Biologically relevant effects of mRNA amplification on gene expression profiles

BACKGROUND: Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. RESULTS: Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. CONCLUSION: This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways

Urinary nitric oxide metabolites and individual blood pressure progression to overt hypertension

Baseline blood pressure (BP) is the strongest known determinant of progression to hypertension, but for an individualized prediction of the incidence of hypertension, the identification of additional biomarkers is crucial. In animal models of hypertension, renal nitric oxide (NO) handling modifies the systemic BP responses prior to the development of hypertension. This study aimed to evaluate whether urinary NO metabolites (NOx) predict the progression of hypertension in normotensive subjects. Among 62 participants enrolled in the Flemish Study on Environment, Genes and Health Outcomes, we assessed progression to hypertension over 4.6 years. In a case-control design, 49 normotensive subjects including 10 subjects with high-normal blood pressure were enrolled of whom 25 remained normotensive (controls), whereas 24 'progressed' to hypertension (progressors). Thirteen hypertensive patients served as negative controls. Urinary NOx concentration, renal function and the urinary excretion of electrolytes were assessed at baseline and follow-up. At baseline, progressors showed higher BP values than controls and urinary NOx concentration was significantly lower in progressors as compared to the normotensive controls (p < 0.01). In all initially normotensive subjects baseline urinary NOx concentration was associated with follow-up BP (r=-0.55, p < 0.001) and the relative increase of BP over time (r=-0.47, p < 0.001). In progressors baseline urinary NOx was associated with follow-up BP (r=-0.52, p < 0.009) and the relative increase of BP over time (r=-0.44, p=0.033). Baseline urinary NOx and BP were independent predictors for the relative BP increase. A urinary NOx threshold of < 130.5 mg/L predicted 75% of all progressors. In context with high-normal baseline BP, 87.5% of all progressors were identified. These findings indicate that urinary NO metabolites are associated with BP development in normotensive subjects. Moreover, urinary NOx predicts the progression to hypertension independent of baseline BP suggesting urinary NOx as a biomarker for individual new-onset hypertension.status: publishe