differential display

Helge Weissig helgew at LJCRF.EDU
Sat Nov 6 14:14:35 EST 1993


>To all netters
>
>We have begun to investigate changes in mRNA populations between
>tissues, experimental conditions, etc. by using the "differential
>display" protocol of Liang & Pardee Science, 1992, vol 257, p967; Liang
>et al NAR 1993, vol 21, p3269). Although the method appears quite
>elegant and the authors clain success we have begun to have our doubts.
>Specifically we are wondering whether the "anchored" oligo d(T)
>primers used in the initial reverse transcription really prime from the
>predicted 5' end of the polyA tail of the appropriate set of mRNAs in
>total RNA or are they annealing more non-specifically? The authors
>appear not to have performed the obvious experiment of demonstrating
>reverse transcription of a given mRNA using the appropriate "anchored"
>primer predicted from the corresponding known cDNA sequence.
>Common "house keeping " genes would be appropriate. This could be done
>for each of the "anchored" oligo d(T) primers. Without this data it is
>possible that these "anchored" RT primers are annealing differentially
>BUT non-specifically.
>
>Has anyone done this experiment? Does anyone using this technique
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>have any comments?
>
>Colin Dolphin
>Biochemistry
>Queen Mary & Westfield College
>London, UK
>c.t.dolphin at qmw.ac.uk


not exactly that one, although my results seem to show that the 3'primer do
not prime specifically:
        I set up the PCR rxn after RT w/ the four different, partially
degenerated 3'primers and using a decamer as 5'primer. The decamer was
chosen from a known sequence of a gene that is expressed in the cells i am
looking at. The result was a very similar, if not identical band pattern
for all four 3'primers. Unfortunately i could not identify my
"control-product" (a band of expected size in only one of the 4 different
rxn sets).
This raises the question where the unspecific priming occured. I guess it
must have been in the RT rxn, or alternatively during the PCR given that
TaqPol accepts residual mRNA (did not RNase treat or extract my RT rxns) as
template (does it?). I'll try to modify the rxn conditions (96, 42, 72, no
incubation times, ramp times about 30-45 sec., done on a PE-C model [fairly
old], dNTPs were @ 0.1mM [quite high], MgCl2 @ 2mM, 0.1 ug mRNA in the RT
rxn.) to see if that gives differential display for the four different
3'primers as well as for my different cell types.

any comments appriciated.

helge



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