2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
P11.115
Expression of genes involved in self-renewal or lineage priming
in mouse embryonic stem cells
J. Au-Young, S. Dadi, D. Keys, K. Y. Lee, J. Sherlock, C. Chen;
Applied Biosystems, Foster City, CA, United States.
Profiling gene expression in embryonic stem cells (ESCs) requires
technology that allows the use of very small samples, yet has the capability
to analyze the expression of many mRNAs simultaneously.
A three step workflow including reverse transcription, multiplex preamplification,
and singleplex PCR in TaqMan® Arrays was used. We
demonstrate that multiplex preamplification-based TaqMan® real-time
PCR can be used to profile gene expression in limited starting material
or single cells. A total of 384 genes which are believed to be
functionally associated with maintenance of the undifferentiated embryonic
stem cell state were studied in mouse embryonic stem cells
(mESCs), differentiated embryoid bodies (mEBs), and embryos. Three
endogenous control genes, Ctnnb1, Actb and Gapd were used to confirm
the uniformity of the PreAmp reaction and to normalize RNA input.
Preamplification of starting cDNA molecules enables detection of
low expressers from nanogram amounts of total RNA input and even
single embryonic stem cells. A novel subset of differentially expressed
mRNAs was established, which can be used as biomarkers to distinguish
the pluripotent state from cells that have undergone differentiation.
Preamplification makes it possible to profile hundreds of genes
from limited starting material, including single cells, reproducibly and
without amplification bias.
P11.116
Exploiting the Full Potential Of sequence trace Files For
structural Variation
O. Lancaster, G. A. Thorisson, A. J. Brookes;
University of Leicester, Leicester, United Kingdom.
Structural variation and copy number variation (CNV) are recently recognized
to be extensive in the human genome. Examples identified so
far typically range in size from 5-200kb and encompass many genes
which play fundamental roles in both disease and evolution. The impact
of structural variation on genome function and disease is likely to
be substantial.
Currently known structural variants have been identified by a range of
technologies - both experimentally and in silico. However, these approaches
are very inefficient at detecting rarer, shorter (