Phillip E. Schwartz
schwartz at bioreagents.com
Thu Oct 23 13:28:51 EST 1997
Harold E Smith wrote:
> I would like to probe a blot of cDNA clones with mRNA isolated from two
> different tissues to identify clones that are differentially expressed.
> I'm fairly certain it's been done before, but I can't find a reference.
> Would someone be so kind as to point me in the right direction?
> Thanks in advance,
> Harold Smith
I agree with Andy Phillips' suggestions of performing either subtractive
hybridization to generate your probe or do the mRNA differential
display. I have used magnetic beads from Dynal (1-800-638-9416; they
have an excellent technical manual and an extensive bibliography) to do
2-4 rounds of subtraction for this and the technique worked beautifully.
I isolated over 200 novel cDNA clones which were differentially
expressed between fetal and adult rat vascular smooth muscle cells. One
thing with differential display that you should be aware of: If the
species you work with is highly inbred (eg. mice, rats, etc.) you will
isolate and analyze numerous cDNA clones which are not differentially
regulated but contain repetitive or transposable element DNA (LINE
elements in rats is one example) in the 5' and/or 3' untranslated
regions. On several attempts at using this approach on rat VSMC mRNA
probably 25-30% of the clones contained repetitive element homologies.
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