Systematic-Methodological Platforms RNA SMP: RNA Project: Analysis of Murine Tumor Models and Human Tumors

Introduction Today, researchers can choose from a broad variety of methods for global transcriptional profiling. Among the different technical approaches, microarray technology has gained a premier position. In principle, microarrays can be produced either by robotic printing (“spotting”) of DNA on a chemically modified glass surface1, or by in situ synthesis of oligonucleotides on a silane-reacted quartz substrate2. Spotted arrays usually contain cDNA-specific PCR amplicons (cDNA arrays), ranging from several hundred to a few thousand base pairs in size. Generally, no more than one amplicon is used to probe a given gene. Although they are technically challenging and require both optimized protocols and workflow, cDNA arrays are typically produced by individual research groups or core facilities. Alternatively, they can be purchased from several commercial suppliers. But after the discovery of frequent discrepancies in the annotation of cDNA clones, investigators began to realize potential drawbacks of this highly advocated technology. In situ synthesis of oligonucleotide probes requires sophisticated equipment for photolithography and solid phase chemistry, which is usually too complex and elaborate for an academic environment. A widespread commercial implementation of this technology is the Affymetrix GeneChip platform, which currently uses 11-16 pairs (11 for the arrays used in this study) of perfect-match and single-basemismatch 25-mer oligonucleotides for each gene. Recently, large collections of longer oligonucleotides (50-80 bases), produced by established suppliers using conventional phosphoramidite chemistry, have become increasingly popular as probes for spotted DNA arrays. Technical advantages of oligonucleotide arrays include a constant DNA concentration across all spots and biophysically optimized sequences, reducing secondary structures, avoiding repetitive sequence motives and providing a fixed range for both Tm and length. All this accounts for more uniform, stable and predictable hybridization conditions. The overall costs for long oligonucleotide arrays will often be lower when labor and other costs associated with cDNA libraries, such as replication, amplification or sequence verification, are regarded. Considering this diversity of approaches and the resulting technical differences, researchers are highly interested in the general accuracy and reliability of microarray data and the cross-platform comparability. Several independent methods like Northern blotting or real-time quantitative PCR (RQPCR) have been used to validate microarray results for a small number of transcripts. Generally, there was good agreement between the corresponding values, affirming the ability to accurately profile gene expression with array-based approaches. Former studies also compared global expression measurements between cDNA arrays and short oligonucleotide arrays or SSH. Recently, Barczak et al. compared results between spotted arrays of 70-mer oligonucleotides and in situ synthesized Affymetrix GeneChip arrays. Using RNA of a cell line and a commercial reference RNA, they found strong correlations of the corresponding data sets. Despite these studies clarifying some fundamental questions, there still remains considerable uncertainty regarding the comparability of data from clinical specimens. As this lack of understanding constitutes a barrier, which keeps researchers from an immense amount of potentially valuable information (via efficient integration of microarray data generated on different array platforms), we conducted a comparison with tumor samples from clinical practice, which evaluates cross-platform reproducibility in a practical setting [1,2]. Results/Project Status cDNAand 70mer oligonucleotide microarrays Human cDNA-microarrays: containing replicate spots of 4211 different gene-specific fragments, representing 2600 different genes with relevance to mitosis, cell cycle control, oncogenesis, or apoptosis [3]. Custom-made cDNA-microarrays: cDNA microarrays which represented approximately 7,000 distinct human genespecific fragments, with particular enrichment for cancerrelevant genes. In addition, each microarray carried genespecific cDNA fragments representing 215 genes located within the chromosomal regions 1p36.13-p36.31 and 19q13.2-q13.33. Human 70mer oligonucleotide microarrays (version 2 plus upgrade): 26,791 gene-specific 70mere oligonucleotides (Human Oligo set 2.1 plus upgrade; Operon) Human 70mer oligonucleotide microarrays (version 4.0): 35,035 oligonucleotide probes, representing approximately 25,100 unique genes and 39,600 transcripts (Human genome oligo set 4.0). Murine 20k cDNA microarray: 20k ArrayTAG collection (LION Bioscience), The 20k ArrayTAGTM included 20,172 sequence-verified cDNA clones (comprising about 10,000 annotated genes) with an average insert size of 200-600 bp. All clones were proved for the absence of repetitive elements and low-complexity regions. Murine 23k cDNA microarray (NIA): The microarray contained "unique" cDNA clones that were derived from preand peri-implantation embryos, E12.5 female gonad / mesonephros, and newborn ovary. Up to 50% of these clones were originated from novel genes expressed during mouse embryogenesis with an average insert size of 1.5 kb