Genomic and cDNA sequences were derived from known sequences (ABCB4: “type”:”entrez-nucleotide”,”attrs”:”text”:”AC005068.2″,”term_id”:”10122135″,”term_text”:”AC005068.2″AC005068.2 selleck chemicals Rucaparib for non-coding exons -3 to 1 and coding exons 2 and 3; “type”:”entrez-nucleotide”,”attrs”:”text”:”AC006154.1″,”term_id”:”4156145″,”term_text”:”AC006154.1″AC006154.1 for exons 4 to 12; AC0005045.2 for exons 13 to 28; and “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_000443.2″,”term_id”:”9961253″,”term_text”:”NM_000443.2″NM_000443.2 for cDNA; ABCB11: GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AC008177.3″,”term_id”:”10716656″,”term_text”:”AC008177.3″AC008177.3 for promoter and exons 1 to 21; “type”:”entrez-nucleotide”,”attrs”:”text”:”AC069165.2″,”term_id”:”8705068″,”term_text”:”AC069165.

2″AC069165.2 for exons 22 to 28 and “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_003742.2″,”term_id”:”21536377″,”term_text”:”NM_003742.2″NM_003742.2 for cDNA). ABCB4 and ABCB11: Sequencing of ABCB4 covered a proximately 8000 bp, including (1) 2000 bp of the upstream promoter region and non-coding exon -3 to 1 and, (2) coding exons 2-28 and 100-350 bp of the intronic sequence around each exon. For ABCB11, sequencing covered 10 000 bp including (1) non-coding exon 1 and 2400 bp of the upstream promoter region and, (2) coding exons 2-28 and 100-350 bp of the intronic sequence around each exon. Primers for genomic DNA were designed to span all exons and at least 100 bp of the flanking intronic sequence at the 5�� and 3�� end of each exon.

The DNA sequence of purified PCR fragments was analyzed on an ABI3700 capillary sequencer (ABI, Weiterstadt, Germany) and assembled using the phredPhrap, Consed and PolyPhred software (University of Washington). Details regarding the primers, optimized PCR conditions and subsequent purification and sequencing of the fragments are available at [email protected]. ABCC2: Three non-synonymous polymorphisms with a potential impact on MRP2 function and expression were chosen for genotyping[25]: 1249G>A variant (V417I, rs2273697), 3563T>A (V1188E, rs17222723) and 4544G>A (C1515Y, rs8187710). Genotyping was performed with the Custom TaqMan SNP Genotyping Assays procedure (Applied Biosystems, Foster City, CA, USA) which contained a sense- and an antisense primer and two probes, labeled with fluorescent reporter dyes, either VIC or 6-Fam at the 5�� end and a non-fluorescent quencher at the 3�� end to distinguish between alleles 1 and 2, respectively.

Primer and probe sequences for individual SNPs are given in Table Table1.1. Probe solution (0.625 ��L) and 12.5 ��L of 2 �� Universal PCR Master Mix (Applied Biosystems) were brought to 25 ��L with 20 ng of genomic DNA. PCR reaction (2 min at 50��C, followed by 10 min at 95��C and 40 cycles of 15 s at 92��C and 1 min at 60��C). Allelic discrimination was processed Drug_discovery with an ABI PRISM 7700 Sequence Detector.