Making the TruSeq RNA-sample prep kit go further?

[James]

Hello all.

We've previously used the mRNA library prep kit, and used 1/4 volumes to make the kit go further and this was fine. What I was wondering was, has anyone done a similar thing with the new Truseq RNA kit, and if so what % of original volumes are you using? I'm not going to be doing the prep in a multiwell plate as we don't have the magnet (or enough samples to process at once), I was going to use a magnet that fits 1.5ml tubes.

Thanks. James

[deganuti]

Hi James,

could you tell me how much is the TruSeq RNA Sample Prep Kit?

Tks

[James]

£2,265.00 makes 48 libraries

[RayLangley]

Hi James,

I haven't tried expanding the TruSeq kits but I did do it with the older kits.

I would prefer keep the input the same. My suggestion is try 1/2 of the master mixes initially and doubling the incubation times for first strand, second strand, end repair, and a tailing. The major problem I see with this is that illumina is not providing extra MP-adapters or primers and they aren't for sell yet. You might be able to get away with using half the volumes for ligation and pcr, but if you do I would reduce your starting input by half as well. If you have really good starting input (RIN > 9) and your poly-a selection technique is good you should be okay.

You could also try talking with tech services about getting more of the primers and adapters. They were nice enough to provide me extra primers and PCR master mix for DSN. If they have enough people requesting they hopefully will start selling.

If you can afford it, I would highly recommend getting the dyna mag 96-well side (Catalog Number 123-31D) from invitrogen ($670). You can work with strip tubes and it is a lot easier to use with multichannel pipettors. The price is totally worth it for the amount of time you save. And I think you can easily run 24 samples at a time with a 12-channel pipettor. The magnet works really nicely with the agencourt beads.

However, for my last experiment (desperate times call for desperate measures) - I found a rare earth bar magnet around here that I tapped the inside of a pipette tip box. For eight samples it worked fine. I think you can find these magnets in old computer hard drives. But I am honestly not certain where they found the one I was using.

Good luck , I would love to hear how it all works.
Ray