Strategic white matter hyperintensity locations associated with post-stroke cognitive impairment: A multicenter study in 1568 stroke patients.

BACKGROUND: Post-stroke cognitive impairment (PSCI) occurs in up to 50% of stroke survivors. Presence of pre-existing vascular brain injury, in particular the extent of white matter hyperintensities (WMH), is associated with worse cognitive outcome after stroke, but the role of WMH location in this association is unclear. AIMS: We determined if WMH in strategic white matter tracts explain cognitive performance after stroke. METHODS: Individual patient data from nine ischemic stroke cohorts with magnetic resonance imaging (MRI) were harmonized through the Meta VCI Map consortium. The association between WMH volumes in strategic tracts and domain-specific cognitive functioning (attention and executive functioning, information processing speed, language and verbal memory) was assessed using linear mixed models and lasso regression. We used a hypothesis-driven design, primarily addressing four white matter tracts known to be strategic in memory clinic patients: the left and right anterior thalamic radiation, forceps major, and left inferior fronto-occipital fasciculus. RESULTS: The total study sample consisted of 1568 patients (39.9% female, mean age = 67.3 years). Total WMH volume was strongly related to cognitive performance on all four cognitive domains. WMH volume in the left anterior thalamic radiation was significantly associated with cognitive performance on attention and executive functioning and information processing speed and WMH volume in the forceps major with information processing speed. The multivariable lasso regression showed that these associations were independent of age, sex, education, and total infarct volume and had larger coefficients than total WMH volume. CONCLUSION: These results show tract-specific relations between WMH volume and cognitive performance after ischemic stroke, independent of total WMH volume. This implies that the concept of strategic lesions in PSCI extends beyond acute infarcts and also involves pre-existing WMH. DATA ACCESS STATEMENT: The Meta VCI Map consortium is dedicated to data sharing, following our guidelines.

White Matter Hyperintensity Volume and Poststroke Cognition: An Individual Patient Data Pooled Analysis of 9 Ischemic Stroke Cohort Studies.

BACKGROUND: White matter hyperintensities (WMH) are associated with cognitive dysfunction after ischemic stroke. Yet, uncertainty remains about affected domains, the role of other preexisting brain injury, and infarct types in the relation between WMH burden and poststroke cognition. We aimed to disentangle these factors in a large sample of patients with ischemic stroke from different cohorts. METHODS: We pooled and harmonized individual patient data (n=1568) from 9 cohorts, through the Meta VCI Map consortium (www.metavcimap.org). Included cohorts comprised patients with available magnetic resonance imaging and multidomain cognitive assessment <15 months poststroke. In this individual patient data meta-analysis, linear mixed models were used to determine the association between WMH volume and domain-specific cognitive functioning (Z scores; attention and executive functioning, processing speed, language and verbal memory) for the total sample and stratified by infarct type. Preexisting brain injury was accounted for in the multivariable models and all analyses were corrected for the study site as a random effect. RESULTS: In the total sample (67 years [SD, 11.5], 40% female), we found a dose-dependent inverse relationship between WMH volume and poststroke cognitive functioning across all 4 cognitive domains (coefficients ranging from -0.09 [SE, 0.04, P=0.01] for verbal memory to -0.19 [SE, 0.03, P<0.001] for attention and executive functioning). This relation was independent of acute infarct volume and the presence of lacunes and old infarcts. In stratified analyses, the relation between WMH volume and domain-specific functioning was also largely independent of infarct type. CONCLUSIONS: In patients with ischemic stroke, increasing WMH volume is independently associated with worse cognitive functioning across all major domains, regardless of old ischemic lesions and infarct type.

[Awakening ptosis]. / Kortdurende ptosis bij het ontwaken.

BACKGROUND: A variable ptosis may point towards serious neurological disorders and is presented to general practitioners, ophthalmologists and neurologists. CASE DESCRIPTION: Two patients presented at the neurology outpatient clinic with a ptosis confined to awakening from sleep. There were no other neurological complaints and neurological examination was normal. The diagnosis 'awakening ptosis' was made. CONCLUSION: Awakening ptosis is a benign, but rare disorder. The exact pathophysiology remains unclear. In the case of a classic clinical picture of awakening ptosis, additional examinations are not indicated.

Disabling Wake Up Stroke without Lesions on Initial Diffusion Weighted Imaging - Case Report and Clinical Implications.

