Comparison of Three RT-PCR Based Methods for Relative Quantification of mRNA

Comparison of three RT-PCR based methods: semi-quantitative, competitive and real--time RT-PCR for relative quantification of mRNA is presented. Aminopeptidase N expressed on human promyeloid HL-60 cell line, at basal and activated state, served as a model for comparison. HL-60 cells were stimulated with IFN- (6 ng/mL) for 72 h at 37 oC, total cellular RNA was isolated, reverse transcribed to cDNA and semi-quantitative, competitive and real-time RT-PCR were performed to obtain the relative levels of mRNA for aminopeptidase N. The data obtained showed that all three RT-PCR based methods gave reliable and comparable results, i.e. approximately twofold increase of aminopeptidase N mRNA on IFN- stimulated HL-60 cells. Thus, in spite of rapid advances made in the area of real-time RT-PCR, end-point RT-PCR such as competitive and semi-quantitative RT- -PCR, although laborious and time consuming, may still remain useful techniques for relative mRNA quantification when small number of samples are to be analyzed

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life

Leber's hereditary optic neuroretinopathy (LHON) associated with mitochondrial DNA point mutation G11778A in two Croatian families

Background: LHON is LEBER's hereditary optic neuroretinopathy with bilateral acute or subacute loss of central vision due to optic atrophy. It is linked to point mutations of mitochondrial DNA. Mitochondrial DNA (mtDNA) is a small circular molecule of 16,5 kB, which encodes the enzymes of the respiratory chain in mitochondria. It is inherited maternally. The most common pathogenic mtDNK point mutations associated with LHON are 3460 G→A, 11778 G→A and 14484 T→C. These mutations are linked with the defects of subunits of complex I (NADH-dehydrogenase-ubiquinone reductase) in mitochondria. The 11778 G→A point mutation has the worst optic outcome (blindness). - - - - - Methods: Complete ophthalmologic examination including best corrected visual acuity on the both eyes according to Snellen chart, ophthalmoscopy, Goldmann perimetry and colour vision with Ishichara plates were done. mtDNA point mutation 11778 G→A was detected in DNA of peripheral blood lymphocytes using PCR and RFLP method. - - - - - Results: This is the first study of Croatian patients with LHON defect associated with mtDNK mutations. Two LHON 11778 G→A families are presented in this paper. The mothers and female relatives are LHON mutants without symtoms, while the sons mutants suffered from blindness. - - - - - Conclusions: Molecular diagnosis may help in explanation of the cause of LHON disease. LHON should not be based solely on clinical description

Use of antibody-coated polyacrylic plastic beads for semiquantittavie determination of cell surface antigens.

Polyacrylic plastic beads coated with specific antibodies, which had previously been shown to bind specifically to cells carrying the related antigen, are shown to react quantitatively with the antigen on individual cells. The number of beads per cell which can be counted by simple microscopy is a measure of the amount of antigen accessible on the cell surface. The method may also be used in combination with other methods for cell surface antigens, such as immunofluorescence, for multiple antigen determination

Fc-receptor-bearing cells in spleen of mice injected with cell-free Ehrlich ascites fluid (EAF).

The kinetics of different cell populations (T and B) and subpopulations (one bearing easily releasable FcR and one bearing stable FcR) was followed in spleens of mice after one single i.p. injection of EAF. The number of FcR bearing cells doubled within 2-7 days after EAF injection. This increase was due to cells temperature sensitive FcR and was accompanied by the doubling of theta positive cells. These results, supported by the demonstration of doubly labeled (theta + FcR +) cells, suggest that EAF injected into normal mice causes the appearance of T-cells expression easily releasable FcR. These cells, according to Fridman et al. (1977) are suppressor cells. Maximal increase of theta positive cells and of cells with temperature sensitive FcR detected in vitro coincided with the maximum of the suppressive activity of EAF detected in vivo