2008 Barcelona - European Society of Human Genetics

Genomics, technology, bioinformatics 0
clinical samples, where the amount of material is limited and analysis
time is an important factor .
P08.49
combined analysis of altered promoter methylation and gene
expression changes in non-small cell lung cancer
N. Tõnisson 1,2,3 , K. Fellenberg 4 , R. Kuner 4 , M. Meister 5 , T. Vooder 2,1 , K. Välk 1 ,
K. Kirotar 1 , A. Metspalu 1,3 , J. D. Hoheisel 6 ;
1 University of Tartu, Tartu, Estonia, 2 Tartu University Hospital, Tartu, Estonia,
3 Estonian Biocentre, Tartu, Estonia, 4 German Cancer Research Center (DKFZ),
Heidelberg, Germany, 5 Thorax Clinic, Heidelberg, Germany, 6 University of TartuGerman
Cancer Research Center (DKFZ), Heidelberg, Germany.
Combined analysis of promoter methylation and gene expression
helps to reveal causative links between the epigenotype and phenotype
of malignant tumours . Methylation changes in cancer are frequent
and relatively stable, therefore they’re also attractive biomarkers for
tumour profiling, as well as potentially for monitoring the efficiency of
anticancer therapy .
We have performed an analysis of 48 genes in 60 surgically treated
lung adeno- and squamous cell carcinoma patients, using in situ synthesized
oligonucleotide microarrays . The samples were analysed for
promoter methylation, as well as the corresponding gene expression
changes . The analysis included known and putative tumour suppressor
genes, genes controlling cell growth and differentiation, antiangiogenetic
factors and genes participating in cell to cell and cell to extracellular
matrix connections . A panel of tumour-free lung tissue was
used as the negative control . The dual level data was analysed with
M-CHIPS platform and showed clear differences between cancer and
cancer-free tissue .
A similar study with more complex setup is underway in a different
clinical center .
P08.50
the new mLPA based approach for HLA-DQA1, -DQB1 typing
E. Bliznetz, N. Galeeva, A. Polyakov;
Research Centre for Medical Genetics, Moscow, Russian Federation.
The genetic complexity of the human major histocompatibility complex
(MHC) has required the using of various molecular typing methods to
identify specific alleles. Sequence specific primers (SSP), sequence
specific oligonucleotide probes (SSOP) and sequence-based typing
(SBT) are a few of the methods that have been utilized in the HLA
community at present . We have designed Multiplex ligation-dependent
probe amplification (MLPA) method based system for HLA-DQA1 and
-DQB1 loci typing . Such MLPA advantages by comparison against others
molecular method as cheapness and simplicity of the technique
and a possibility to determine chromosome haplotype are governing
for HLA typing method . Our MLPA based system reveals 8 DQA1 alleles
in one tube and 19 DQB1 alleles in one tube . A working procedure
is consisting of three steps; the site-specific oligonucleotide
probes ligation, multiplex amplification with common primers pair and
PCR fragments assessment . For comparison, our earlier SSP based
system reveals the same alleles; the working procedure includes 2-4
PCR/Multiplex PCR during HLA-DQA1 typing and 5-8 PCR/Multiplex
PCR during HLA-DQB1 typing per person . In our option MLPA method
is excellent additional molecular HLA typing method .
P08.51
NAsBA product labeling for following microarray experiments
O. Scheler 1 , B. Glynn 2 , S. Parkel 1,3 , M. Maher 2 , A. Kurg 1,3 ;
1 University of Tartu, Institute of Molecular and Cell Biology, Department of
Biotechnology, Tartu, Estonia, 2 National University of Ireland, The National
Diagnostics Centre, DNA Diagnostics Laboratory, Galway, Ireland, 3 Estonian
Biocentre, Tartu, Estonia.
DNA microarrays that enable quick and simultaneous investigation of
many different biological targets are steadily becoming everyday tools
in genetics and molecular diagnostics . Environmental and medical
samples can easily be identified and investigated by specific probe
microarrays that are complementary to the suitable marker sequences
in analyzed solution . Biological markers of interest are usually amplified
and labeled prior to the microarray hybridization experiment.
RNA targets can efficiently and exclusively be amplified using nucleic
acid sequence based amplification (NASBA), even in the presence of
genomic DNA . NASBA is an isothermal method that has previously
been applied for the detection of different pathogens and mRNAs .