An 86-year-old lady presented after waking up with left sided paresis and neglect. Right hemispheral stroke was suspected, but initial MRI with Diffusion Weighted Imaging (DWI) was negative and there was no large vessel occlusion. We accordingly withheld intravenous thrombolysis. She did not improve clinically and follow-up MRI after three days showed a marked lesion compatible with acute ischemic stroke in the right middle cerebral artery territory. This case shows that even with a disabling stroke in the anterior circulation initial DWI may be negative. Former studies established that thrombolysis can be safe in these cases when there is no doubt about the clinical diagnosis of acute ischemic stroke.

Natural course of scoliosis and lifetime risk of scoliosis surgery in spinal muscular atrophy.

OBJECTIVE: To investigate the natural course of scoliosis and to estimate lifetime probability of scoliosis surgery in spinal muscular atrophy (SMA). METHODS: We analyzed cross-sectional data from 283 patients from our population-based cohort study. Additional longitudinal data on scoliosis progression and spinal surgery were collected from 36 consecutive patients who received scoliosis surgery at our center. RESULTS: The lifetime probability of receiving scoliosis surgery was ≈80% in SMA types 1c and 2. Patients with type 2 who only learned to sit (type 2a) were significantly younger at time of surgery than those who learned to sit and stand (type 2b). The lifetime risk of surgery was lower in type 3a (40%) and strongly associated with age at loss of ambulation: 71% in patients losing ambulation before 10 years of age vs 22% losing ambulation after the age of 10 years (p = 0.005). In type 3a, preserving the ability to walk 1 year longer corresponded to a 15% decrease in lifetime risk of scoliosis surgery (hazard ratio 0.852, p = 0.017). Scoliosis development was characterized by initial slow progression, followed by acceleration in the 1.5- to 2-year period before surgery. CONCLUSION: The lifetime probability of scoliosis surgery is high in SMA types 1c and 2 and depends on age at loss of ambulation in type 3. Motor milestones such as standing that are not part of the standard classification system are of additional predictive value. Our data may act as a reference to assess long-term effects of new SMA-specific therapies.

Advance directives, proxy opinions, and treatment restrictions in patients with severe stroke.

BACKGROUND: Patients with severe stroke often do not have the capacity to participate in discussions on treatment restrictions because of a reduced level of consciousness, aphasia, or another cognitive disorder. We assessed the role of advance directives and proxy opinions in the decision-making process of incapacitated patients. METHODS: Sixty patients with severe functional dependence (Barthel Index ≤6) at day four after ischemic stroke or intracerebral hemorrhage were included in a prospective two-center cohort study. The decision-making process with respect to treatment restrictions was assessed by means of a semi-structured questionnaire administered to the treating physician at the day of inclusion. RESULTS: Forty-nine patients (82%) did not have the capacity to participate in the decision-making process. In eight patients, there was no discussion on treatment restrictions and full care was installed. In 41 patients, the decision whether to install treatment restrictions was discussed with proxies. One patient had a written advance directive. In the remaining 40 patients, proxies based their opinion on previously expressed wishes of the patient (18 patients) or advised in the best interest of the patient (22 patients). In 36 of 41 patients, treatment restrictions were installed after agreement between physician and proxy. At six months, 23 of 49 patients had survived. In only three of them the decision on treatment restrictions was based on previously expressed wishes. Remarkably, two of these survivors could not recall any of their alleged previously expressed wishes. CONCLUSIONS: Treatment restrictions were installed in the majority of incapacitated patients after stroke. Proxy opinions frequently served as the best way to respect the patients' autonomy, but their accuracy remains unclear.

Predictive accuracy of physicians' estimates of outcome after severe stroke.

