Small RNA extraction - iRNA (Apr/03/2006 )
Hi everyone. I have been trying to isolate smallRNA from a virus infected nicotiana for my RNA interference experiments. First i try with a protocol i was given and which consisted in 10 MLiCl and phenol extractions, then i tried with a kit but till now i couldn't extract a good amount of it. I am needing around 2 or 3 microg/microl. Does anyone know a good kit or way to get such amount? Thanks in advance...
If you want perform detection such as northern blot, I’d feel that Trizol is good and the total RNA can used for North-blot directly and no necessary to isolate small RNA.
If you prefer small size RNA, then you might precipitate isolated small RNA by ethanol first then dissolve by DEPC water to obtain such concentration.
There are many kits for small RNA isolation (might namely for miRNA isolation)
here is one of Qiagene’s for your reference:
i don't trust columns fr siRNA purif. I think tri reagent or trizol supplemented by glycogen in ethanol precip step is better.
Hi, thanks for the advice. Just another question. When you use trizol you extract the total RNA i think. But if i just want the small RNA then i need to make another purification. ethanol purification is enough? With column Method i can notget a good concentration of small RNA... or maybe just extracting the total RNA and the runing it in a acrylamida gel and cutting the band and purifiying it would be the bestway to get a good amount of it.?..
after trizol extract, you can apply this method to separate small to big rnas.
see this reference :
Profiling microRNA expression using sensitive cDNA probes and filter arrays, Mouldy Sioud and Øystein Røsok, BioTechniques 37:574-580 (October 2004)
Thank you very much...i will try again
The mirVana kit from Ambion is very reliable and flexible. It can be used for Total recovery, >200nt recovery and <200nt recovery. I've used it specifically for miRNAs and it is very robust and consistent. The columns are very effiecient. For example, you can also rescue small RNA fractions from organic aqeaous phases just by adding ethanol and running it through the column.
Hi again, i have been reading a little and i was sourprise when i read in one site that 10:1 phenol:chlorophorm yielded more sRNA than 1:1....Did anyone listen about it?
can you give the adress of it?
Sorry for the delay! the site is
(in the second paragraph after the one starting with the world NEW)hehehe
Did you hear about it before?