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PubMed1995

Hutchinson-Gilford progeria fibroblasts exhibit metabolically normal uridine uptake and RNA synthetic rates.

The accelerated aging disease Hutchinson-Gilford progeria syndrome displays altered messenger RNA levels in cultured fibroblasts, yet little is known of effects on transcription by RNA polymerases other than RNA polymerase II. Total RNA metabolism was examined by incubation of Hutchinson-Gilford progeria fibroblasts with [5-3H]uridine in asynchronous culture. Uptake of radiolabel was quantitative and was incorporated preferentially (99 +/- 0.3%) into newly synthesized RNA. Progeria and control cultures showed comparable rates of uptake of radiolabel, time courses of RNA synthesis, and relative intensity profiles of newly synthesized ribosomal RNA. These characteristics held over a greater than ten-fold range of cell densities (5 x 10(3) to 8 x 10(4) cells/cm2). Progeria and control fibroblasts thus have comparable metabolic capacities for uridine uptake and net RNA production, emphasizing the relative specificity of transcriptional changes previously identified in progeria cells.

Biological Transport↗
PubMed1992

BC200 RNA in normal human neocortex, non-Alzheimer dementia (NAD), and senile dementia of the Alzheimer type (AD).

BC200 RNA is a polyadenylated 200 nucleotide primate brain-specific transcript with 80% homology to the left monomer of the human Alu family of repetitive elements. Whether this transcription product contributes anything to normal brain gene function or is a residue of post transcriptional processing of brain heterogeneous nuclear RNA (hnRNA) is uncertain. However, the high abundance, tissue-specific expression and nucleotide sequence characteristics of BC200 RNA suggests that the generation of this small RNA is associated with some brain cell function. Sustained levels of the BC200 RNA transcript may be indicative of a genetically competent and normally functioning cerebral neocortex. In this investigation, we have measured the abundance of the BC200 RNA transcript in total RNA isolated from 18 temporal neocortices (Brodman area 22) of brains with no pathology and those affected with neurodegenerative disease. Neocortices were examined from 3 neurologically normal brains, 5 non-Alzheimer demented [NAD; 3 Huntington's chorea (HC), 1 amyotrophic lateral sclerosis (ALS) and 1 dementia unclassified] and 10 Alzheimer disease (AD) affected brains. Our results indicate a strong BC200 presence in both the normal brains and NAD affected neocortices, but a 70 per cent reduction in BC200 signal strength in AD afflicted brains. These results may be related to the observation that Alzheimer brains exhibit marked deficits in the abundance of neuron-specific DNA transcripts; these deficits are consistent with the idea that AD is characterized by an impairment in the primary generation of brain gene transcription products.

Alzheimer Disease↗
PubMed1979

Repression of rRNA synthesis induced by disaggregation in Dictyostelium discoideum.

Cells disaggregated from the slugs of Dictyostelium discoideum continued to incorporate [3H]uridine or [3H]uracil, though at a lower rate than interphase amoebae. Disaggregation brought about a marked increase in the proportion of labeled poly(A)-containing RNA to labeled total RNA. The proportion reached the maximum in disaggregated cells after 1 h of incubation, but remained high even after 5 h of incubation. Reconstruction of slugs from the disaggregated cells did not bring about a decrease but a further increase in the proportion. The real proportion estimated from the minimum period of labeling reached almost 100% in the disaggregated cells, in contrast to about 30% in the interphase amoebae. The increase in the proportion is attributed to the disaggregation-induced repression of rRNA synthesis, which was supported by sucrose density gradient analyses of RNA synthesized in the disaggregated cells. The possibility that rRNA synthesis in this organism is regulated by the loss of cell contact is discussed.

