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Help: Small RNA (siRNA) isolation from cultured cells - (Jan/06/2008 )

A couple of questions:

1) I am working on processing of long dsRNA into siRNA in mammalian cells. After transfection, I need to isolate and purify the super-short siRNA from cell lysates. Any good protocols to recommend? or any kits?

2) How to separate (or purify) the 21nt 5'-32P labeled DNA oligos from the T4 PNK radio-labeling reaction mix (separate from the un-incorporated 32P-ATP)?

I don't have much experience on RNA work. I would greatly appreciate any suggestions or help!!!

-Pennmed-

for the first question :
trizol or trireagent are the most used liquid systems.
also a recent "technique" goes more and more used. Using a RNA extraction kit, people keep the flow through fraction of the loading step. It contains the small RNAs
directly pellet them with salts and PEG and do trizol procedure.


for your second question :
i use the nucleotide removal kit from quiagen.
Once i monitored the radioactivity in my elution and on the column, and I fond 2 elutions steps are the best regarding recovery.

-fred_33-

QUOTE (Pennmed @ Jan 6 2008, 10:00 PM)
A couple of questions:

1) I am working on processing of long dsRNA into siRNA in mammalian cells. After transfection, I need to isolate and purify the super-short siRNA from cell lysates. Any good protocols to recommend? or any kits?

2) How to separate (or purify) the 21nt 5'-32P labeled DNA oligos from the T4 PNK radio-labeling reaction mix (separate from the un-incorporated 32P-ATP)?

I don't have much experience on RNA work. I would greatly appreciate any suggestions or help!!!


With AquaRNA kit (www.aquaplasmid.com), you could first pellet gDNA/large RNA/protein in 40% isopropanol and then recover the supernatant, which contains small RNAs, and then add some more isopropanol to 60% final concentration to pellet the small RNA. This should work for getting small RNAs from other TRUE total RNA preps (column based total RNA kits usually do not recover small RNA) as well after some trial-and-error. You may get rid of 5.8s, 5s, and tRNA with polyacrylamide gel, but it would be hard to purify your siRNA from similar sized miRNA. At least you could run a northern to show you got your siRNA expressed and processed to the 21-22nt fragments in the cell.

[attachment=4075:Small_RNA.JPG]

-chessplayer-

Good evening. I have some questions regarding about TRIzol reagent. From the protocol they have stated that After homogenization and before addition of chloroform, samples can be stored at -60◦C to -70◦C for at least one month. Have anyone tried that before? The second question is can any other reagent replace Isopropyl alcohol during RNA precipitation?

Thank you guys!!

-darreng-

QUOTE (darreng @ Jan 28 2008, 02:58 AM)
Good evening. I have some questions regarding about TRIzol reagent. From the protocol they have stated that After homogenization and before addition of chloroform, samples can be stored at -60◦C to -70◦C for at least one month. Have anyone tried that before? The second question is can any other reagent replace Isopropyl alcohol during RNA precipitation?

Thank you guys!!


You could use 3 vol of ethanol instead of 1 vol of isopropanol to precipitate the RNA.

-chessplayer-

i would prefer the butanol rather ethanol as ethanol needs salts to be more efficent and the use of salts during trizol procedure makes the washing step harder.
Butanol may be a longer process but recovery is better.

-fred_33-