Capturing >60nt fragments

[smarajit]

I am making a library for which it is essential to purify any RNA which is greater than 60-70nt and get rid of anything below that. Is there any column or bead based system that can purify all fragment above this cut-off without adding any size bias?

[kwaraska]

Could you make the library and then size select out anything too small rather than trying to size select the RNA?

[smarajit]

Actually, I am planning to do it after linker ligation. I can definitely select out anything smaller than that but I could not find any system to do that. Most systems purify RNA >200nt. I could use Millipore's cut-off filters but haven't heard if anybody used them before for this purpose

[kwaraska]

After linker ligation, what size are they? The TruSeq adapters add 120 so you would be looking at a reasonable size to select under.

Otherwise, why not go all the way to RT and such? Size select just before final PCR?

[rnaeye]

why can't you use gel purification?

[smarajit]

Gel purification will be difficult in my case because I'm not looking for a particular size.

I've been researching about this and contacted several companies, here is what I found. Clontech has a set of gel filtration columns called "CHROMASPIN" columns. They come in different cut-off sizes, like CHROMASPIN 10, 100, 200, 400, 1000 etc. These columns are RNase free and available in TE or DEPC water. Apart from these, the Qiagen RNeasy (MinElute kit) columns can also be used for size selecting RNA. These columns bind to everything >200nt according to standard protocol, however, smaller RNA can also be bound by increasing the volume of ethanol. The Agencourt RNA clean XP beads are good for high yield, but the bind to relatively larger RNAs. I'm sure there are other methods available like the Amicon's filtration devices, however, I found the CHROMASPIN columns the most useful if they work well