非表示:
キーワード:
-
要旨:
Background: Studies of protein association with DNA on a genome wide
scale are possible through methods like ChIP-Chip or ChIP-Seq. Massive
problems with false positive signals in our own experiments motivated us
to revise the standard ChIP-Chip protocol. Analysis of chromosome wide
binding of the alternative sigma factor sigma(32) in Escherichia coli
with this new protocol resulted in detection of only a subset of binding
sites found in a previous study by Wade and colleagues. We suggested
that the remainder of binding sites detected in the previous study are
likely to be false positives. In a recent article the Wade group claimed
that our conclusion is wrong and that the disputed sites are genuine
sigma(32) binding sites. They further claimed that the non-detection of
these sites in our study was due to low data quality.
Results/discussion: We respond to the criticism of Wade and colleagues
and discuss some general questions of ChIP-based studies. We outline why
the quality of our data is sufficient to derive meaningful results.
Specific points are: (i) the modifications we introduced into the
standard ChIP-Chip protocol do not necessarily result in a low dynamic
range, (ii) correlation between ChIP-Chip replicates should not be
calculated based on the whole data set as done in transcript analysis,
(iii) control experiments are essential for identifying false positives.
Suggestions are made how ChIP-based methods could be further optimized
and which alternative approaches can be used to strengthen conclusions.
Conclusion: We appreciate the ongoing discussion about the ChIP-Chip
method and hope that it helps other scientist to analyze and interpret
their results. The modifications we introduced into the ChIP-Chip
protocol are a first step towards reducing false positive signals but
there is certainly potential for further optimization. The discussion
about the sigma(32) binding sites in question highlights the need for
alternative approaches and further investigation of appropriate methods
for verification.
