RNA extraction - Which is which? (May/07/2009 )
Hi all,
I have extracted RNA from leaves and I'm aware that there would
usually be extra bands apart from 28S and 16S. Anyway the question is
which ones are the 28s and 16S bands. Also what are the extra bands?
Thanks! ( PS:I have attached my gel from the three samples extracted showing 5 bands each).
http://www.ambion.com/techlib/append/supp/rna_gel.html
Moondust on May 7 2009, 07:13 PM said:
I have extracted RNA from leaves and I'm aware that there would
usually be extra bands apart from 28S and 16S. Anyway the question is
which ones are the 28s and 16S bands. Also what are the extra bands?
Thanks! ( PS:I have attached my gel from the three samples extracted showing 5 bands each).
It wouldn't be bad if you'd used RNase inhibitor. It would minimize the risk of RNA degradation.
I don't think that your samples are degraded. For me they look fine.
The best way to find out which bands are the 28S and 16S woud be to use a RNA ladder.
Hi!
I've got a similar problem. I get from purified E. coli a 16S band (1500nt), a 23S (3000nt) band, and above that, a band that is approximately 4000-6000nt. I cannot figure out what that extra one is. I've got a RNA dye, that shows only RNA (I've tested this with a DNA ladder, which doesn't show) so the extra band must be RNA. Otherwise the bands are quite sharp and I wouldn't think a degraded RNA would show that high and that sharp a band. Does anybody know what it might be? Cheers! Emilia.
Emilia,
If your RNA is not degraded but you get a clear band, it could be that your samples are contaminated with eukaryotic RNA. They have a 28S band. I have had that problem with fungal contamination of cultures.
cheers
Emilia Viitanen on May 13 2009, 04:44 PM said:
I've got a similar problem. I get from purified E. coli a 16S band (1500nt), a 23S (3000nt) band, and above that, a band that is approximately 4000-6000nt. I cannot figure out what that extra one is. I've got a RNA dye, that shows only RNA (I've tested this with a DNA ladder, which doesn't show) so the extra band must be RNA. Otherwise the bands are quite sharp and I wouldn't think a degraded RNA would show that high and that sharp a band. Does anybody know what it might be? Cheers! Emilia.
Hi! And thanks! 
But I'm not sure if it is contamination, cos wouldn't I then see a 18S band too? I've included my gel picture here. There's E. coli, empty lane, E. coli, RNA ladder (from bottom to up it goes, 1000nt, 1500nt, 2000nt, 3000nt, 4000nt and 6000nt), E. coli, empty lane, E. coli, and a fungus. The fungus has a 18S band app. 2000nt and a 28S band app. 4000nt. I'm really baffled about the third band in E. coli. And cos it is a RNA dye, it cannot be DNA... Weird! But thanks for the suggestion, I'll see if it is fungus after all. Cheers, Emilia.
Emilia,
If your RNA is not degraded but you get a clear band, it could be that your samples are contaminated with eukaryotic RNA. They have a 28S band. I have had that problem with fungal contamination of cultures.
cheers