INTRODUCTION: End-of-life decisions after stroke should be guided by accurate estimates of the patient's prognosis. We assessed the accuracy of physicians' estimates regarding mortality, functional outcome, and quality of life in patients with severe stroke. METHODS: Treating physicians predicted mortality, functional outcome (modified Rankin scale (mRS)), and quality of life (visual analogue scale (VAS)) at six months in patients with major disabling stroke who had a Barthel Index ≤6 (of 20) at day four. Unfavorable functional outcome was defined as mRS >3, non-satisfactory quality of life as VAS <60. Patients were followed-up at six months after stroke. We compared physicians' estimates with actual outcomes. RESULTS: Sixty patients were included, with a mean age of 72 years. Of fifteen patients who were predicted to die, one actually survived at six months (positive predictive value (PPV), 0.93; 95% CI, 0.66-0.99). Of thirty patients who survived, one was predicted to die (false positive rate (FPR), 0.03; 95%CI 0.00-0.20). Of forty-six patients who were predicted to have an unfavorable outcome, four had a favorable outcome (PPV, 0.93; 95% CI, 0.81-0.98; FPR, 0.30; 95% CI; 0.08-0.65). Prediction of non-satisfactory quality of life was less accurate (PPV, 0.63; 95% CI, 0.26-0.90; FPR, 0.18; 95% CI 0.05-0.44). CONCLUSIONS: In patients with severe stroke, treating physicians' estimation of the risk of mortality or unfavorable functional outcome at six months is relatively inaccurate. Prediction of quality of life is even more imprecise.

Treatment restrictions in patients with severe stroke are associated with an increased risk of death.

INTRODUCTION: Treatment restrictions in the first 2 days after intracerebral haemorrhage have been independently associated with an increased risk of early death. It is unknown whether these restrictions also affect mortality if these are installed several days after stroke onset. PATIENTS AND METHODS: Sixty patients with severe functional dependence at day 4 after ischaemic stroke or intracerebral haemorrhage were included in this prospective two-centre cohort study. The presence of treatment restrictions was assessed at the day of inclusion. Information about mortality, functional outcome (modified Rankin scale) score and quality of life (visual analogue scale) was recorded 6 months after stroke onset. Poor outcome was defined as modified Rankin scale >3. Satisfactory quality of life was defined as visual analogue scale ≥ 60. RESULTS: At 6 months, 30 patients had died, 19 survivors had a poor functional outcome and 9 patients had a poor quality of life. Treatment restrictions were independently associated with mortality at 6 months (adjusted relative risk, 1.30; 95% confidence interval, 1.06-1.59; p = 0.01), but not with functional outcome. DISCUSSION: Our findings were observed in 60 selected patients with severe stroke. CONCLUSION: The instalment of treatment restrictions by itself may increase the risk of death after stroke, even if the first 4 days have passed. In future stroke studies, this potential confounder should be taken into account. Quality of life was satisfactory in the majority of the survivors, despite considerable disability.

Short-term erythropoietin treatment does not substantially modulate monocyte transcriptomes of patients with combined heart and renal failure.

BACKGROUND: Combined heart and renal failure is associated with high cardiovascular morbidity and mortality. Anti-oxidant and anti-inflammatory, non-hematopoietic effects of erythropoietin (EPO) treatment have been proposed. Monocytes may act as biosensors of the systemic environment. We hypothesized that monocyte transcriptomes of patients with cardiorenal syndrome (CRS) reflect the pathophysiology of the CRS and respond to short-term EPO treatment at a recommended dose for treatment of renal anemia. METHODS: Patients with CRS and anemia (n = 18) included in the EPOCARES trial were matched to healthy controls (n = 12). Patients were randomized to receive 50 IU/kg/week EPO or not. RNA from CD14(+)-monocytes was subjected to genome wide expression analysis (Illumina) at baseline and 18 days (3 EPO injections) after enrolment. Transcriptomes from patients were compared to healthy controls and effect of EPO treatment was evaluated within patients. RESULTS: In CRS patients, expression of 471 genes, including inflammation and oxidative stress related genes was different from healthy controls. Cluster analysis did not separate patients from healthy controls. The 6 patients with the highest hsCRP levels had more differentially expressed genes than the 6 patients with the lowest hsCRP levels. Analysis of the variation in log(2) ratios of all individual 18 patients indicated that 4 of the 18 patients were different from the controls, whereas the other 14 were quite similar. After short-term EPO treatment, every patient clustered to his or her own baseline transcriptome. Two week EPO administration only marginally affected expression profiles on average, however, individual gene responses were variable. CONCLUSIONS: In stable, treated CRS patients with mild anemia, monocyte transcriptomes were modestly altered, and indicated imprints of inflammation and oxidative stress. EPO treatment with a fixed dose has hematopoietic effects, had no appreciable beneficial actions on monocyte transcription profiles, however, could also not be associated with undesirable transcriptional responses.

Patterns of expression of DNA repair genes and relapse from melanoma.

