Custom taqman expression assay

Genetic analysis was performed at the Institute for Biochemistry and Molecular Genetics, Faculty of Medicine, at the University of Ljubljana (Slovenia). Genomic DNA was extracted from venous blood samples using FlexiGene DNA kit 250 (Qiagen, Hilden, Germany), following the manufacturer instructions. The LPA genotyping and analysis of LPA kringel repeats was performed with TaqMan Universal PCR Master Mix and the QuantStudio 7 Flex real-time PCR system (all Applied Biosystems, Foster City, CA, USA). The rs10455872 and rs3798220 LPA polymorphisms were analysed using TaqMan SNP genotyping assays (C_30016089_10, C_25930271_10; Applied Biosystems, Foster City, CA, USA). Ten percent of the samples were reanalysed to monitor the quality of the genotyping. The LPA kringel repeats were analysed in triplicate using multiplex qPCR with a custom TaqMan expression assay for exon 5 of the LPA gene, and TaqMan Copy Number Reference Assay for the RNAseP gene that was used as a single copy reference gene (both Applied Biosystems, Foster City, CA, USA). If there were any discrepancies of >0.25 in the Ct values in replicates for either assay in individual samples, the sample was reanalysed. The relative number of KIV-2 repeats representing average value of repeats on both alleles in the individual patients was determined using the CopyCaller v2.1 software (Applied Biosystems, Foster, CA, USA).

Total RNA was prepared using RNeasy Mini RNA kit (Qiagen, Hilden, Germany) and genomic DNA was removed using RNase-free DNase I set (Qiagen). Levels of t-PA, Sp1, Sp2, Sp3, Sp4, KLF2, KLF4, KLF5, KLF6, KLF9, KLF10, KLF13, KLF15, p300, and PCAF mRNA were analyzed with real-time PCR, performed on an Applied Biosystems 7500 Fast Real-Time PCR System using cDNA and Taqman reagents obtained from Applied Biosystems. t-PA mRNA was analyzed with a Custom Taqman Expression Assay (Applied Biosystems) using the following sequences: t-PA forward primer: 5′-GGC CTT GTC TCC TTT CTA TTC G-3′, t-PA reverse primer: 5′-AGC GGC TGG ATG GGT ACA G-3′, and t-PA probe 5′-TGA CAT GAG CCT TCA GCC GCT-3′. Confirmation of knock-down of respective Sp/KLF factor and PCAF was performed using Taqman Gene Expression Assays (Applied Biosystems) and of p300 using a QuantiTect Primer Assay from Qiagen (all listed in Online Resource 1). HPRT was used as endogenous internal standard in all experiments except in KLF9 studies where GUSB was used.