Nucleic Acids Research, Vol 26, Issue 17 3883-3891, Copyright © 1998 by Oxford University Press
T Trenkle, J Welsh, B Jung, F Mathieu-Daude and M McClelland
A method is presented in which the reduced complexity and non-
stoichiometric amplification intrinsic to RNA arbitrarily primed PCR
fingerprinting (RAP-PCR) is used to advantage to generate probes for
differential screening of cDNA arrays. RAP-PCR fingerprints were converted
to probes for human cDNA clones arrayed as Escherichia coli colonies on
nylon membranes. Each array contained 18 432 cDNA clones from the IMAGE
consortium. Hybridization to approximately 1000 cDNA clones was detected
using each RAP-PCR probe. Different RAP-PCR fingerprints gave hybridization
patterns having very little overlap (<3%) with each other or with
hybridization patterns from total cDNA probes. Consequently, repeated
application of RAP-PCR probes allows a greater fraction of the message
population to be screened on this type of array than can be achieved with a
radiolabeled total cDNA probe. This method was applied to RNA from HaCaT
keratinocytes treated with epidermal growth factor. Two RAP-PCR probes
detected hybridization to 2000 clones, from which 22 candidate
differentially expressed genes were observed. Differential expression was
tested for 15 of these clones using RT-PCR and 13 were confirmed. The use
of this cDNA array to analyze RAP-PCR fingerprints allowed for an increase
in detection of 10-20-fold over the conventional denaturing polyacrylamide
gel approach to RAP-PCR or differential display. Throughput is vastly
improved by the reduction in cloning and sequencing afforded by the use of
arrays. Also, repeated cloning and sequencing of the same gene or of genes
already known to be regulated in the system of interest is minimized. The
procedure we describe uses inexpensive arrays of plasmid clones spotted as
E.coli colonies to detect differential expression, but these reduced
complexity probes should also prove useful on arrays of PCR- amplified
fragments and on oligonucleotide chips. Genesobserved in this manuscript:
H11520, U35048, R48633, H28735, M13918, H12999, H05639, X79781, M31627,
H23972, AB000712, R75916, U66894, AF067817.
ARTICLES
Non-stoichiometric reduced complexity probes for cDNA arrays
Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121, USA.
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