Other investigators may have received portions of these tissue samples. Patient diagnostic and treatment information were made available for each tissue. Tissues were collected as snap frozen specimens stored at -80°C. Sample preparation and genomic DNA isolation Each snap frozen tissue was sectioned on a bed of dry ice to ensure minimal thawing during sample preparation. An approximately 30-50 mg piece of tissue was cut and an adjacent piece of tissue was removed for formalin fixation and paraffin embedding

for subsequent histological processing. Genomic DNA was isolated from tissue samples via homogenization in ice cold lysis buffer [10 mM Tris pH 8.0, 0.1 M ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS), 100 μg/mL Proteinase MLN0128 datasheet K, 25 μg/mL RNAase]. Subsequent phenol-chloroform extraction was carried out as previously described [24]. Integrity and concentration of each resulting DNA sample was assessed buy IWR-1 by agarose gel electrophoresis. Sequencing primer design The known coding region of SOSTDC1 is contained within two exons. Other potentially transcribed areas have been identified in the University of California Santa Clara Genome database [25–27]. Two of these potential exons occur upstream of the coding region and an additional exon occurs between the known coding exons for a total

of five putative exons or regulatory regions at this locus (see Additional file 1). Primers were designed for direct sequencing for a total of 13 pairs of direct sequencing primers (see Additional file Vasopressin Receptor 2). All primers were synthesized by Integrated DNA Technologies (IDT). PCR amplification and direct sequencing Each direct sequencing primer pair was used to amplify all five putative regions of interest in each normal and tumor sample via PCR. PCR was performed in 40 μL reactions using 60 ng of genomic DNA, 15 pmol of both the forward and reverse primer, 4-5U of Taq polymerase (Life Technologies), 1.5 mM MgCl2, 200 μM dNTPs. Depending on prior reaction optimization, general cycling conditions were:

94°C 4 min, followed by 25-30 cycles at 94°C for 1 min, Tanneal for 1 min, and at 72°C for 1 min; and finishing with a single extension cycle at 72°C for 5 min. PCR products were purified using the Quickstep 96-well PCR purification kit (Edge Biosystems). DNA sequencing was performed using the ABI BigDye Terminator sequencing kit (Applied Biosystems, Inc.) Each 10 μL sequencing reaction contained 10-50 ng of purified PCR product, 1.5 pmoles of sequencing primer, 1 μL of BigDye Terminator mix, 1.5 μL of 5 × sequencing dilution buffer (400 mM Tris pH 9.0, 10 mM MgCl2) and water to volume. Cycling conditions were 94°C for 1 min; 25 cycles at 94°C for 30 sec, 50°C for 30 sec, and 60°C for 4 min; and finishing with a single 72°C extension step for 5 min. The sequencing reactions were run on an ABI 3730XL DNA sequencer and data were analyzed using Sequencher software (GeneCodes, Version 4.7).