PURPOSE: To use gene expression profiling of formalin-fixed primary melanoma samples to detect expression patterns that are predictive of relapse and response to chemotherapy. EXPERIMENTAL DESIGN: Gene expression profiles were identified in samples from two studies (472 tumors). Gene expression data for 502 cancer-related genes from these studies were combined for analysis. RESULTS: Increased expression of DNA repair genes most strongly predicted relapse and was associated with thicker tumors. Increased expression of RAD51 was the most predictive of relapse-free survival in unadjusted analysis (hazard ratio, 2.98; P = 8.80 × 10(-6)). RAD52 (hazard ratio, 4.73; P = 0.0004) and TOP2A (hazard ratio, 3.06; P = 0.009) were independent predictors of relapse-free survival in multivariable analysis. These associations persisted when the analysis was further adjusted for demographic and histologic features of prognostic importance (RAD52 P = 0.01; TOP2A P = 0.02). Using principal component analysis, expression of DNA repair genes was summarized into one variable. Genes whose expression correlated with this variable were predominantly associated with the cell cycle and DNA repair. In 42 patients treated with chemotherapy, DNA repair gene expression was greater in tumors from patients who progressed on treatment. Further data supportive of a role for increased expression of DNA repair genes as predictive biomarkers are reported, which were generated using multiplex PCR. CONCLUSIONS: Overexpression of DNA repair genes (predominantly those involved in double-strand break repair) was associated with relapse. These data support the hypothesis that melanoma progression requires maintenance of genetic stability and give insight into mechanisms of melanoma drug resistance and potential therapies.

Gene expression profiling of paraffin-embedded primary melanoma using the DASL assay identifies increased osteopontin expression as predictive of reduced relapse-free survival.

PURPOSE: Gene expression studies in melanoma have been few because tumors are small and cryopreservation is rarely possible. The purpose of this study was to evaluate the Illumina DASL Array Human Cancer Panel for gene expression studies in formalin-fixed melanoma primary tumors and to identify prognostic biomarkers. EXPERIMENTAL DESIGN: Primary tumors from two studies were sampled using a tissue microarray needle. Study 1: 254 tumors from a melanoma cohort recruited from 2000 to 2006. Study 2: 218 tumors from a case-control study of patients undergoing sentinel node biopsy. RESULTS: RNA was obtained from 76% of blocks; 1.4% of samples failed analysis (transcripts from <250 of the 502 genes on the DASL chip detected). Increasing age of the block and increased melanin in the tumor were associated with reduced number of genes detected. The gene whose expression was most differentially expressed in association with relapse-free survival in study 1 was osteopontin (SPP1; P = 2.11 x 10(-6)) and supportive evidence for this was obtained in study 2 used as a validation set (P = 0.006; unadjusted data). Osteopontin level in study 1 remained a significant predictor of relapse-free survival when data were adjusted for age, sex, tumor site, and histologic predictors of relapse. Genes whose expression correlated most strongly with osteopontin were PBX1, BIRC5 (survivin), and HLF. CONCLUSION: Expression data were obtained from 74% of primary melanomas and provided confirmatory evidence that osteopontin expression is a prognostic biomarker. These results suggest that predictive biomarker studies may be possible using stored blocks from mature clinical trials.

Glutathione peroxidase 2 and aquaporin 8 as new markers for colonic inflammation in experimental colitis and inflammatory bowel diseases: an important role for H2O2?

