Search for novel non-coding RNAs in prostate carcinoma cells Christine Schulz AG RNomics.

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Presentation transcript:

Search for novel non-coding RNAs in prostate carcinoma cells Christine Schulz AG RNomics

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

Prostate Cancer 2 nd leading cause of cancer death in men Very poor prognosis for metastatic androgen-independent form No accurate diagnostic markers to distinguish different stages Prostatectomy Chemotherapy Androgen deprivation therapy androgen independent Progression androgen dependent Chemotherapy

Identification of novel differentially expressed ncRNAs in prostate cancer Diagnostic markers (differentiation between androgen- dependent and independent form) Drug targets

Identification of novel ncRNAs using Tiling Arrays Exonic region Intronic region Intergenic region Oligonucleotide coverage (25- mer probes) 25-mer oligonucleotide probes cover exonic, intronic and intergenic regions of chromosomes 21 and 22 (repeats are removed) Architecture of Human GeneChip ® Tiling Arrays (chr 21 and 22) Protein-coding gene

Cellular models for prostate cancer LNCaPPC3DU-145RWPE-1 Androgen responsiveness yes no no yes Tiling array Androgen- dependent prostate cancer Androgen- independent prostate cancer Epithelial prostate tissue

Experimental Approach Cell culture, RNA isolation ds cDNA synthesis Fragmentation and labeling Hybridisation Scan 1. Tiling Array (LNCaP vs PC3) 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)

Shown examples: Results – overview Regions for experimental verification by RT-PCR 10 Intronic 3 Intergenic 5 Overlapping intron-exon boundaries 2 Differentially expressed regions (tiling array evaluation) 18 2 Primer design not possible (repetetive sequences, no unique sequences)

LNCaP PC3 DU-145 RWPE-1 AntisenseSense- Primer- RTβ-actin 430 nt LNCaPPC3 DU-145 RWPE-1 β-actin Random primedStrand-specific Intronic transcripts: Candidate 1 (chr 21) RNAz Evofold PC3 LNCaP RefSeq (+) RefSeq (-) mRNA-BLAST: mRNAs (95%: and ) ncRNA-BLAST: human scAlu RNA (90%: and 80%: ) ORF: 237 nt 6701 nt Cell adhesion molecule

288 nt LNCaPPC3 DU-145 RWPE-1 β-actin LNCaP PC3 DU-145 RWPE-1 AntisenseSense- Primer- RTβ-actin Random primedStrand-specific Intronic transcripts: Candidate 2 (chr 21) RNAz Evofold PC3 LNCaP RefSeq (+) RefSeq (-) ORF: 150 nt 550 nt esterase

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

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