The transition, located at 325 bp downstream of exon 10, was found serendipitously because the primer we designed for the amplification of exon 10 was positioned very deep inside intron 10. We usually use the primer which we designed originally for the F8 analysis. It was difficult to design the primer pair that amplifies exon 10 in our examination. Therefore, as a result of careful selection, the primer positions were decided at deep inside of the intron. The genetic abnormalities which cause haemophilia A are usually detected in Erismodegib in vivo F8. However,
in about 2% of Haemophilia A patients, no genetic abnormality can be found, even after complete sequencing of F8 including the promoter and the 3′-UTR regions. Because F8 is very large, 186 kb long, the range which can usually be analysed is restricted to the coding region including flanking splice sites and is less than one-tenth of the entire F8 gene. The remainder regions, representing almost all of the intronic sequences, are unanalysed. Therefore,
in cases where a gene abnormality has not been detected there is the possibility that some abnormalities are hidden in the intronic regions which remain unanalysed. The F8 gene is mainly expressed in sinusoidal endothelial cells and Kupffer cells in the liver . However, trace amount levels this website of F8 mRNA, ectopic mRNA, exist in blood cells and can be analysed by RT-PCR amplification [10, 15, 16]. The analysis of the ectopic mRNA obtained from blood is available to observe the state of splicing, and this analysis is widely used to screen for genetic abnormalities. If the mutation exists deep inside the intron, it will give some influence on the transcript. Therefore, examination of the mRNA is very effective to detect unknown genetic mutations or MCE公司 rearrangements. Furthermore, the analysis of ectopic mRNA is also effective to examine the influence that detected gene abnormalities exert on the splice.
In fact, the mutation that we found was confirmed to cause the splice abnormality by analysing ectopic mRNA. Although predictive software analysis  suggested that this patient’s mutation may cause splicing abnormalities, there was no further evidence to prove this. We analysed ectopic mRNA by using the method that had been reported by El-Maarri et al. . This method utilizes the nested PCR technique and is suitable for detection of small amounts of mRNA. At first, the F8 is divided into four regions, exon 1–8, 8–14, 14–21 and 19–26, and is amplified. Then, each of the first amplification products are further divided into two regions and amplified again.