OBJECTIVE: Different mouse models of inflammatory bowel diseases (IBD) demonstrate various aspects of the pathophysiology of IBD. We looked for overlapping gene expression profiles in three different mouse models of experimental colitis and analysed whether these overlapping genes are of help to find new genes that could be used as general markers in human IBD. METHODS: Using Agilent mouse TOX oligonucleotide microarrays, we analysed the gene expression profiles in three widely used models of experimental colitis: 2,4,6-trinitrobenzene sulphonic acid, dextran sodium sulfate and CD4CD45RB transfer and looked for overlapping gene expression in these models. Overlapping genes were analysed using Lightcycler (Roche Diagnostics, Mannheim, Germany) in biopsy materials from human IBD and control tissue. RESULTS: Compared with control mice in dextran sodium sulfate, 2,4,6-trinitrobenzene sulphonic acid and the CD45RB transfer colitis mice five known genes, extracellular proteinase inhibitor (Expi), glutathione peroxidase 2 (Gpx2), mast cell protease 1 (Mcpt1), resistin-like beta (Retnlb) and sulphatase 2 (Sulf2), and two unknown genes were upregulated and the two genes aquaporin 8 (Aqp8) and kallikrein 5 (Klk5) were downregulated in all three models. In human Crohn's disease and ulcerative colitis biopsies, one of the upregulated glutathione peroxidase (Gpx2) and one of the downregulated Aqp8 genes in the mouse models were also differentially expressed in affected colonic tissue of patients with IBD. CONCLUSION: Experimental mouse models are suitable models for the search of new markers for human IBD. As both Gpx2 and Aqp8 are involved in H2O2 metabolism (Gpx2 as a radical scavenger whereas Aqp8 facilitates its diffusion), upregulation of Gpx2 and downregulation of Aqp8 could be a mechanism to defend against severe oxidative stress and indicate that H2O2 is a universal mediator in the inflammatory process in the colon. This provides a focus on homeostasis of the antioxidant pathway and its importance in IBD.

Muramyl dipeptide-induced differential gene expression in NOD2 mutant and wild-type Crohn's disease patient-derived dendritic cells.

BACKGROUND: Mutations in the gene encoding the nucleotide-binding oligomerization domain 2 (NOD2) protein are associated with Crohn's disease (CD), but the mechanism underlying this is not completely understood. To study the mechanism of CD resulting from NOD2 mutations, we analyzed NOD2-dependent whole-genome expression profiles of patient-derived antigen-presenting cells. PATIENTS AND METHODS: Monocyte-derived dendritic cells (DCs) from CD carriers of double-dose NOD2 mutations, wild-type CD patients, and wild-type healthy volunteers were stimulated with the NOD2 ligand muramyl dipeptide. Whole-genome microarrays were used to assess the differential gene expression. The clustering of significantly changed genes was analyzed by online gene ontology mapping software. RESULTS: In the DCs from the wild-type CD patient group, 683 genes were significantly changed, with most of the genes clustering in the pathways of inflammatory response. In addition, a significant number of genes clustered in the apoptosis regulation-related pathway. In the DCs from the healthy volunteer group, only 50 genes were significantly changed, predominantly those belonging to the response to pathogen pathway. Analysis of differentially expressed gene ontology pathways in the DCs from the NOD2 mutant CD patient group showed that the transcription of pathogen response genes was absent. In this group, 298 genes were significantly changed, predominantly clustering in the negative apoptosis regulation and cell organization and biogenesis pathways. CONCLUSIONS: Our results suggest that NOD2 mutations may result in perpetuation of mucosal inflammation through insufficient pathogen elimination. Further, these observations implicate a possible role of defective regulation of dendritic cell apoptosis in CD pathogenesis.

Bone morphogenetic protein 4 expressed in esophagitis induces a columnar phenotype in esophageal squamous cells.

BACKGROUND & AIMS: Barrett's esophagus (BE) is a metaplastic condition in which normal squamous esophageal epithelium is replaced by columnar epithelium. It is proposed that one of the possible mechanisms is dedifferentiation of squamous epithelium into columnar epithelium. The pathophysiology through which this metaplasia occurs is unknown. A recent study by serial analysis of gene expression showed that bone morphogenetic protein 4 (BMP-4) is uniquely expressed in BE. In this study, the role of the BMP pathway in the metaplastic transformation of normal squamous cells into columnar cells was examined. METHODS: Tissues from patients with esophagitis and BE and in an esophagitis-BE rat model were examined for the activation of the BMP pathway. Short-term cultures of primary normal squamous esophageal cells were treated with BMP-4, and cell biological changes were examined by Western blot analysis, immunohistochemistry, and microarrays. RESULTS: In both human and rat tissues, the BMP pathway proved to be activated in esophagitis and BE. Upon incubation of squamous cell cultures with BMP-4, the cytokeratin expression pattern showed a shift that was consistent with columnar epithelium. Involvement of the BMP pathway was suggested by up-regulation of Phosphorylated-Smad 1/5/8 (P-Smad 1/5/8) that was effectively blocked by Noggin, a BMP antagonist. Comparison of the gene expression profiles of squamous cells, BMP-4-treated squamous cells, and BE cells showed a significant shift in the profile of the BMP-4-treated squamous cells toward that of the cultured BE cells. CONCLUSIONS: These results suggest that the BMP pathway could play a role in the transformation of normal esophageal squamous cells into columnar cells.