Cell Division↗
PubMed1980

Synthesis of complementary DNAs to partially purified mRNAs coding for the storage proteins of Pisum sativum (L).

mRNAs in developing cotyledons of Pisum sativum (L) coding for the major storage proteins, vicilin and legumin, were partially purified and characterized. Both vicilin (47 000 and 50 000 daltons) and legumin (60 000) precursor subunits were translated in the reticulocyte lysate cell-free system when driven by 18 S poly(A)+-RNA. Total poly(A)+-RNA, purified twice by oligo(dT)-cellulose chromatography, migrated on dissociating gels on electrophoresis as a polydisperse peak with three maxima at 18, 14 and 12 S. This mRNA preparation was used as a template for the synthesis of single- and double-stranded cDNAs under optimized reaction conditions. Analysis of single-stranded cDNA on dissociating gels showed a polydisperse profile, with three major components of molecular weights 3.7 x 10(5), 2.3 X 10(5) and 2.0 X 10(5). The double-stranded cDNA preparation contained some contaminating single strands, some partially double-stranded molecules formed by continuation of looped first strands, and some molecules which were primarily single strands protected by partial second strands. Restriction endonucleases cut the double-stranded cDNA preparation into several discrete fragments.

Base Composition↗
PubMed1977

Number and distribution of polyadenylated RNA sequences in yeast.

The poly(A)-containing RNA, isolated from the budding yeast Saccharomyces cerevisiae, has been characterized with regard to the number and distribution of sequences by a kinetic analysis of RNA-cDNA hybridization. In agreement with results previously obtained on metazoan eucaryotes (Bishop et al., 1974), discrete complexity classes were observed. There exist low, medium, and high complexity classes which contain approximately 20, 400, and 2400 sequences, respectively. This measurements of the number of sequences has been verified by hybridization with single copy DNA. 20% of the single copy fraction of the yeast genome is rendered double-stranded by poly(A)-containing RNA. Assuming asymmetric transcription, this is equivalent to approximately 4000 poly(A)-containing sequences, verifying the results obtained with RNA-cDNA hybridization. In addition, the first-order kinetics of the hybridization with single copy DNA verified the notion that most of the sequence complexity is present at the same intracellular concentration. The same number and distribution of sequences were found in poly(A)-containing polysomal RNA and in total RNA, suggesting that most or all of the sequence complexity is on polysomes and is adenylated. The results indicate that RNA-cDNA hybridization is an accurate method for determining sequence complexity values and that yeast, grown under vegetative conditions, has 3000-4000 different mRNA sequences.

Base Sequence↗
PubMed1992

Differentiation of potyviruses and their strains by hybridization with the 3' non-coding region of the viral genome.

Nucleic acid hybridization with the 3' non-coding region of the potyvirus genome as the probe was shown to be a relatively simple means of distinguishing between distinct potyviruses and their strains. Comparisons of the nucleotide sequences of potyvirus genomes (ignoring gaps) showed that the degree of identity between equivalent genes of strains was greater than 96%, while between distinct potyviruses the identity ranged from 42% to 65%, suggesting that any extended sequence could be considered representative of the whole genome and be suitable as a diagnostic probe. The comparisons however, also revealed that some parts of the genome, but not the 3' non-coding region, had local regions of high sequence identity that could lead to cross-hybridization between distinct potyviruses. For this reason, and because its location immediately upstream of the poly(A) tail makes it the most accessible region for the purpose of cloning and sequencing, the 3' non-coding sequence should be most suitable for use as a diagnostic probe. Successful hybridizations (using radiolabeled, polymerase chain reaction-amplified 3' non-coding sequences) have been achieved by probing recombinant clones, purified potyviral RNA, partially purified total RNA from infected plants, and a crude extract of infected plant tissue. The method has been used to support the proposals that watermelon mosaic virus 2 and soybean mosaic virus-N are both strains of the same virus, and to discriminate between several isolates previously believed to be strains of sugarcane mosaic virus. The method should have wide application as a means of differentiating distinct potyviruses from strains.

Blotting, Southern↗
PubMed1988

Insulin action in early embryonic life: anti-insulin receptor antibodies retard chicken embryo growth but not muscle differentiation in vivo.

