Re: Flow RNA assays
Bill Hyun (hyun@dmc.ucsf.EDU)
Wed, 5 Apr 1995 11:12:58 -0700
>Hi kids!
>
>There have been a few postings asking about RNA recovery from
>flow sorted cells. This could be pretty embarrassing, but I
>have to ask, has anybody tried a method we published way back
>in the MolBiol dark ages of 1989 (Cytometry 10:199-204)?
>
>Our research priorities shifted very soon after we developed
>the technique so I don't have much more experience than what
>the manuscript describes, but at the time I thought it was
>pretty cool (cool=sensitive, linear, relevant). It's easy,
>seemed to work consistently and over a nice dynamic range. I
>was sure it would revolutionize the field (I often have that
>effect), but I haven't seen squat.
>
>Any experience? If the truth is too brutal you might send me
>a note directly, but I have always wondered.
>
>Thanks, Jack.Dunne@Syntex.com
Hey Daddy Jack,
With cunning recall, I remember the landmark Dunne paper describing a "sort-blot" technique where cells were sorted onto nitrocellulose for mRNA quantitation. We used this technique to confirm a more traditional approach of sorting lots of cells for a Northern analysis of heat shock protein mRNA in transformed CHO cells. Both approaches correlated quite well, with the Dunne sort-blot method requiring shorter sort times. The major problems I remember were handling the filter pieces, getting them to sit in the sort stream path and confining the sort-blot spot. Attempts to modify the procedure from autoradiography to a fluorescent probe we could measure on an image cytometer failed. If someone would like to try this promising, yet unrealized, technique I would be happy to rumage for old experimental notes.
-Bill
William Hyun, Director
Laboratory for Cell Analysis, Box 0808
University of California, San Francisco
San Francisco, CA 94143-0808
voice (415)476-2632
FAX (415)476-8218
email hyun@dmc.ucsf.edu
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
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