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

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