Herbert Auer

Chipping away at the chip bias: RNA degradation in microarray analysis

Measurement of gene expression is based on the assumption that an analyzed RNA sample closely represents the amount of transcripts in vivo. Transcripts show stability differences of up to two orders of magnitude in vivo1, raising the possibility that partial degradation during cell lysis could cause a variable extent of bias in quantification of different transcripts. One of the most effective tools for characterizing RNA integrity is capillary electrophoresis, in which RNA degradation is indicated by an altered 28S/18S rRNA signal ratio2. In the software of the commonly used system (Bioanalyzer 2100, Agilent), quantification of 18S and 28S rRNA is compromised by the fact that this calculation is based on area measurements that are heavily dependent on definitions of start and end points of peaks (Fig. 1a). Even accurate determination of this ratio is not sufficient to detect degradation efficiently (Fig. 1b). We developed a mathematical model that results in an objective number for quantitative characterization of RNA degradation. Aside from three prominent peaks (small RNAs, 18S and 28S rRNA), a chromatogram of the size distribution of cellular RNAs shows a broad range of molecular weights with much weaker signals. With increasing degradation, heights of 18S and 28S peaks gradually decrease and additional ‘degradation peak signals’ appear in a molecular weight range between small RNAs and the 18S peak (Fig. 1b). The ratio of the average degradation peak signal to the 18S peak signal multiplied by 100 will hereafter be referred to as the degradation factor. This analysis has been tested on 19 tissues of seven organisms, and it is a reproducible parameter for degradation of mammalian RNA (Supplementary Table 1 online). As an example, 12 repeated measurements of the same sample yielded an average degradation factor of 27.14 with a standard deviation of 1.06. Degradometer software for calculation of the degradation factor can be downloaded from http://www.dnaarrays.org. If one RNA sample was intact and the other was degraded during isolation, up to three-quarters of the differential gene expression measured was due solely to differences in RNA integrity between two samples (Fig. 1d). Supplementary Figure 1 online shows changes in mRNA levels caused by alteration of RNA integrity. This effect was independent of the algorithm applied to raw data analysis (Supplementary Tables 2, 3 and 4 online). For GAPD and ACTB, two transcripts for which signal intensities from 3′ and 5′ portions are frequently measured in microarray analysis, there is a positive correlation between the 3′/5′ ratio and the degradation factor of samples (Fig. 1c). This correlation is tissue-dependent (Supplementary Fig. 2 online).The smaller the difference in degradation factors between samples, the more closely the measured expression differences reflect biological differences (Fig. 1d). Aside from general RNase activity by members of the RNase A family3, RNase L, an enzyme activated in apoptotic Chipping away at the chip bias: RNA degradation in microarray analysis C O R R E S P O N D E N C E

Gene expression and functional evidence of epithelial-to-mesenchymal transition in papillary thyroid carcinoma invasion

Papillary thyroid carcinomas (PTCs) that invade into local structures are associated with a poor prognosis, but the mechanisms for PTC invasion are incompletely defined, limiting the development of new therapies. To characterize biological processes involved in PTC invasion, we analyzed the gene expression profiles of microscopically dissected intratumoral samples from central and invasive regions of seven widely invasive PTCs and normal thyroid tissue by oligonucleotide microarray and performed confirmatory expression and functional studies. In comparison with the central regions of primary PTCs, the invasive fronts overexpressed TGF β, NFκB and integrin pathway members, and regulators of small G proteins and CDC42. Moreover, reduced levels of mRNAs encoding proteins involved in cell–cell adhesion and communication were identified, consistent with epithelial-to-mesenchymal transition (EMT). To confirm that aggressive PTCs were characterized by EMT, 34 additional PTCs were examined for expression of vimentin, a hallmark of EMT. Overexpression of vimentin was associated with PTC invasion and nodal metastasis. Functional, in vitro studies demonstrated that vimentin was required both for the development and maintenance of a mesenchymal morphology and invasiveness in thyroid cancer cells. We conclude that EMT is common in PTC invasion and that vimentin regulates thyroid cancer EMT in vitro.

