Significantly greater than background, and relative gene expression was quantified using the 2 DCT method [37]. The 2 DCT method is also known as the comparative CT method. WNT4 expression was calculated using the following equation: DDCT = DCT, WNT4 CT, Title Loaded From File reference gene. These CT value was normalized to theRNA IsolationTotal cellular RNA was isolated from frozen tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s recommendations. The quantity and quality of total RNA wasChicken WNT4 in the Female Reproductive Tractsendogenous reference genes. For comparing WNT4 expression between untreated and DES-treated oviducts in chickens, the relative quantification of gene expression was normalized to the CT value of the untreated oviduct.In Situ Hybridization AnalysisLocation of WNT4 mRNA in sections (5 mm) of chicken oviducts and ovaries was determined by in situ hybridization analysis as described 16574785 Title Loaded From File previously [21]. Briefly, for hybridization probes, PCR products were generated from cDNA primers used for RT-PCR analysis. The products were gel-extracted and cloned into pGEM-T vector (Promega). After verification of the sequences, plasmids containing gene sequences were amplified with T7- and SP6-specific primers (T7:59-TGT AAT ACG ACT CAC TAT AGG G-39; SP6:59-CTA TTT AGG TGA CAC TAT AGA AT-39). Then digoxigenin (DIG)-labeled RNA probes were transcribed using a DIG RNA labeling kit (Roche Applied Science, Indianapolis, IN). Tissues were collected, fixed in 4 paraformaldehyde, embedded in paraffin, sectioned at 5 mm and sections placed on APES-treated (silanized) slides. The sections were then deparaffinized in xylene and rehydrated to diethylpyrocarbonate (DEPC)-treated water through a graded series of alcohol. The sections were treated with 1 Triton X-100 in PBS for 20 min and washed twice in DEPC-treated PBS. The sections were then digested in 5 mg/ml Proteinase K (Sigma) in TE buffer (100 mM Tris-HCl, 50 mM EDTA, pH 8.0) at 37uC. After postfixation in 4 paraformaldehyde, sections were incubated twice for 5 min each in DEPC-treated PBS and incubated in TEA buffer [0.1M triethanolamine containing 0.25 (v/v) acetic anhydride]. The sections were incubated in a prehybridization mixture containing 50 formamide and 4X standard saline citrate (SSC) for at least 10 min at room temperature. After prehybridization, the sections were incubated with a hybridization mixture containing 40 formamide, 4X SSC, 10 dextran sulfate sodium salt, 10 mM DTT, 1 mg/ml yeast tRNA, 1mg/ml salmon sperm DNA, 0.02 Ficoll, 0.02 polyvinylpyrrolidone, 0.2mg/ml RNase-free bovine serum albumin and denatured DIG-labeled cRNA probe overnight at 42uC in a humidified chamber. After hybridization, sections were washed for 15 min in 2X SSC at 37uC, 15 min in 1X SSC at 37uC, 30 min in NTE buffer (10 mM Tris, 500 mM NaCl and 1mM EDTA) at 37uC and 30 min in 0.1X SSC at 37uC. After blocking with 2 normal sheep serum(Santa Cruz Biotechnology, Inc., Santa Cruz, CA), the sections were incubated overnight with sheep anti-DIG antibody conjugated to alkaline phosphatase (Roche, Indianapolis, IN). The signal was visualized by exposure 23977191 to a solution containing 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate, 0.4 mM nitroblue tetrazolium, and 2 mM levamisole (Sigma Chemical Co., St. Louis, MO).MicroRNA Target Validation AssayThe 39-UTR of WNT4 was subcloned into pcDNA3eGFP (Clontech, Mountain View, CA) to generate the eGFP-miRNA target 39-UTR fusion construct. Fo.Significantly greater than background, and relative gene expression was quantified using the 2 DCT method [37]. The 2 DCT method is also known as the comparative CT method. WNT4 expression was calculated using the following equation: DDCT = DCT, WNT4 CT, reference gene. These CT value was normalized to theRNA IsolationTotal cellular RNA was isolated from frozen tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s recommendations. The quantity and quality of total RNA wasChicken WNT4 in the Female Reproductive Tractsendogenous reference genes. For comparing WNT4 expression between untreated and DES-treated oviducts in chickens, the relative quantification of gene expression was normalized to the CT value of the untreated oviduct.In Situ Hybridization AnalysisLocation of WNT4 mRNA in sections (5 mm) of chicken oviducts and ovaries was determined by in situ hybridization analysis as described 16574785 previously [21]. Briefly, for hybridization probes, PCR products were generated from cDNA primers used for RT-PCR analysis. The products were gel-extracted and cloned into pGEM-T vector (Promega). After verification of the sequences, plasmids containing gene sequences were amplified with T7- and SP6-specific primers (T7:59-TGT AAT ACG ACT CAC TAT AGG G-39; SP6:59-CTA TTT AGG TGA CAC TAT AGA AT-39). Then digoxigenin (DIG)-labeled RNA probes were transcribed using a DIG RNA labeling kit (Roche Applied Science, Indianapolis, IN). Tissues were collected, fixed in 4 paraformaldehyde, embedded in paraffin, sectioned at 5 mm and sections placed on APES-treated (silanized) slides. The sections were then deparaffinized in xylene and rehydrated to diethylpyrocarbonate (DEPC)-treated water through a graded series of alcohol. The sections were treated with 1 Triton X-100 in PBS for 20 min and washed twice in DEPC-treated PBS. The sections were then digested in 5 mg/ml Proteinase K (Sigma) in TE buffer (100 mM Tris-HCl, 50 mM EDTA, pH 8.0) at 37uC. After postfixation in 4 paraformaldehyde, sections were incubated twice for 5 min each in DEPC-treated PBS and incubated in TEA buffer [0.1M triethanolamine containing 0.25 (v/v) acetic anhydride]. The sections were incubated in a prehybridization mixture containing 50 formamide and 4X standard saline citrate (SSC) for at least 10 min at room temperature. After prehybridization, the sections were incubated with a hybridization mixture containing 40 formamide, 4X SSC, 10 dextran sulfate sodium salt, 10 mM DTT, 1 mg/ml yeast tRNA, 1mg/ml salmon sperm DNA, 0.02 Ficoll, 0.02 polyvinylpyrrolidone, 0.2mg/ml RNase-free bovine serum albumin and denatured DIG-labeled cRNA probe overnight at 42uC in a humidified chamber. After hybridization, sections were washed for 15 min in 2X SSC at 37uC, 15 min in 1X SSC at 37uC, 30 min in NTE buffer (10 mM Tris, 500 mM NaCl and 1mM EDTA) at 37uC and 30 min in 0.1X SSC at 37uC. After blocking with 2 normal sheep serum(Santa Cruz Biotechnology, Inc., Santa Cruz, CA), the sections were incubated overnight with sheep anti-DIG antibody conjugated to alkaline phosphatase (Roche, Indianapolis, IN). The signal was visualized by exposure 23977191 to a solution containing 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate, 0.4 mM nitroblue tetrazolium, and 2 mM levamisole (Sigma Chemical Co., St. Louis, MO).MicroRNA Target Validation AssayThe 39-UTR of WNT4 was subcloned into pcDNA3eGFP (Clontech, Mountain View, CA) to generate the eGFP-miRNA target 39-UTR fusion construct. Fo.
