isolation of small RNAs from culture supernatant - (Apr/03/2007 )
I'm still a new-ish grad student and have a question about micro/si RNA isolation. Basically i want to see if there are any small RNAs floating around in my culture supernatant. Is there an 'easy' way to do this? I'm using a lot of starting material, anywhere from 15-300 mL (yup, milliliters) of material. I'm concerned about using less than this amount as the stuff (protein, RNA) in the supe itself is pretty dilute, and if I haven't figured out a way to concentrate it without losing the small RNAs as well.
Are there cells in culture supernatant? If not, it would be challenging
I'm not sure I understand - you want to see if there is any secreted RNA in the supernatant? My best guess is to see if it is possible to do an ethanol precipitation and chloroform: phenol wash, try and purify for RNA and concentrate. Not sure if all the other reagents in the culture medium will have an adverse effect on the precipitation step though...maybe you can try culturing cells in less medium to start with?
Am I even close to what you want to do?
It is very interesting, perhaps cells can comunicate each other by sending miRNAs.
well, so yes there are cells in the culture supernatant (at least that's what we hope).
basically, we have a wild hypothesis (i use that term in a very loose sense) that the intracellular organism we're studying can secrete si or micro RNAs to affect its host cell. we can cultivate the parasite in vitro (no host cells) and were hoping to pull out any RNA floating in the supe.
unfortunately, the amount of media for the culture is a set amount (the organisms don't grow in less culture media). i know that doing a mass percipitaiton of nucleic acid in the media will not rule out things like dying parasites lysing and releasing their cytoplasmic contents. i just want to see if there is ANY rna there.
i'll definitely try the ethanol percip (thanks miRNA man) and see how it goes. Thanks for the input and if anyone out there has any other suggestions they are greatly appreciated.
one more q: is urea-PAGE the best way to resolve 20-100nt oligos? and if so, how do i differentiate a specific oligo from degraded junk...would i just see a distinct band in a background of fuzz?
If you hope to investigate possible miRNAs released by cells to supernatant, you must get ride of any cells in supernatant before RNA purification.
For detection of small RNA, denatured PAGE is best way to resolve small RNAs. In detection of RNAs purified from culture cells, the small size rRNA or tRNA might show distinct bands, while small RNA in size of ~ 20nt is hardly visualized as specific band by EB staining. As to your expected RNA from supernatant, it is not sure... For differentiated specific RNA oligo, you’d better using northern hybridization or RT-PCR.
as water said, you have to get rid of supernatant cells, any DNA fragments, and proteins if it is possible
you can apply SDS-PAGE
i think of something : can't you use complementary RNA or DNA fragments ligated with a marker (reporter)
Hi guys, thanks for all your help. unfortunately this is a brand new direction for my lab (so none of us really know what we're doing) and I really really appreciate any advice i get.
so here's an update
i ran out isolate RNA from both the supe and cell pellet on a 1.5% agarose gel:
-rna from the pellet had the usual mRNA/rRNA bands plus a fuzzy band below 100bp.
-rna from the supe gave me NO large fragments (so no mRNA/rRNA stuff) but a distinct band around 200bp and a fuzzy band less than about 100-200bp.
i then ran these samples out on a 9% urea-page gel stained with EtBr
-so the pellet: the small fuzzy band here resolved to a distinct around 80bp (using a DNA marker...i'm waiting for the RNA marker)
-finally the supe: nothing there. not even fuzziness.
? should i use a higher % PAGE gel? use a different DNA detection method (sybr green etc)? I'm currently planning to re-extract rna from the supe in case it degraded, as well as extract more than 600uL of supe in case there wasn't enough to be seen on the PAGE gel.
ps. the complementary DNA/RNA + report stuff sounds great (but we don't konw the sequence of what we're looking for) ... how would that work, does that mean i would have to translate whatever fuzz is there to cDNA and clone it out/add reporter element ... ?
i mean something like what is done with microarrays, use labeled fluorescent probes which would hybridize with their complements and can be distinguished by colors...but as u said the problem is that u don't know the sequence of what u're looking for
Usually, people using 12% PAGE/Urea for seperate small size RNAs, but you could not expect to see miRNA bands. For confirmation, you'd better use northern blot or RT-PCR to detect the existance of well know or abundant miRNA, or think about cloning approach.
Any way, your idea that miRNA might function as signal between cells is very attractive..