subtractive hybridization or differential display ?
bjanssen at bio.tamu.edu
Thu Sep 7 11:10:37 EST 1995
In article <42mg23$cki at fu-berlin.de>, bongr at mail.grumed.fu-berlin.de wrote:
> I' m looking for a method for identification of differentially
> expressed genes. Subtractive hybridization, differential
> display, RAP-PCR ... Which one is the "best" (feasibility, strengths
> and weacknesses)? Could you advise me a good working
> commercial kit? Remarks and advice are welcome.
> Thank you in advance for your help.
We've used both differential screening and differential display to look
for genes expressed during fruit development. Differential screening
works but is slow and tedious, the major problem we've had with
differential screening is that about half to two thirds of the putative
specific cDNAs turn out to be non-specific on northern analysis, which is
a lot of work for a "useless" gene. Because of the limitations in
sensitivity using labelled cDNA probes differential screening tends to
identify abudant cDNAs, this is not always a bad thing.
Differential display seems to give a higher frequency of specific genes
when you do the final analysis on northerns. Differential display (as far
as I know) seems to be able to identify rare specific messages as well.
The down side of differential display is the hassle of then going back to
a cDNA library to get a full length clone (but sometimes you need to do
that with differential screening as well). I'm afraid I'm not sure I'd
call one method better than the other, they are both good and result in
isolation of specific genes. If you want an abudant specific gene I'd
probably go for differential screening, if you're looking for a rare
message (eg a TF) then use differential display.
As for kits, we've used lambda Zap (stratagene, M13 based excision) and
superscript (Gibco BRL, cre-lox based excision) to get good libraries (RNA
quality is the critical factor). Definately make a phage library,
screening colony libraries is a pain in the butt (make sure the bacterial
strain gives you good plaque morphology, ie not SURE cells) and phage
libraries are more stable in our hands anyway.
All these comments are based on my experience and reflect my abilities or
lack thereof, there will be people who like other systems/methods better.
The other problem is that I did this stuff a year ago so my experience is
probably out of date :). Ask around and use whatever works best for you.
It's also a lot easier if there is someone nearby who can physically show
you rather than just tell you.
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