Reverse Northern

Paul N Hengen pnh at ncifcrf.gov
Thu Sep 26 11:54:46 EST 1996


Gene Huh (gshuh at garnet.berkeley.edu) wrote:

| 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? 

Gene, I found the reference to this technique. Thanks.
It was in the 20 October 1995 special Genome issue of Science...

--

TITLE [TI]
    Quantitative monitoring of gene expression patterns with a
    complementary DNA microarray

AUTHOR [AU]
    Schena M; Shalon D; Davis RW; Brown PO

ADDRESS [AD]
    Department of Biochemistry, Beckman Center, Stanford University Medical
    Center, CA 94305, USA.

JOURNAL [JL]
    Science 270: 467-70 (1995)

ABSTRACT [AB]
    A high-capacity system was developed to monitor the expression of many
    genes in parallel. Microarrays prepared by high-speed robotic printing
    of complementary DNAs on glass were used for quantitative expression
    measurements of the corresponding genes. Because of the small format and
    high density of the arrays, hybridization volumes of 2 microliters
    could be used that enabled detection of rare transcripts in probe
    mixtures derived from 2 micrograms of total cellular messenger RNA.
    Differential expression measurements of 45 Arabidopsis genes were made
    by means of simultaneous, two-color fluorescence hybridization.

--

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

| 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?  

I would argue that the extra step of T7 transcription would alter the bias even
more than PCR because of readthrough. Anyone know the percentage of "longer
than normal" transcripts from T7 promoter driven probe RNA synthesis in vitro?
I could see that any cDNA on the array with some homology to the vector
sequence beyond the cloned fragment might cause a false high expression signal
because the probe would stick there.

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

I've also heard that DNA-RNA hybrids are structurally more stable than
DNA-DNA, but no one has given me a reasonable explanation for this.
Please enlighten me :-)

-Paul.

--
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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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