RT-PCR analysis of wild-type and mutant fibrinogen Bβ-chain mRNAs.

(A) Agarose (2%)-gel electrophoresis of RT-PCR products, amplified from total RNA extracted from HeLa cells after transfection either with IVS6 + 13C > T or IVS7 + 1G > T mutant minigenes. M indicates molecular weight marker (pUC8HaeIII). (B) Analysis of the same RT-PCR products as in panel A, labeled by fluorescent hot-stop technique, separated on an automated DNA sequencer, and analyzed by GeneScan software. In order, from left to right, are shown GeneScan Analysis windows of the capillary-electrophoretic runs of RT-PCR products for the wild-type, IVS7 + 1G > T, and IVS6 + 13C > T minigenes, respectively. Molecular weight standard peaks are in gray; peaks corresponding to the labeled fragments, indicated by arrows, are in black. GeneScan fluorescence units (x-axis) and length of molecular weight fragments (y-axis) are reported. (C) Fragment size and relative amounts of fluorochrome-labeled RT-PCR products, indicated by arrows in panel B, are shown. Molecular weights and peak areas were evaluated by means of GeneScan software. The percentage values correspond to the peak areas, setting the sum of RT-PCR black areas of each experiment equal to 100%.