Biomedical subjects

Paolo Fardin

Publications and source records attributed to Paolo Fardin.

4 recordsLinked to original sources

PubMed2007

Normalization of low-density microarray using external spike-in controls: analysis of macrophage cell lines expression profile.

BACKGROUND: The normalization of DNA microarrays allows comparison among samples by adjusting for individual hybridization intensities. The approaches most commonly used are global normalization methods that are based on the expression of all genes on the slide and on the modulation of a small proportion of genes. Alternative approaches must be developed for microarrays where the proportion of modulated genes and their distribution are unknown and they may be biased towards up- or down-modulated trends. RESULTS: The aim of the work is to study the use of spike-in controls to normalize low-density microarrays. Our test-array was designed to analyze gene modulation in response to hypoxia (a condition of low oxygen tension) in a macrophage cell line. RNA was extracted from controls and cells exposed to hypoxia, mixed with spike RNA, labeled and hybridized to our test-array. We used eight bacterial RNAs as source of spikes. The test-array contained the oligonucleotides specific for 178 mouse genes and those specific for the eight spikes. We assessed the quality of the spike signals, the reproducibility of the results and, in general, the nature of the variability. The small values of the coefficients of variation revealed high reproducibility of our platform either in replicated spots or in technical replicates. We demonstrated that the spike-in system was suitable for normalizing our platform and determining the threshold for discriminating the hypoxia modulated genes. We assessed the application of the spike-in normalization method to microarrays in which the distribution of the expression values was symmetric or asymmetric. We found that this system is accurate, reproducible and comparable to other normalization methods when the distribution of the expression values is symmetric. In contrast, we found that the use of the spike-in normalization method is superior and necessary when the distribution of the gene expression is asymmetric and biased towards up-regulated genes. CONCLUSION: We demonstrate that spike-in controls based normalization is a reliable and reproducible method that has the major advantage to be applicable also to biased platform where the distribution of the up- and down-regulated genes is asymmetric as it may occur in diagnostic chips.

Action Potentials↗

PubMed2006

Hypoxia modifies the transcriptome of primary human monocytes: modulation of novel immune-related genes and identification of CC-chemokine ligand 20 as a new hypoxia-inducible gene.

Peripheral blood monocytes migrate to and accumulate in hypoxic areas of inflammatory and tumor lesions. To characterize the molecular bases underlying monocyte functions within a hypoxic microenvironment, we investigated the transcriptional profile induced by hypoxia in primary human monocytes using high-density oligonucleotide microarrays. Profound changes in the gene expression pattern were detected following 16 h exposure to 1% O(2), with 536 and 677 sequences showing at least a 1.5-fold increase and decrease, respectively. Validation of this analysis was provided by quantitative RT-PCR confirmation of expression differences of selected genes. Among modulated genes, 74 were known hypoxia-responsive genes, whereas the majority were new genes whose responsiveness to hypoxia had not been previously described. The hypoxic transcriptome was characterized by the modulation of a significant cluster of genes with immunological relevance. These included scavenger receptors (CD163, STAB1, C1qR1, MSR1, MARCO, TLR7), immunoregulatory, costimulatory, and adhesion molecules (CD32, CD64, CD69, CD89, CMRF-35H, ITGB5, LAIR1, LIR9), chemokines/cytokines and receptors (CCL23, CCL15, CCL8, CCR1, CCR2, RDC1, IL-23A, IL-6ST). Furthermore, we provided conclusive evidence of hypoxic induction of CCL20, a chemoattractant for immature dendritic cells, activated/memory T lymphocytes, and naive B cells. CCL20 mRNA up-regulation was paralleled by increased protein expression and secretion. This study represents the first transcriptome analysis of hypoxic primary human monocytes, which provides novel insights into monocyte functional behavior within ischemic/hypoxic tissues. CCL20 up-regulation by hypoxia may constitute an important mechanism to promote recruitment of specific leukocyte subsets at pathological sites and may have implications for the pathogenesis of chronic inflammatory diseases.

Cell Hypoxia↗

PubMed2006

Growth arrest-inducing genes are activated in Dbl-transformed mouse fibroblasts.

The Dbl oncogene is a guanine nucleotide exchange factor for Rho GTPases and its activity has been linked to the regulation of gene transcription. Dbl oncogene expression in NIH3T3 cells leads to changes in morphological and proliferative properties of these cells, inducing a highly transformed phenotype. To gain insights into Dbl oncogene-induced transformation we compared gene expression profiles between Dbl oncogene-transformed and parental NIH3T3 cells by cDNA microarray. We found that Dbl oncogene expression is associated with gene expression modulation involving upregulation of 51 genes and downregulation of 49 genes. Five of the overexpressed genes identified are known to exert antiproliferative functions. Our observations suggest that the expression of Dbl oncogene in NIH3T3 may lead to the induction of genes associated with cell cycle arrest, possibly through the activation of stress-induced kinases.

Animals↗

PubMed2002

Analysis of HLA-G expression in breast cancer tissues.

Among the different mechanisms by which cancer can elude the immune system, alterations in the expression of human leukocyte antigen (HLA) class I molecules on tumor cells may play a crucial role by impairing the HLA molecules interaction with T and natural killer (NK) cells specific receptors. More recently, aberrant expression of HLA-G has been described in different tumor tissues in addition to HLA class I downregulation. The HLA-G molecule is a nonclassical HLA class I antigen selectively expressed by trophoblast and thymic epithelial cells. Several studies reported that the HLA-G function might represent an additional mechanism of tumor immune escape, mainly inhibiting NK and cytotoxic T-cell activity. Here we report the analysis of HLA-G expression both at RNA level by reverse transcriptase-polymerase chain reaction and at protein level by Western blot and immunohistochemistry in 25 breast cancer patient tissues. The aim of this study was to elucidate the HLA-G gene expression pattern in breast tumor tissues and correlate it with HLA class I alterations. Our results demonstrated that HLA-G molecules expression was never found even in a group of patients revealing HLA class I total loss, and that HLA-G is not expressed in breast cancer tissue with a low-tumor grade (G1-G2) and minimal stromal contamination.

Blotting, Western↗