Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study.
BACKGROUND
Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology.


RESULTS
The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results.


CONCLUSIONS
This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance.
Cao
Wuxiong
W
Aventis Pharmaceuticals, Bridgewater, NJ 08887, USA. wuxiong.cao@aventis.com
Epstein
Charles
C
Liu
Hong
H
DeLoughery
Craig
C
Ge
Nanxiang
N
Lin
Jieyi
J
Diao
Rong
R
Cao
Hui
H
Long
Fan
F
Zhang
Xin
X
Chen
Yangde
Y
Wright
Paul S
PS
Busch
Steve
S
Wenck
Michelle
M
Wong
Karen
K
Saltzman
Alan G
AG
Tang
Zhihua
Z
Liu
Li
L
Zilberstein
Asher
A
D003713:Dendritic Cells / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012313:RNA