Insulin receptors are present in chicken embryos at day 2 of development and insulin stimulates embryonic growth and differentiation. Most important, anti-insulin antibodies cause either death or developmental retardation in chicken embryos of that age. To determine if the embryo's endogenous insulin acts through its own receptor, we compared the effects of anti-insulin antibodies to the effects of anti-insulin receptor antibodies on growth and differentiation indexes in the chicken embryo. While the anti-insulin antibody caused a dose-dependent decrease in growth parameters like weight, total protein, DNA, RNA, total creatine kinase activity and a marker of differentiation, the creatine kinase-MB, the anti-insulin receptor antibody decreased all parameters except the creatine kinase-MB. Many, but not all, of the effects of insulin in early embryos, thus, are mediated through the insulin receptor.

Animals↗
PubMed1999

Increased expression of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage and P450 17alpha-hydroxylase enzymes in ovarian hyperthecosis.

OBJECTIVE: To investigate whether the increased ovarian androgen synthesis in hyperthecosis is due to increased expression of the steroidogenic enzymes essential for androgen synthesis. DESIGN: Controlled study to investigate concentration of steroidogenic enzymes in the ovarian stroma of women with hyperthecosis of the ovaries. SETTING: Academic research environment. PATIENT(S): Three women with ovarian hyperthecosis and eight with normal ovulatory cycles. INTERVENTION(S): Ovarian stromal tissues were obtained from women with hyperthecosis and women with normal ovaries. Diagnosis of hyperthecosis was confirmed by histologic examination of the ovaries. Steroid levels were measured in the ovarian vein serum of one patient with hyperthecosis. MAIN OUTCOME MEASURE(S): Tissues were frozen immediately in liquid nitrogen and kept frozen until RNA was extracted. Total RNA was examined by Northern blot analysis using 32P-labeled complementary DNA (cDNA) probes encoding human P450scc and P450(17alpha) enzymes. RESULT(S): P450scc and P450(17alpha) messenger RNAs (mRNAs) were detected in the normal ovarian stroma and stromal hyperthecosis. Compared with normal ovarian stroma, P450scc mRNA was increased twofold and P450(17alpha) mRNA was increased threefold in stromal hyperthecosis. CONCLUSION(S): [1] Ovarian stroma is probably the site of androgen production in ovarian hyperthecosis. [2] Increased stromal androgen synthesis in hyperthecosis could be due to increased expression of the enzymes P450scc and P450(17alpha) in the ovarian stroma. [3] Markedly increased concentrations of 17alpha-hydroxyprogesterone in the ovarian vein serum indicate possible dysregulation of P450(17alpha) in ovarian hyperthecosis.

Adult↗
PubMed1997

Expression of midkine and pleiotropin in ovarian tumors.

OBJECTIVE: To compare the expression of midkine and pleiotropin in malignant ovarian tumors with that in normal and benign ovarian tissue. METHODS: Total RNA was isolated from 23 samples of normal ovaries, 15 benign ovarian tumors, and 36 malignant ovarian tumors. Midkine and pleiotropin gene expression was examined by using Northern blot analysis. To confirm the localization of midkine expression, in situ hybridization and immunohistochemical analyses were performed. The truncated midkine messenger RNA was examined using polymerase chain reaction with complementary DNA synthesized from total RNA with reverse transcriptase. RESULTS: Expression of midkine gene was observed in 19 of 23 normal ovary samples and in 51 of 53 ovarian tumors (13 of 15 benign, both of the two borderline tumors, and all 36 malignant tumors). Pleiotropin gene was expressed in six normal ovaries and in 24 tumors (nine benign, two borderline, and 13 malignant tumors). The expression of midkine in germ cell tumors was significantly lower than in epithelial tumors, whereas expression in malignant epithelial tumors was significantly higher than in benign ones. In germ cell tumors, two samples with differentiated neural tissues showed high levels of pleiotropin gene expression. In situ hybridization and immunohistochemical analysis showed strong expression of midkine in cancer cells. The truncated midkine messenger RNA was not found in any of the normal, benign, or malignant tissues examined. CONCLUSION: These results suggest an association between midkine and carcinogenesis. Expression of pleiotropin is more restricted, and high levels of its expression may be correlated with neural differentiation.

Adult↗
PubMed1976

Calculation of absolute rates of RNA synthesis, accumulation, and degradation in tobacco callus in vivo.

