Balkan Journal of Medical Genetics

TWENTY-FOUR GENES ARE UPREGULATED IN PATIENTS WITH HYPOSPADIAS
Karabulut R1, Turkyilmaz Z1, Sonmez K1, Kumas G2, Ergun SG2, Ergun MA2,*, Basaklar AC1
*Corresponding Author: Mehmet A. Ergun, M.D., Ph.D., Department of Medical Genetics, Gazi University Faculty of Medicine, Besevler, Ankara, Turkey; Tel.: +90 312 2024637; Fax. +90 312 2024635; E-mail: aliergun@gazi.edu.tr
page: 39

MATERIALS AND METHODS

Patients. A total of eight patients with isolated distal (subcoronal) hypospadias (mean age 6.8; range 2-10 years) and five healthy circumcised controls (mean age 6.5; range 2-10 years) were enrolled in this study. The penile skin tissue specimens obtained at surgery during hypospadias repair or elective circumcision were divided into two groups: children with hypospadias (n = 8), and normal controls (n = 5). Informed consent was obtained from the parents/guardians of all the children. Samples were flash frozen with liquid nitrogen and stored at –80 °C until further processing. The gene expression studies were assessed using Genechip® Primeview™ Human Gene Expression Array (Affymetrix, Santa Clara, CA, USA) which contain more than 53,000 probes including over 36,000 transcripts and variants. RNA Isolation. Samples were distrupted and powdered under liquid nitrogen with pestle and mortar. Tissues were transferred into microcentrifuge tubes and lysated with 1 mL of TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). All samples were homogenized using Qiashredder (Qiagen, Valencia, CA, USA); 0.2 mL chloroform was added to homogenized samples and centrifuged at 12,000g for 15 min. Upper aqueous phase was transferred into a new microcentrifuge tube and isolation of the highpurity total RNA was perfomed using the RNeasy® Mini Kit (Qiagen) following the manufacturer’s specifications. The quantity and purity of RNA was determined using the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at 260 nm. Gene Expression Studies. Genechip® Primeview ™ Human Gene Expression Array (Affymetrix) was used for gene expression studies. Five hundred ng of total RNA was reverse transcribed, amplified and biotin-labelled with Genechip 3’IVT (in vitro transcription) Express Kit (Affy-metrix) according to manufacturer’s instructions. aRNAs were then purified with magnetic beads and after fragmentation of purified biotinylated aRNAs, samples were loaded to Genechips for subsequent hybridization. Afterwards, Genechips were washed and stained on the Fluidics station with specified protocol. Statistical Analysis. Signal intensities were acquired by Genechip Scanner 3000 7G (Affymetrix) to generate cell intensity files (CEL). Statistical analysis was performed using Partek Genomics Suite software (Partek Inc., St. Louis, MO, USA). Robust multi-array average (RMA) algorithm was used for data normalization. One-way analysis of variance (ANOVA) was used when >2 groups were compared, followed the by t-test. The statistical significance level was set at false discovery rate ((FDR) p <0.05 to minimize false identificaion of genes. Greater than 2-fold changes were analyzed for up or down regulated genes. Hierarchical clustering based on genes and samples was performed with Partek Genomics Suite Software (Partek Inc.).

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