Tweet
Hi,
I am planing my Illumina RNAseq experiments and since my samples are hard to come by and yield only less than 100 ng total RNA, I need to choose sample preparation steps with care.
The best candidates so far are the NuGEN Ovation RNA-Seq or the 3'-DGE (I haven't decided yet, whether I want to do more than just DGE). Yet, these kits are pretty expensive and it would be great to figure out a cheaper way. As far as I understood, one big advantage of the Ovation RNA-Seq kit is that you don't need to perform any fragmentation steps, because the amplification of your mRNA yields only short amplicons of ~200 bp on average.
Another option might be the Ovation Pico WTA System, yet this is not intended for RNAseq.
Does anybody know, if there is a way to adjust the WTA System to get it working with Illumina RNAseq? What makes the WTA System not applicable for RNAseq?
Are there any other (hopefully cheaper) kits I could use for the linear amplification & the library prep?
So far, I found three relevant papers going in a right direction:
#1 Sengupta et al 2010- the protocol presented here requires gel runs for size selection steps, which I don't really fancy doing.
#2 Tariq et al 2011
#3 Lott et al 2011
The first and the third paper describe more or less handmade workflows that are not based on single kits. This might in fact be much cheaper, but I expect that it would require quite some time to get the protocols running in my lab. This might be worth a shot, though.
I am planing my Illumina RNAseq experiments and since my samples are hard to come by and yield only less than 100 ng total RNA, I need to choose sample preparation steps with care.
The best candidates so far are the NuGEN Ovation RNA-Seq or the 3'-DGE (I haven't decided yet, whether I want to do more than just DGE). Yet, these kits are pretty expensive and it would be great to figure out a cheaper way. As far as I understood, one big advantage of the Ovation RNA-Seq kit is that you don't need to perform any fragmentation steps, because the amplification of your mRNA yields only short amplicons of ~200 bp on average.
Another option might be the Ovation Pico WTA System, yet this is not intended for RNAseq.
Does anybody know, if there is a way to adjust the WTA System to get it working with Illumina RNAseq? What makes the WTA System not applicable for RNAseq?
Are there any other (hopefully cheaper) kits I could use for the linear amplification & the library prep?
So far, I found three relevant papers going in a right direction:
#1 Sengupta et al 2010- the protocol presented here requires gel runs for size selection steps, which I don't really fancy doing.
#2 Tariq et al 2011
#3 Lott et al 2011
The first and the third paper describe more or less handmade workflows that are not based on single kits. This might in fact be much cheaper, but I expect that it would require quite some time to get the protocols running in my lab. This might be worth a shot, though.