Uptake and incorporation data were used to calculate an absolute rate of ribonucleic acid (RNA) synthesis by assuming that incorporation of radioactive nucleosides into RNA was a first-order function of the nucleoside triphosphate cellular pool. Homogenized tissue was separated into an RNA and cellular pool fraction. Methods were developed to directly measure the specific radioactivity of specific nucleosides in each fraction. Exogenous [3H]uridine was taken up by the cells, converted to I[3H]uridine triphosphate ([3H]UTP) and [3H]cytidine triphosphate ([3H]CTP), and incorporated into RNA. All of the radioactivity in RNA was contained in either uridine or cytidine. The average rate of RNA synthesis (20 mg of RNA (g of RNA)-1h-1) was calculated from changes in the specific radioactivity of uridine or cytidine in RNA and UTP or CTP in the pool. Since ribosomal RNA does not turn over in exponentially growing tissue, its rate of synthesis was measured (14 mg g-1h-1) and compared with the rate of accumulation of RNA (12 mg g-1h-1). The similarity between ribosomal RNA synthesis and total RNA accumulation confirmed the assumption that these methods measured an absolute rate of RNA synthesis.

Cytosine Nucleotides↗
PubMed2001

An invasive cleavage assay for direct quantitation of specific RNAs.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.

Base Sequence↗
PubMed2002

Expression, modulation and signalling of IL-17 receptor in fibroblast-like synoviocytes of patients with rheumatoid arthritis.

Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL-17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared to patients with osteoarthritis. In contrast to the restricted expression of IL-17, the IL-17 receptor (IL-17R/CDw217) is expressed ubiquitously. Using a real-time RT-PCR assay, we detected similar absolute levels of IL-17R mRNA expression in fibroblast-like synoviocytes (SFC) from patients with RA (mean 9 pg/microg total RNA; ranged from 0.1 pg to 96 pg IL-17R mRNA/microg total RNA) compared to synoviocytes of non-RA patients. Analysis of the IL-17R surface expression confirmed the results obtained for IL-17R mRNA expression. Exposure of SFC to IL-17 led to a mRNA induction of CXC chemokines IL-8, GRO-alpha and GRO-beta. An anti-IL-17 antibody blocked these effects of IL-17. The MAPK p38 appears necessary for the regulation of IL-8, GRO-alpha and GRO-beta expression as shown by inhibition with SB203580. The inhibitors genistein (tyrosine kinase inhibitor) and calphostin C (inhibitor of protein kinase C) reduced significantly the IL-17-stimulated mRNA expression of IL-8, GRO-alpha and GRO-beta in SFC, whereas PD98059 (inhibitor of MEK-1/2) was without effect. Pharmacological drugs used in therapy of RA, such as cyclosporin and methotrexate, induced a fourfold increase of IL-17R mRNA expression and augmented the IL-17-stimulated IL-8 expression. Our results support the hypothesis that IL-17/IL-17R may play a significant role in the pathogenesis of RA contributing to an unbalanced production of cytokines as well as participating in connective tissue remodelling.

Antirheumatic Agents↗
PubMed2004

Aldosterone resistance in kidney transplantation is in part induced by a down-regulation of mineralocorticoid receptor expression.

