PowerPointPrsentation

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Search for novel non-coding RNAs in prostate
carcinoma cells
Christine Schulz AG RNomics
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Non-coding RNAs (ncRNAs)
classical ncRNAs tRNA rRNA
regulatory ncRNAs miRNA (microRNA)
siRNA (small interfering
RNA) snoRNA
(short nuclear RNA)
snRNA (small nucleolar RNA)
piRNA (piwi-associated
RNA) long ncRNA roles
in regulation of gene expression
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Prostate Cancer

• 2nd leading cause of cancer death in men

Very poor prognosis for metastatic
androgen-independent form No accurate diagnostic
markers to distinguish different stages
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Identification of novel differentially expressed
ncRNAs in prostate cancer
Drug targets
Diagnostic markers (differentiation between
androgen-dependent and independent form)
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Identification of novel ncRNAs using Tiling Arrays
Architecture of Human GeneChip Tiling Arrays
(chr 21 and 22)
Protein-coding gene
Exonic region
Intergenic region
Intronic region
Oligonucleotide coverage (25-mer probes)

• 25-mer oligonucleotide probes cover exonic,
  intronic and intergenic regions of chromosomes 21
  and 22 (repeats are removed)

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Cellular models for prostate cancer
LNCaP
PC3
DU-145
RWPE-1
Tiling array
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Experimental Approach
1. Tiling Array (LNCaP vs PC3)
Scan
Cell culture, RNA isolation
ds cDNA synthesis
Fragmentation and labeling
Hybridisation
2. Bioinformatical evaluation

• differential expression
• BLAST (RefSeqmRNA, Noncode, RNAdb) and ORF search

3. Verification of tiling array signals by RT-PCR

• Random primed and strand-specific reverse
  transcription PCR (RT-PCR)

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Results overview
Differentially expressed regions (tiling array
evaluation) 18
Primer design not possible (repetetive sequences,
no unique sequences)
Regions for experimental verification by RT-PCR
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Intronic 3
Intergenic 5
Overlapping intron-exon boundaries 2
Shown examples
2
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Intronic transcripts Candidate 1 (chr 21)
RNAz
mRNA-BLAST mRNAs (95 1613-1921 and
4573-4887) ncRNA-BLAST human scAlu RNA
(90 1619-1732 and 80 4768-4875) ORF 237 nt
Evofold
PC3
LNCaP
Cell adhesion molecule
RefSeq ()
RefSeq (-)
6701 nt
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Intronic transcripts Candidate 2 (chr 21)
RNAz
ORF 150 nt
Evofold
PC3
LNCaP
esterase
RefSeq ()
RefSeq (-)
550 nt
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Summary/Future Prospects
Transcripts for experimental verification by
RT-PCR 10
Differential expression at expected genomic
locus 2 (intronic)
Identity with transcripts of other genomic loci
or mRNAs 4
Further investigation necessary 3
Not detectable 1

• RACE-PCR
• Detection in other tissues

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Acknowledgements
Group Leaders Dr. Antje K. Kretzschmar Dr. Jörg
Hackermüller
Senior Scientific Advisors Prof. Dr. Friedemann
Horn Prof. Dr. Peter Stadler
Group Members Sebastian Schulz Kerstin
Ullmann Stephan Schreiber Katharina
Schutt Richard Schlegel Dr. Kristin Reiche