples of DNAfectin and/or RNAfectin to test out. Yay for risk free optimization!

Edited by biotechgirl, 12 January 2011 - 04:01 PM.

[steffi333]

rkay447, on 12 January 2011 - 10:58 AM, said:

Just get a GFP vector and compare the transfection efficiency with both reagents.  It's easy to just check the cells the next day for GFP expression to see if one reagent works better than others.  You really should make sure that this cell line can be transfected at a high enough efficiency for your intended purpose.  Some cell lines just don't transfect well and you may find that this cell line will not be appropriate for your desired experiment!!  I would ensure that this cell line will work before ordering siRNA.

Yeah we have a GFP plasmid which is known to work in other cell lines so I'm attempting a lipofectamine 2000 transfection with that in the next few days before I order anything.

Thanks for the advice

[steffi333]

biotechgirl, on 12 January 2011 - 04:00 PM, said:

I recommend using DNAfectin or RNAfectin, there are 4 types and you choose which one based on the cell type you are transfecting. It has much lower toxicity than Lipofectamine and is also much cheaper. 100% full guarantee. http://www.abmgood.c...n=3770&dsn=3876

I should add that you can request samples of DNAfectin and/or RNAfectin to test out. Yay for risk free optimization!

Thanks for the recommendation! We have lipofectamine 2000 readily available in the lab for now, but if that doesn't work/it commits cellular genocide I will definitely give this a go!