Reverse Northern

Gene Huh gshuh at garnet.berkeley.edu
Thu Sep 26 02:31:57 EST 1996


In article <52924t$2up2 at ncisun1-nf0.ncifcrf.gov>, pnh at ncifcrf.gov (Paul N
Hengen) wrote:
> 
> I agree with you that the term "expression profiling" seems better in this
> context, but it is really a general term and could be mistaken for many
> things. Does anyone have the exact reference from J. Eberwein, or the one
                                                            ^^^
> mentioned that was published in Nucleic Acids Research? Does anyone know
> of a better primary reference for the method?

   So, after going home and getting the Neuron reference, I found to my
horror that  I had misspelled Eberwine.  My apologies to those searching
thru medline for the wrong name.  D'oh!  With thanks to Matthew Frosch,
who pointed this out.

   So, here's the Neuron reference I mentioned previously:

Mackler, S. A., et al.  1992.  Stimulus-induced coordinate changes in mRNA
abundance in single postsynaptic hippocampal CA1 neurons.  Neuron 9:
539-548.

   And as for the NAR reference:

Bernard, K., et al. 1996.  Multiplex messenger assay: simultaneous,
quantitative measurement of expression of many genes in the context of T
cell activation.  Nucleic Acids Res.  24: 1435-1442.

The latter reference employs colony spotting, rather than direct DNA dot
blotting.  The probe in this case was radiolabeled cDNA that was directly
reverse-transcribed from total RNA.  I have not encountered a definitive
primary reference, but it seems to me that the general concept has been
around for years and has been exploited by those who have carried out
differential cDNA library screening.  Wasn't the T-cell receptor cloned in
this way?

One other reference that you may already have on hand, Paul, is a Science
paper published not long ago from Patrick Brown's lab at Stanford.  I
guess one could call the technique in this paper a Micro-Reverse Northern
;););), as cDNA clones were spotted on a microarray chip and then probed
with fluorescently labeled cDNA.  The paper illustrated the utility of the
technique by identifying genes in Arabidopsis that were differentially
regulated (by auxin, perhaps, but I forget).  Anybody have the reference
handy? 

> Technically speaking, is there any advantage to making the RNA probe
> through T7 RNA polymerase first?
> 
> -Paul.

   I suspect that the point in making an RNA intermediate during probe
synthesis is that T7 RNA polymerase can transcribe a given template
multiple times in a single cycle of aRNA synthesis, thus providing a
degree of amplification not provided by a single cycle of, say, PCR. 
Perhaps this leads to less bias in the cDNA probe than if PCR were used?  

   In addition, perhaps an RNA probe may bind more tightly?  

Hope this helps
Gene Huh
gshuh at garnet.berkeley.edu



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