mRNA Analysis Total RNA was extracted from ESC cultures according to the single step acid-phenol guanidinium method [36] using Trizol reagent (Invitrogen, Carlsbad, CA). mRNA was enriched from total RNA using the RNeasy kit (Qiagen free overnight delivery Inc., Valencia, CA) according to the manufacturer’s instructions. cDNA were synthesized using High-Capacity cDNA Transcription kit (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions. Real time RT-PCR reactions were performed and analyzed using the ABI Prism? 7900HT Sequence Detection System (Applied Biosystems) and SDS Plate Utility Software (version 2.1). cDNA samples were assessed for genes of interest and the housekeeping gene, GAPDH, in independent reactions using the Taqman Universal PCR master mix in combination with commercially available primers and probes consisting of Assays-on-Demand? Gene Expression kits (Applied Biosystems) following the manufacturer’s instructions.
Expression kits included: UP1A, Mm01176597_g1; UP1B, Mm00769504_m1 UP2, Mm00447665_m1; UP3A, Mm00447665_m1, UP3B, Mm00558406_m1, GAPDH, Mm99999915_g1; OCT-4, Mm00658129_gH; cytokeratin 1, Mm00492992_g1; cytokeratin 10, Mm03009921_m1; cytokeratin 18, Mm01601706_g1; cytokeratin 20, Mm00508106_m1; GATA4, Mm03053570_s1; GATA5: Mm00484692_m1; GATA6, Mm00802636_m1; SOX7, Mm00776876_m1; SOX17, Mm00488363_m1; FOXA1, Mm00484713_m1; FOXA2, Mm00839704_mH; ��-fetoprotein, Mm00431715_m1; PEM, Mm00476718_m1; p63, Mm00495788_m1; CXCR4, Mm01292123_m1; HOXA13, Mm00433967_m1; smooth muscle myosin heavy chain (SM-MHC), Mm00443013_m1; nestin, Mm03053244_s1; PDX1, Mm00435565_m1; and Nkx3.
1 Mm00440479_m1. For each cDNA sample, the threshold cycle (Ct) was defined as the cycle number at which amplification of the target gene was within the linear range of the reaction. Relative expression levels for each gene of interest were calculated by normalizing the target gene transcript level (Ct) to the respective GAPDH level as described previously (2?����Ct formula, Perkin Elmer User Bulletin #2).
Immunocytochemistry and Immunohistochemistry Cells were fixed with neutral buffered formalin and UP, p63, CK20, ��-actin, and GATA4/6 expression was detected by immunofluorescence using the following primary antibodies: GSK-3 anti-pan-uroplakin [rabbit antisera raised against total bovine uroplakin extracts, TT Sun from New York University, 1100 dilution], anti-p63 [Santa Cruz Biotechnology, Santa Cruz, CA, anti mouse, 1200 dilution], anti-CK20 [Santa Cruz Biotechnology, anti-rabbit, 1200 dilution], anti-��-actin [Sigma-Aldrich, anti-mouse, 1200] anti-GATA4 [Santa Cruz Biotechnology, anti-rabbit, 1200] and GATA6 [Santa Cruz Biotechnology, anti-rabbit, 1200] followed by incubation with species-matched Cy3, FITC, or Texas Red-conjugated secondary antibodies.