extraction of endogeneous mirna and sirna - (Jun/05/2005 )
Hello All: I am trying to find whether there is a difference in the types of sirnas/mirnas present in two types of cells. Has somebody got some experience or good suggestions to give?
for the extraction trizol or Tri reagent are best ones...
In order to find the differences, i was wondering is it possible to do a substractive hybridization?
Precipitation will bring down all RNAs, large and small. Since a large abundance of the RNA is not what you are looking for (tRNA and rRNA), you can do an enrichment of just small RNAs.
If you already have RNeasy kits in your lab, you can call QIAGEN TechService and they can tell you how to bind and elute the smaller sized RNAs.
If you have budget for another kit, Ambion does sell a kit for enrichment of small RNAs.
Hi Cali Girl,
Can you share with us what Qiagen says about using RNeasy kit for small RNA isolation. I know from literature that generally Trizol or Trireagent is used for RNA isolation for detection of microRNA. Not sure if column can bind small RNAs.
cali girl means probably using columns that bind large RNAs and do not retain small RNAs. I've used centricon colums in order to eliminate RNA more than 120ntd (when searching siRNA enrichment). after this step, you need to precipitate RNAs and resuspend in smaller volume.
i know the cut off for such colums differs and you surely may find the appropriate one for your experiments.
Hi, sorry, been busy...
Yes, normal protocols for any column-based method will cut off RNAs smaller than 100bp or 200bp, depending on composition of binding buffer.
RNeasy does have an alternate protocol for binding of small RNAs to column, but I think you have to call their techservice to get this.
Or, Ambion has "microRNA isolation kits"
Thanks to all of you for the suggestions. I rellay appreciate them. I have one more question. Could anyone please tell me an easy way to characterize the siRNA/miRNA if I find any in my cells?