Data Availability StatementThe data helping the conclusions of this article are included within the article and its additional file. decreased toward the proximal magnum. In quail oviduct tissue, the gene expression pattern of oviduct/epithelial markers was similar to chicken. The markers of progenitors/stemness in hen oviduct (Musashi-1 and CD44 glycoprotein) had the highest relative expression in the infundibulum and decreased toward the proximal magnum. In quail, we found significant expression of four progenitor markers (LGR5 gene, SRY sex determining region Y-box?9, OCT4/cPOUV gene, and CD44 glycoprotein) that were largely present in the distal oviduct. After in vitro culture of oviduct cells, the gene expression pattern has changed. High secretive potential of magnum-derived cells diminished by using decreased abundance of mRNA. On the other hand, chicken oviduct cells originating from the infundibulum gained ability to express and Among progenitor markers, both hen and quail cells expressed high level of SOX9, LGR5 and Musashi-1. Conclusion Analysis of tissue material revealed gradual increase/decrease pattern in majority of the oviduct markers in both species. This pattern changed after the oviductal cells have been cultured in vitro. The results can provide molecular tools to validate the phenotype of in vitro biological models from reproductive tissue. Electronic supplementary material The online version of this article (10.1186/s12861-018-0168-2) contains supplementary material, which is available to authorized users. and in vitro. We propose a panel of epithelial genetic markers to determine the progenitor/epithelial cell pattern in selected compartments of the oviduct (Fig.?1). In particular, we have aimed to reveal which of the avian oviduct compartments (infundibulum (INF), distal magnum (DM), or proximal magnum (PM)) carry known progenitor signaturesfor 5?min at room temperature (RT). Cell pellets Afuresertib were resuspended in 0.5?mL RNAfix (EURx, Gdansk, Poland) to preserve cells prior to RNA isolation. RNA was extracted using the universal RNA purification kit (EURx, Gdansk, Afuresertib Poland) according to manufacturers recommendation. RNA was quantified using spectrophotometry and RNA quality by gel electrophoresis. RT-qPCR analysis Reverse transcription was performed with Maxima First Strand cDNA synthesis kit for RT-qPCR (Thermo Scientific/Fermentas, Vilnius, Lithuania). cDNA was diluted to a final concentration of 70?ng/L and stored at ?20C. Reverse transcription-quantitative polymerase Afuresertib chain reaction (RT-qPCR) was performed in a total volume of 10?L, including Maxima SYBR Green qPCR Get better at Blend (Thermo Scientific/Fermentas, Vilnius, Lithuania), 1?M of every primer (ahead Afuresertib and change), and 2?L of diluted cDNA (140?ng). Primer sequences (Desk?1) were produced from the books or made with NCBI Primer Blast, predicated on cDNA research sequences [17]. Thermal bicycling was carried out in LightCycler II 480 (Roche Applied Technology, Basel, Switzerland). qPCR thermal profile contains preliminary denaturation at 95?C for 20?min, accompanied by 40?cycles of amplification including 15?s of denaturation in 95?C, 20?s of annealing in 58?C, and 20?s of elongation in 72?C. After conclusion of the amplification response, a melting curve was produced to check for the Mouse monoclonal to CRTC2 specificity of RT-qPCR. For this function, the temperature was risen to 98?C with continuous fluorescence dimension. Desk 1 Primer sequences found in RT-qPCR research B C quail (research, muscle samples through the same birds had been used. For in vitro study, the chicken macrophage-like cell line [19] was used as a calibrator. Ct was then calculated using the equation: Ct sample C Ct calibrator. Fold change of the gene expression was calculated as: sequence with human LGR5, but the same protein sequence shows 95% identity with human VAV3 GDP/GTP exchange factor. For a quail, only 900 proteins are annotated in existing UniProt databases. Thus, when a gap in quail database [22] limits the interpretation Afuresertib of a sequence, a relevant genomic alignment onto chicken was performed [23]. Depending on the database used (ENSEMBL, NCBI, and/or UniProt), sequences of the genes selected for this study had 89%C100% similarity. Thereby, gene expression assays developed were comparable between both species The overall gene expression of the markers analyzed in both species (hen and quail) and sample types (tissue and in vitro) is presented in Table?3. All twelve genes were expressed only in.