BACKGROUND: After renal transplantation immunosuppressive drugs-like cyclosporin A (CsA) and FK506 induce either hypoaldosteronism or pseudo-hypoaldosteronism presenting with hyperkalemia and metabolic acidosis. We investigated the relationship between renal allograft function under CsA therapy and plasma aldosterone concentration, potassium- and water homeostasis and mineralocorticoid receptor (MR) expression level in peripheral leukocytes. METHODS: We studied 21 renal transplant patients under CsA therapy and 12 healthy controls. Transplant recipients were studied before and under fludrocortisone treatment. Using quantitative reverse-phase polymerase chain reaction (RT-PCR) specific for the MR, we analyzed the level of expression of MR in peripheral leukocytes. RESULTS: In acidotic transplant recipients (HCO(3) 18.5 +/- 1.2 mM) renal function was only slightly impaired with 2.0 +/- 0.2 mg creatinine/dL when compared with 1.8 +/- 0.3 mg/dL (ns) in non-acidotic patients (HCO(3) 23.0 +/- 2.8 mM). Mean plasma aldosterone levels in renal transplant recipients did not differ from control levels (150 +/- 33 pg/mL vs. 148 +/- 33 pg/mL, ns). In contrast, the expression level of MR in peripheral leukocytes of renal transplant recipients treated with CsA was significantly decreased when compared with healthy controls without renal disease (120 +/- 78 vs. 423 +/- 73 RNA molecules/0.5 microg total RNA, p < 0.01). The level of expression of MR in renal transplant recipients did not differ between acidotic patients and non-acidotic patients (ns). The application of fludrocortisone reversed hyperkalemia and metabolic acidosis without significant effect on MR expression. CONCLUSIONS: The present data demonstrate that hyperkalemia and metabolic acidosis following CsA treatment in kidney transplantation might be associated with a down-regulation of MR expression on peripheral leukocytes. Electrolyte imbalance is reversible on application of fludrocortisone. This observation supports fludrocortisone treatment in transplant patients with severe electrolyte disturbances.

Acidosis↗
PubMed1993

Coordinated decreases in rRNA gene transcription factors and rRNA synthesis during muscle cell differentiation.

rRNA synthesis decreases significantly during the differentiation of rat L6 myoblasts to myotubes. Nuclear run-on assays demonstrated that the decrease was attributable to decreased rates of rRNA gene transcription. Immunoblot analysis indicated a marked reduction in amounts of the RNA polymerase I transcription factors UBF1 and UBF2 (upstream binding factors 1 and 2, respectively). The levels of these factors dropped in parallel with the down-shift in rRNA gene transcription. The amount of UBF does not fall due to a general decrease in cellular protein, as myosin heavy-chain protein accumulates markedly during this same time. RNA blots of total RNA isolated from myoblasts and differentiating myotubes showed a decrease in the mRNA for UBF, at the same time the mRNA for myogenin was accumulating. The down-shift in UBF mRNA levels preceded the decrease in the protein levels for UBF. There have been reports that the acute response of the rRNA gene transcription system to physiological signals in many systems involves an RNA polymerase I-associated factor. However, our results imply that the regulation of rRNA gene DNA transcription in response to physiological processes, such as differentiation, may involve multiple regulatory pathways.

Adenosine Triphosphate↗
PubMed2006

Amplification of sense-stranded prokaryotic RNA.

Microarray expression analysis has proven to be a valuable methodology. In eukaryotic systems where RNA is limiting, established protocols for amplification of mRNA, which rely on the poly(A) tails, are well established. In contrast, the difficulty in amplifying prokaryotic mRNA has limited the application of microarrays to microbiology. Here we present a method for the Linear Amplification of Prokaryotic Transcripts (LAPT) that is efficient and unbiased. The overhang tailing activity of Moloney murine leukemia virus reverse transcriptase is used to add the T7 promoter to cDNAs during reverse transcription. The promoter addition is uncoupled from the initial priming event allowing the promoter to be attached to the 5' end of the RNA transcript. This enables the amplification of sense-stranded RNA that is representative of the complexity and distribution of the original transcript pool. In microarray assays amplified prokaryotic RNA (10 ng total RNA starting material) showed good Spearman correlations to an unamplified control sample. Using genome-directed primers to bias addition of a T7-promoter to bacterial transcripts allowed amplification of prokaryotic transcripts in the presence of mammalian RNA (at a eukaryotic/prokaryotic RNA ratio of 500 to 1). This technology should facilitate the study of prokaryotic transcriptomes in situations, such as in vivo studies or mixed microbial populations, where the prokaryotic RNA amount is limited and/or the nontarget/target RNA ratios is high.

Animals↗
PubMed1995

Myotonic dystrophy: evidence for a possible dominant-negative RNA mutation.

