Web based pcr array data analysis system

Total RNA was extracted from the GT and reproductive tract (including gubernaculum, testis, epididymis, and vas‐defenses, GTEV) of E15.5 male mouse embryos of different groups, based on the TRIzol method according to the operation manual (Invitrogen, Cat: 10296010). RNA quality was assessed by a Nanodrop spectroscopy and the ratios of 28 s and 18 s ribosomal RNA after gel electrophoresis. The cDNA was synthesized from 500 ng total RNA using iScript Reverse Transcription Supermix (Bio‐rad, Hercules, CA). Pathway‐specific mRNA expression was determined by using the Hedgehog signaling PCR Array (PAMM‐078Z, Qiagen) and the CFX96 Real‐time PCR system (Bio‐rad) according to the manufacturer's instructions. The Web‐Based PCR Array Data Analysis system (Qiagen) was used to analyze PCR array results. The primers of hormone receptors, androgen biosynthesis pathway, and testicular descent related genes were designed using PrimerQuest Tool (Integrated DNA Technologies, Inc.) and validated, and primer sequences were listed in Table S2. The details for gene expression analyses and other information were summarized in supplementary materials.

The one‐way analysis of variance (ANOVA) with Dunnett's T3 post hoc analysis (Analytics Software & Solutions) was applied to determine if ATZ exposure produced significant effects on the evaluated parameters, including AGD, glans penis length, body, and testis weight. Comparisons between control and treatment groups in the incidences of male urogenital mating protuberance (MUMP) shape, urethral meatus position (hypospadias), and undescended testicle (cryptorchidism) were conducted via the Mann–Whitney tests (PASW Statistics 18.0). The effects on hormone levels and gene expression level (fold) changes were also determined using ANOVA except for the PCR array data. For each parameter, an average value per liter was included for statistical analysis. Data were logarithmically transformed to approximate a normal distribution prior to ANOVA analyses. The PCR array data analysis was done using Web‐Based PCR Array Data Analysis system (Qiagen). The level of significance was set at α = 0.05.

Quantitative real-time polymerase chain reaction (RT-PCR) was performed according to a modification of previously described methods (30, 38). MT-treated and control female ICR mouse GTs were dissected at E16.5, two GTs from each litter were randomly selected for RNA extraction, and three litters of mice from each group were used. Total RNA was extracted using TRIzol method according to the operation manual (Invitrogen), and RNA quantity (>100 ng/μL) and purity (260/280 > 2.0, 260/230 > 1.65) were determined by using a Nanodrop. RNA integrity (28S/18S ratio > 1.5) was assessed by gel electrophoresis. A quantity of 500 ng of high-quality RNA for each sample (n = 3) was converted into cDNA by using the RT² First Strand cDNA Kit (Qiagen, Hilden, Germany). Pathway-specific gene expression was determined using the Wnt signaling PCR Array (PAMM-043Z, Qiagen, Hilden, Germany) and the CFX96 Real-time PCR system (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. The Web-Based PCR Array Data Analysis system (Qiagen, Hilden, Germany) was used to analyze PCR array results.