[QUESTION] Is it possible to incorperate Tri Reagent (Trizol) with the Mirvana m - (Oct/26/2012 )

Hello all,

I am a grad student starting to work on my thesis. Recently, I transiently transfected a cell line with a miRNA plasmid, and let it grow. I wanted to extract microRNA from the cell so that I can run qPCR at a later date, but due to time constraints of classes I decided to just lyse the cells open with Tri Reagent (Trizol). We use this in lab when we preform Total RNA extraction. So I figured it would be alright to do the same again with the cells that I wanted to extract microRNA from. The protocols seemed very similar in my opinion. The problem is now that I want to start extracting microRNA so that I can run qPCR, i kind of cant.

I am using a Mirvana miRNA Extraction Kit from Ambion. The first stem was to spin the cells down and count them and then add the appropriate amount of Lysis/Binding Solution. Then to add a Homogenate Addative. Then the rest of the protocol somewhat follows the similar procedure in our lab for Total RNA isolation.

My question is am I going to be able to follow the protocol offered in the mirVana microRNA isolation kit, or did I just ruin very good cells? Is there a way of getting around the Tri Reagent?

Thank You in advance for any and all help.

I don't think the two will be compatible, but you can usually contact the compan(ies) and ask if they have work arounds.

You should be able to precipitate the miRNA from the trizol, but it probably won't be very efficient.

That is what I was afraid of. I knew the first step when I added the Lysis/Binding Solution would lyse the cells like Trizol does, but I also figured it had something bind to the miRNA given its name, which would not allow it to elute through the filters they provided.

I was thinking that I just go ahead and start with adding the Lysis/Binding Solution and continue the protocol from there, since the cells are already lysed I am hoping there would be no adverse effects and that the binding part will just do its job, I just hope that Trizol does nothing to mess up the reaction.

I will definitely call the companies up to see if they may have a work around. I was kind of hoping that someone here ran into the same problem, but I guess not.

Thank you bob1.

The binding solution may have it's name since it provides special requirements for appropriate binding. AFAIK you have two options.

1. Leave the mirVana and do miRNA isolation from Trizol, since there are known issues with precipitation of small RNAs with the ethanol step, you have to find a special protocol for Trizol miRNA isolation. Try search the forums in the first place. The yield will be probably lower, but those protocols are already tested.

2. Continue with Trizol lysate using mirVana kit. mirVana also uses phenol-chlorophorm extraction. But you can't from the first step with lysis solution, because now you already have phenol in your samples! Do the first step of Trizol isolation (adding chlorophorm) and follow Trizol protocol to obtain aqueous phase. Now use this phase as noted in the F.I. Total RNA Isolation Procedure part of mirVana protocol, and continue now on to columns and so.
There may be some issues with the fact, that some specific stuff should be present in the aqueous phase which is now not, and it's hard to tell whether it will affect the binding of RNA. It may not at all or it may seriously, leading to a low yield again.

So, you decide.