Why are you concerned with the aggregation of the cells in RNA later? If all you are doing is pelleting and extracing the RNA, I don't see why you would need to use beads to lyse the cells.

As for lysing ... what are you doing now? The Ambion and the Qiagen RNA isolation kits have all the buffers included.

The samples are whole blood in RNA later. Well, more like RBC + WBC (plasma was taken out before storing in ARNlater). And we need the RNA from the WBC. Therefore homogenize, break cells and get the ARN out of cells... does that make sense?

For extraction I am using QIAGEN RNEasy mini Kit

So, for whole blood, you have to lyse the RBCs first, and then the WBC. So if you look at all the whole blood isolation kits from Ambion or Qiagen, there are two lysis steps.

There are a lot of buffers for RBC lysis. Once that is done, you can add any of the phenol based RNA isolation product (like RNAzol) to the pellet and that'll lyse everything.

Are you trying to isolate the WBCs without lysing the RBCs? I'm not sure that is possible at this stage...

If you are using the Qiagen kit, it should work.

Maybe I need some clarification. Where in the qiagen steps are you having issues with?

Troubles In the homogenization of the samples.

The samples (In EDTA tubes: RBC+WBC in RNAlater) have been store for 5 years (-20C). Once thawn, even if vortexing a lot, the composition of the sample looks aggregated, pieces or clots like.

So, if I take for example 200-300ul to start the extraction, these 200ul wont be representative because of this "clots" that may content more or less cells...

For so, I was suggested to use glass beads to break cells, mix everything, homogenise. But never use bead beating before.

I suppose I have to take out the RNAlater first, then add the beads + lysis buffer (or something similar to re-suspend the sample), then bead beat and from that, take what I need for extracting RNA... More clear now? =)

I won't worry about the clumps before centrifugation. And yes, the clumps can cause you to overload the column.

And, you are correct, you still have to pellet the cells first, remove the RNAlater, before you can lyse the cells with the beads. It is not recommended to use the beads in the RNAlater due to RNA degradation.

So... I would think that you would want to spin down the entire sample first, remove the RNAlater, before following the Qiagen kit.

If you want to try the beads, which I don't think you will need, as the lysis buffer supplied by the kit is more then sufficient to lyse the pellet... For beads, there really is not a "speed", you just add it in with a RNA stablization solution (this can contain actual lysis solution, but it doesn't have to as the beads is the physical method to break the cell membranes), and vortex for a few rounds (probably 2 or 3). Usually about 15 to 30 seconds each round.

Hope this helps.

Hope other people have more insights.

Thanks Shen-An for helping me out...

I have used the lysis buffer before and follow the protocol as it is, but the clots, and aggregations doesn't seem to break completely, interfering with the column and also working with a non homogenise sample (cells with ARN could be there or not).

I think that I need to: Remove ARNlater--> add lysis buffer or any RNA stabilisation solution (any suggestion? could it be DEPC treated water?)

-->add beads and mix --> from that, take the volume needed for extraction -->extract with the kit.

If I do that, can the rest of the sample that I won't be using be store once already taken out the ARNlater? Store at -80? It would be mixed with lysis buffer/RNA stablization and the beads...

For a lysis/RNA stablizing solution, your best bet especially if you want to save the remaining samples for use later, something like the Tri Reagent or TriZol(which will lyse your cells and stablize the RNA). If I'm not mistaken, you can freeze your samples in the Tri Reagent. If not, I definately know you can freeze samples in RNAzol.

I'll see if we can buy one of those. And double check if one can freeze samples in Trizol...

Thanks A LOT!!!