Figure 2.
VWF mRNA transcription analysis. (A) The schematic scale of the VWF coding region (exons 2-52) with the position of designed primers for amplification of the full-length VWF cDNA and corresponding amplicons segments. Agarose gel electrophoresis image displays the 10 overlapping RT-PCR products of VWF using total RNA from the index patient (IP) as a template. (B) Comparative levels of IP-ECFCs VWF mRNA quantified by real-time PCR, using primer/probe combinations directing 3 different sites in VWF cDNA, across exons 4 and 5, 11 and 12, and 43 through 45. In the first set, the forward primer and the probe both were designed to target sequences in exon 4, and a reverse primer was designed across the exons 4-5 junction of VWF cDNA. In the second set, the forward and the reverse primers were directed at exons 11 and 12, respectively, and the fluorogenic probe targeted a sequence across the junction of the exons 11 and 12. In the third set, a fluorogenic probe as well as forward and reverse primers were targeting sequences in exon 44, exons 43 and 44, and 44 and 45 junctions, respectively. The measurements were performed based on the comparative CT (ΔΔ CT) method. Measurements of VWF mRNA levels were normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or actin β (ACTB) mRNA. (C) Agarose gel electrophoresis of multiplex RT-PCR products amplified using primers designed across the junction of exons 2-3 (forward) and exons 51-52 (reverse) along with control internal primers (forward and reverse primers targeting sites in exon 12-13 junctions and exon 18, respectively). RT-PCR products of RNA obtained from the IP demonstrate a larger product (2820 bp) corresponding to the abnormal deleted VWF transcript derived from deleted VWF allele (del4-34) and a smaller fragment (979 bp) relevant to the normal transcript (lane 1). However, RT-PCR using RNA from healthy control as the template shows only the smaller normal fragment (lane 2). Molecular weight marker: GeneRuler 1kb ladder (Thermo Scientific, Germany).

VWF mRNA transcription analysis. (A) The schematic scale of the VWF coding region (exons 2-52) with the position of designed primers for amplification of the full-length VWF cDNA and corresponding amplicons segments. Agarose gel electrophoresis image displays the 10 overlapping RT-PCR products of VWF using total RNA from the index patient (IP) as a template. (B) Comparative levels of IP-ECFCs VWF mRNA quantified by real-time PCR, using primer/probe combinations directing 3 different sites in VWF cDNA, across exons 4 and 5, 11 and 12, and 43 through 45. In the first set, the forward primer and the probe both were designed to target sequences in exon 4, and a reverse primer was designed across the exons 4-5 junction of VWF cDNA. In the second set, the forward and the reverse primers were directed at exons 11 and 12, respectively, and the fluorogenic probe targeted a sequence across the junction of the exons 11 and 12. In the third set, a fluorogenic probe as well as forward and reverse primers were targeting sequences in exon 44, exons 43 and 44, and 44 and 45 junctions, respectively. The measurements were performed based on the comparative CT (ΔΔ CT) method. Measurements of VWF mRNA levels were normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or actin β (ACTB) mRNA. (C) Agarose gel electrophoresis of multiplex RT-PCR products amplified using primers designed across the junction of exons 2-3 (forward) and exons 51-52 (reverse) along with control internal primers (forward and reverse primers targeting sites in exon 12-13 junctions and exon 18, respectively). RT-PCR products of RNA obtained from the IP demonstrate a larger product (2820 bp) corresponding to the abnormal deleted VWF transcript derived from deleted VWF allele (del4-34) and a smaller fragment (979 bp) relevant to the normal transcript (lane 1). However, RT-PCR using RNA from healthy control as the template shows only the smaller normal fragment (lane 2). Molecular weight marker: GeneRuler 1kb ladder (Thermo Scientific, Germany).

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