Role of cancer-associated stromal fibroblasts in metastatic colon cancer to the liver and their expression profiles.

The cancer microenvironment and interaction between cancer and stromal cells play critical roles in tumor development and progression. The molecular features of cancer stroma are less well understood than those of cancer cells. Cancer-associated stromal fibroblasts are the predominant component of stroma associated with colon cancer and its functions remain unclear. Fibroblast cell cultures were established from metastatic colon cancer in liver, liver away from the metastatic lesions, and skin from three patients with metastatic colorectal cancer. We generated expression profiles of cancer-associated fibroblasts using oligochip arrays and compared them to those of uninvolved fibroblasts. The conditioned media from the cancer-associated fibroblast cultures enhanced proliferation of colon cancer cell line HCT116 to a greater extent than cultures from uninvolved fibroblasts. In microarray expression analysis, cancer-associated fibroblasts clustered tightly into one group and skin fibroblasts into another. Approximately 170 of 22 000 genes were up-regulated in cancer-associated fibroblasts (fold change>2, P<0.05) as compared to skin fibroblasts, including many genes encoding cell adhesion molecules, growth factors, and COX2. By immunohistochemistry in-vivo, we confirmed COX2 and TGFB2 expression in cancer-associated fibroblasts in metastatic colon cancer. The distinct molecular expression profiles of cancer-associated fibroblasts in colon cancer metastasis support the notion that these fibroblasts form a favorable microenvironment for cancer cells.

Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development.

The events regulating human preimplantation development are still largely unknown owing to a scarcity of material, ethical and legal limitations and a lack of reliable techniques to faithfully amplify the transcriptome of a single cell. Nonetheless, human embryology is gathering renewed interest due to its close relationship with both stem cell biology and epigenetic reprogramming to pluripotency and their importance in regenerative medicine. Carefully timed genome-wide transcript analyses of single oocytes and embryos uncovered a series of successive waves of embryonic transcriptional initiation that start as early as the 2-cell stage. In addition, we identified the hierarchical activation of genes involved in the regulation of pluripotency. Finally, we developed HumER, a database of human preimplantation gene expression, to serve the scientific community. Importantly, our work links early transcription in the human embryo with the correct execution of the pluripotency program later in development and paves the way for the identification of factors to improve epigenetic reprogramming.

20q11.1 amplification in giant‐cell tumor of bone: Array CGH, FISH, and association with outcome

The goal of this study was to identify recurrent regions of genomic gain or loss in giant‐cell tumor of bone (GCTb). Array comparative genomic hybridization (aCGH) was performed for 20 frozen tumor samples of GCTb. A separate subset of 59 GCTb with outcome data was used for validation. The most frequent region of change identified by aCGH was gain of a 1‐Mbp region at 20q11.1. In the validation arm of 59 cases the minimal common region of copy number gain at 20q11.1, seen in 54% of the samples, was BAC clone RP11‐4O9, which contained the genes TPX2 and BCL2L1. For most cases, amplification was restricted to the mononuclear component and was not present in the multinucleated giant cells. Southern blot for TPX2 and BCL2L1 identified the former as the gene with the highest level of amplification for these two proposed candidate genes of importance. Immunohistochemistry for TPX2 expression correlated with amplification, while BCL2L1 expression was not identified. Kaplan–Meier curves for progression‐free survival showed a statistically significant difference for cases with 20q11.1 amplification (P = 0.0001). Univariate analysis involving Cox proportional hazards models did not show a significant difference for initial treatment type (curettage versus resection) (P = 0.575), age (≤50 vs. >50) (P = 0.543), or sex (P = 0.268), but did correlate with 20q11.1 amplification (P = 0.001). By multivariate analysis, it was found that 20q11.1 amplification (P = 0.001) was the only factor to reach statistical significance. 20q11.1 amplification can be used as a marker of prognostic importance in GCTb. We propose TPX2 as a candidate oncogene in the core‐amplified region at 20q11.1.

HOXB4's road map to stem cell expansion

Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent “universal” targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-α. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo. The concept of TNF-α inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4–6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.