Comparative analysis of colonic gene expression of three experimental colitis models mimicking inflammatory bowel disease.

BACKGROUND: Mouse models of inflammatory bowel diseases (IBD) are used to unravel the pathophysiology of IBD and to study new treatment modalities, but their relationship to Crohn's disease (CD) or ulcerative colitis (UC) is speculative. METHODS: Using Agilent mouse TOX oligonucleotide microarrays, we analyzed colonic gene expression profiles in three widely used models of experimental colitis. In 2 of the models (TNBS and DSS-induced colitis), exogenous agents induce the colitis. In the third model the colitis is induced after transfer of a T-cell population (CD4(+)CD45RB(high) T cells) that lacks regulatory cells into an immunodeficient host. RESULTS: Compared with control mice, in DSS, TNBS, and the CD45RB transfer colitis mice, 387, 21, and 582 genes were more than 2-fold upregulated in the intestinal mucosa. Analyses of exclusively shared gene expression profiles between the different models revealed that DSS/transfer colitis share 69 concordantly upregulated genes, DSS/TNBS 6, and TNBS/transfer colitis 1. Seven genes were upregulated in all three models. The CD45RB transfer model expression profile included the most genes that are known to be upregulated in IBD. Of 32 genes that are known to change transcriptional activity in IBD (TNF, IFN-gamma, Ltbeta, IL-6, IL-16, IL-18R1, IL-22, CCR2, 7, CCL2, 3, 4, 5, 7, 11, 17, 20, CXCR3, CXCL1, 5, 10, Mmp3, 7,9, 14, Timp1, Reg3gamma, and Pap, S-100a8, S-100a9, Abcb1, and Ptgs2), 2/32 are upregulated in TNBS, 15/32 are upregulated or downregulated in DSS and 30/32 are upregulated or downregulated in the CD45RB transfer colitis. CONCLUSION: The pattern of gene expression in the CD45RB transfer model most closely reflects altered gene expression in IBD.

A global test for groups of genes: testing association with a clinical outcome.

MOTIVATION: This paper presents a global test to be used for the analysis of microarray data. Using this test it can be determined whether the global expression pattern of a group of genes is significantly related to some clinical outcome of interest. Groups of genes may be any size from a single gene to all genes on the chip (e.g. known pathways, specific areas of the genome or clusters from a cluster analysis). RESULT: The test allows groups of genes of different size to be compared, because the test gives one p-value for the group, not a p-value for each gene. Researchers can use the test to investigate hypotheses based on theory or past research or to mine gene ontology databases for interesting pathways. Multiple testing problems do not occur unless many groups are tested. Special attention is given to visualizations of the test result, focussing on the associations between samples and showing the impact of individual genes on the test result. AVAILABILITY: An R-package globaltest is available from http://www.bioconductor.org

Fluorescent labelling of cRNA for microarray applications.

Microarrays of oligonucleotide expression libraries can be hybridised with either cDNA, generated from mRNA during reverse transcription, or cRNA, generated in an Eberwine mRNA amplification procedure. While methods for fluorescent labelling of cDNA have been thoroughly investigated, methods for cRNA labelling have not. To this purpose, we developed an aminoallyl-UTP (aa-UTP) driven cRNA labelling protocol and compared it in expression profiling studies using spotted 7.5 K 65mer murine oligonucleotide arrays with labelling via direct incorporation of Cy-UTPs. The presence of dimethylsulfoxide during coupling of aa-modified cRNA with N-hydroxysuccinimide-modified, fluorescent Cy dyes greatly enhanced the labelling efficiency, as analysed by spectrophotometry and fluorescent hybridisation signals. Indirect labelling using aa-UTP resulted in 2- to 3-fold higher degrees of labelling and fluorescent signals than labelling by direct incorporation of Cy-UTP. By variation of the aa-UTP:UTP ratio, a clear optimal degree of labelling was found (1 dye per 20-25 nt). Incorporation of more label increased Cy3 signal but lowered Cy5 fluorescence. This effect is probably due to quenching, which is more prominent for Cy5 than for Cy3. In conclusion, the currently developed method is an efficient, robust and inexpensive technique for fluorescent labelling of cRNA and allows sensitive detection of gene expression profiles on oligonucleotide microarrays.