RT-PCR evaluation of MtF mRNA expression in peripheral blood reticulocytes.

DNAase-treated mRNA preparations were reverse transcribed and PCR amplified, and amplification products were then evaluated by electrophoresis. Panel A shows an RT-PCR study using MtF specific-oligonucleotides (45 cycles); in lanes 2, 5, and 6, a fragment of 204 bp (indicated by the arrow) is clearly observable. The fragment was digested by BamHI to confirm the identity of the product as MtF. Panel B shows the negative findings of the control study without RT treatment. The arrow indicates the expected position of the 204-bp amplicon. Panel C shows an RT-PCR study using oligonucleotides specific for glyceraldehyde phosphate dehydrogenase (GAPDH); in all lanes but one (the negative control), a band of 200 bp (indicated by the arrow) is visible. Lanes 1 and 3: heterozygous women with the ALAS2 Cys395Tyr mutation.12 Lane 2: hemizygous man with the ALAS2 Cys395Tyr mutation and clinically manifest XLSA.12 Lanes 4 and 5: hemizygous men (brothers) with the ALAS2 Arg560His mutation and absent phenotypic expression of the disease (lane 4) or clinically manifest XLSA (lane 5).14 Lane 6: HeLa cells expressing the MtF gene after transfection. Lane 7: water. Lane 8: molecular markers.