ADOA3R as a Therapeutic Target in Experimental Colitis: Proof by Validated High-density Oligonucleotide Microarray Analysis

&NA; Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)‐(3‐iodobenzyl)‐adenosine‐5‐N‐methyluronamide (IB‐MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine‐based drugs in experimental inflammatory bowel disease. High‐density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS‐treated (7 days), and oral IB‐MECA‐treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin‐trap monitoring of free radicals. Remarkable colitis‐induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis‐related dysregulation. Downregulation occurs in membrane transporter, mitogen‐activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox‐sensitive genes. Oral IB‐MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB‐MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB‐MECA blocked the colitis‐induced upregulation (≤20‐fold) of Bzrp, P2X1R, P2X4R, P2X7R, P2Y2R, P2Y6R, and A2aR/A2bR but not A1R or A3R genes or downregulated P2X2R, P2Y1R, and P2Y4R. Real‐time SYBR green polymerase chain reaction validated gene chip data for both induction of colitis and treatment with IB‐MECA for >90% of genes tested (33 of 37 genes). We conclude that our validated high‐density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine‐based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease.

Aneuploidy-induced delaminating cells drive tumorigenesis in Drosophila epithelia.

Genomic instability has been observed in essentially all sporadic carcinomas. Here we use Drosophila epithelial cells to address the role of chromosomal instability in cancer development as they have proved useful for elucidating the molecular mechanisms underlying tumorigenic growth. We first show that chromosomal instability leads to an apoptotic response. Interestingly, this response is p53 independent, as opposed to mammalian cells, and depends on the activation of the c-Jun N-terminal kinase (JNK) signaling cascade. When prevented from undergoing programmed cell death (PCD), chromosomal instability induces neoplasic overgrowth. These tumor-like tissues are able to grow extensively and metastasize when transplanted into the abdomen of adult hosts. Detailed analysis of the tumors allows us to identify a delaminating cell population as the critical one in driving tumorigenesis. Cells loose their apical–basal polarity, mislocalize DE-cadherin, and delaminate from the main epithelium. A JNK-dependent transcriptional program is activated specifically in delaminating cells and drives nonautonomous tissue overgrowth, basement membrane degradation, and invasiveness. These findings unravel a general and rapid tumorigenic potential of genomic instability, as opposed to its proposed role as a source of mutability to select specific tumor-prone aneuploid cells, and open unique avenues toward the understanding of the role of genomic instability in human cancer.

Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform

DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation. However, accurate and reproducible quantification of DNA methylation remains challenging. In this report, we describe Bio-COBRA (combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform), as a novel approach to quantitative DNA methylation analysis. The combination of a well-established method, COBRA, which interrogates DNA methylation via the restriction enzyme analysis of PCR-amplified bisulfite treated DNAs, with the Bioanalyzer platform allows for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. The sensitivity and reproducibility of Bio-COBRA make it a valuable tool for the analysis of DNA methylation in clinical samples, which could aid in the development of diagnostic and prognostic parameters with respect to disease detection and management.

Accurate Expression Profiling of Very Small Cell Populations

Background Expression profiling, the measurement of all transcripts of a cell or tissue type, is currently the most comprehensive method to describe their physiological states. Given that accurate profiling methods currently available require RNA amounts found in thousands to millions of cells, many fields of biology working with specialized cell types cannot use these techniques because available cell numbers are limited. Currently available alternative methods for expression profiling from nanograms of RNA or from very small cell populations lack a broad validation of results to provide accurate information about the measured transcripts. Methods and Findings We provide evidence that currently available methods for expression profiling of very small cell populations are prone to technical noise and therefore cannot be used efficiently as discovery tools. Furthermore, we present Pico Profiling, a new expression profiling method from as few as ten cells, and we show that this approach is as informative as standard techniques from thousands to millions of cells. The central component of Pico Profiling is Whole Transcriptome Amplification (WTA), which generates expression profiles that are highly comparable to those produced by others, at different times, by standard protocols or by Real-time PCR. We provide a complete workflow from RNA isolation to analysis of expression profiles. Conclusions Pico Profiling, as presented here, allows generating an accurate expression profile from cell populations as small as ten cells.