Several labeling protocols for many amplification methods have been
described in literature previously, but none of them are for NASBA
amplification products. Commercial NASBA amplification method was
modified to meet the needs of microarray experiment. Extra aminoallyl-UTPs
were added to the NASBA solution in order to add fluorescent
marker Cy3 to the aminoallyl modified RNA products after the reaction.
Two different commercial aaUTP reagents (aaUTP-Na and aaUTP-Li
salts) were investigated over wide concentration range (0,125mM to
8mM) . Strongest microarray signal intensities were achived with 2mM
final concentration of aaUTP-Li salt used in NASBA reaction. Modified
NASBA method enabled accurately detect pathogens from solution
containing total RNA from as low as 0,1-1 CFU .
P08.52
Identification of mir-21 (MIRN21) targets in breast cancer cells
by gene expression profiling
D. Selcuklu1,2 , M. Donoghue1 , C. Spillane1 , C. Yakicier3 , E. Erson2 ;
1 2 University College Cork, Cork, Ireland, Middle East Technical University, Ankara,
Turkey, 3Bilkent University, Ankara, Turkey.
Increasing numbers of microRNAs have been shown to target cancer
related genes . Our aim was to identify mir-21 targets which are regulated
through mRNA cleavage, deadenylation, or other processes that
can lead to mRNA up- or down-regulation . Our experimental approach
involved silencing of endogenous mir-21 expression by 2’-Ome-inhibitors
(anti-mir-21) in MCF7 cells . Total RNAs from anti-mir-21 transfected,
control oligo transfected and untreated cells were further
processed and hybridized to Affymetrix Human Genome U133 Plus
2 .0 arrays . Statistical and clustering analyses were conducted using
Bioconductor to identify the effects of mir-21 silencing on the transcriptome
. Using p1 .5 as cut-off values, 134 transcripts
were differentially expressed only in anti-21 treated samples .
The transcriptome responsive to mir-21 silencing was analysed further
by GO (gene ontology) based clustering of functionally related genes
and biochemical pathway analysis (GO, KEGG) to gain better understanding
of mir-21 function in cancer pathways . Over 25 % of the mir-
21 sensitive genes were involved in regulation of transcription, 9 .62 %
in apoptosis and regulation of programmed cell death, while the rest
were involved in a range of other biological processes . Amongst these,
some genes were linked to apoptosis by insulin signalling, cell cycle
regulation by TGF-BETA and MAPK signalling pathways suggesting a
potential involvement of mir-21 in regulatory control of these cancerrelated
pathways. Further studies are underway for verification candidate
targets identified. These studies will facilitate the identification of
new cancer biomarkers regulated by microRNAs that could be used for
better diagnostic and therapeutics for breast cancer .
P08.53
A Pipeline for Designing custom taqman® Assays for small
RNA Genes
L. Wong, Y. Wang, Y. Liang, D. Ridzon, C. Chen;
Applied Biosystems, Inc., Foster City, CA, United States.
MicroRNAs (miRNAs) are a new class of non-coding RNAs that mediate
post-transcriptional gene silencing . A growing number of novel
miRNAs and other small RNA genes are being discovered and there
is a significant need for custom assays to determine the level of their
expression . An automated bioinformatics pipeline has been developed
to design TaqMan® MicroRNA Assays to allow quantitation of miRNA
expression by real-time PCR . To date, over 2,000 assays have been
designed using the pipeline for miRNAs listed in miRBase (release
10 .0) . The pipeline has been wet-lab validated for mammalian miRNAs
showing assay performance success of greater than 90% based on assay
linearity and no template control (NTC) background signal . As research
interest in small non-coding RNAs is rapidly expanding beyond
miRNAs, the capability of the pipeline to design assays for other small
RNAs including small interfering RNAs (siRNAs), short hairpin RNAs
(shRNAs), and PIWI-interacting RNAs (piRNAs) is extremely valuable .
Assays for over 100 endogenous siRNAs have been designed using
the same design pipeline as for miRNAs . Preliminary data shows
similar performance as miRNA assays with minimal background in the
presence of tissues and cell lines for these siRNA quantitation assays .
In conclusion, we have developed a robust pipeline to successfully
design TaqMan miRNA assays . Recent data demonstrates the ability