The trinucleotide expansion mutation causing myotonic dystrophy is in the 3' untranslated region of a protein kinase gene. The molecular mechanisms by which the expanded repeat causes the clinically variable and multisystemic disease, myotonic dystrophy, are not understood. It has been particularly difficult to rationalize the dominant inheritance with the fact that the expansion mutation lies outside of the protein-encoding gene elements, and should not be translated into protein. Here we use muscle biopsies from classical adult-onset myotonic dystrophy patients to study the accumulation of transcripts from both the normal and expanded DM kinase genes in patient muscle, and compare the results to normal and myopathic controls. We found relatively small decreases of DM kinase RNA in the total RNA pool from muscle; however, these reductions were not disease specific. Analysis of poly(A)+ RNA showed dramatic decreases of both the mutant and normal DM kinase RNAs, and these changes were disease-specific. Our findings are consistent with a novel molecular pathogenetic mechanism for myotonic dystrophy: both the normal and expanded DM kinase genes are transcribed in patient muscle, but the abnormal expansion-containing RNA has a dominant effect on RNA metabolism by preventing the accumulation of poly(A)+ RNA. The ability of the expansion mutation to alter accumulation of poly(A)+ RNA in trans suggests that myotonic dystrophy may be the first example of a dominant-negative mutation manifested at the RNA level.

Adolescent↗
PubMed1997

An autocrine/paracrine role of human decidual relaxin. II. Stromelysin-1 (MMP-3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1).

Interstitial collagen types I and III are the predominant collagens in the amniotic and chorionic connective tissues. However, this matrix also contains proteoglycans, fibronectin, laminin, and elastin, which together with the collagens may undergo partial degradation prior to fetal membrane rupture at term. In this study, stromelysin (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) were immunolocalized in fetal membranes obtained at term prior to labor. MMP-3 stained the cells of the amniotic epithelium, fibroblasts and macrophages of the amniotic and chorionic matrix, and those of the chorionic cytotrophoblast; there was no staining in the maternal decidua. TIMP-1 showed a similar staining pattern, except that the staining was darker in some amniotic epithelial cells and was present in the maternal decidua. The maternal decidua produces the two human relaxins H1 and H2; the latter, when incubated with explants of human fetal membranes, caused a dose-dependent and significant increase in expression of the MMP-3 gene and its secreted protein into the media. A significant effect of relaxin H2 on 92-kDa gelatinase (MMP-9) gene expression was also shown--an effect requiring poly(A)+ RNA rather than total RNA. Both relaxin H1 and H2 caused a significant increase in secretion of MMP-9 protein and its enzyme activity in the media. The magnitude of the effects of the two relaxins was similar, in contrast to findings from other biological studies in which relaxin H2 was shown to be more active. Neither of the relaxins had any effect on 72-kDa gelatinase (MMP-2) activity or on the TIMP-1 protein or its activity. This study suggests that local relaxins may be involved in the degradation of the complex fetal membrane extracellular matrix and may cause activation of an enzyme cascade resulting in fully activated MMP-9. Such effects could be important in the degradative pathways occurring in the amnion and chorion in the peripartal period.

Blotting, Northern↗
PubMed2004

Nonuniform hybridization: a potential source of error in oligonucleotide-chip experiments with low amounts of starting material.

Low amounts of starting material are a significant limitation of gene-expression profiling of microprepared pathologic specimens. Linear RNA amplification has become the method of choice to overcome this problem. Thus, transcriptomal analyses by oligonucleotide-chips or cDNA microarrays are now feasible with labeled complementary RNA generated from total RNA samples in the lower nanogram range. However, in case of oligonucleotide-chips, it has been underestimated so far that individual complementary RNA molecules are shorter in length than and display a 3' bias in comparison to the sequence stretch represented by oligonucleotides on the chip. This can lead to incorrect interpretation of raw data. We have analyzed this problem testing ex vivo-microprepared endothelial cells with Affymetrix GeneChips U133A. Only a small subset of housekeeping genes showed adequate uniform hybridization. We developed a software tool for objective evaluation of oligonucleotide-chips based on automated analysis of as well as normalization to this subset of housekeeping genes. We analyzed the gene expression profile of microprepared lymphatic vascular endothelial cells. We show that optimized normalization prevented exclusion of angiopoietin-2, a lymphatic endothelial marker, from the lymphovascular transcriptome.

Endothelial Cells↗