Primer extension analysis?
logan at msdos.montpellier.inra.fr
Fri Jun 24 10:58:31 EST 1994
I need advice - having spent ages trying to quantify message induction by
Northern hybridisation and discovering that it is only ~2 fold for a single
message type, I am now considering Primer extension analysis using a primer that
would identify 2 other closely related messages hoping that their combined
induction would be more significant and make studies on things that affect the
level of induction easier.
Questions are these:
1. How sensitive is the method (relative to Northerns)?
2. Can I separate out unincorporated primers by some other means than a gel
without introducing another possible source of errors.i.e. binding to something
like DEAE-cellulose paper, or TCA precipitation?
3. How reliable is quantitation using a primer that might be slightly
heterologous to the RNA of interest. i.e. I have a probe from one plant, but the
system is easier to induce and study in another closely related one! I realise
this is a problem for the Northern analysis too, but when examining the single
message type I have high stringency conditions that do not allow the detection
of the closely related ones in the same plant. Is it reasonable to quantify when
that would suggest that the level of homology for this message between the two
different plant types is very high?
4. How do you standardise (the equivalent of the "constitutive" probe) the RNA
in the different reactions, there is a lot more rRNA in leaves than roots?
If this all sounds confusing it probably is because I am feeling confused having
tried looking through various technique books. I would now like someone with
some experience to give me some straight answers! If you can help please e-mail
me at the address below, if there seem to be other confused people out there I
will post a summary.
Thanks in advance
Logan at montpellier.inra.fr
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