available Taqman assay (Applied Biosystems). Fluorescence outputs were quantified in real time using a 7900HT Fast Real Time PCR System and the data were analyzed using SDS software v.2.2.2 (Applied Biosystems). One way analysis of variance was used to determine the correlation of alternative transcript abundance with the rs950169 and rs2135551 genotypes in human brain tissue. Statistical analyses were performed both separately in control and Alzheimer's disease prefrontal cortex samples, and as a combined subject analysis. A genomic DNA fragment of 4028 bp from the ADAMTSL3 gene that included exons 29 and 30 with flanking intron sequences was PCR-amplified from a reference genomic DNA using the following primers: gggaattcAAGGGCAGATACCCCAAAGT and taggatccCGCTTGCTCTTCCAACTACC. Subsequently, the PCR fragment was subcloned into pSPL3 (GibcoBRL) as a minigene. The minor allele of rs950169 was generated in the minigene by mutagenesis (QuikChange Mutagenesis kit, Stratagene) and the sequences were confirmed by DNA sequencing. The minigenes were transfected into HEK293 cells using Lipofectamine2000 (Invitrogen). After the 48 h transfection, RNA was extracted using RNeasy kit (Qiagen) and converted into cDNA using High-Capacity cDNA Archive Kit (Applied Biosystems). Alternative splicing of exon 29 and exon 30 was detected by Taqman assays and agarose gel.
