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  • Küçük Resim
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    Comparison of five different rna isolation methods from equine endometrium for gene transcription analysis
    (Veteriner Fakultesi Dergisi, 2010) Kurar E.; Atli M.O.; Güzeloğlu A.; Özşensoy Y.; Semacan A.
    In this study, five different isolation protocols to extract total RNA from biopsies of equine endometrium were compared in terms of quality and quantity of RNA samples with respect to downstream gene transcription analysis, such as Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Three phenol-chloroform based protocols (TRIzol, TRItidy, EZ-RNA) and two column based protocols (UltraClean™ and E.Z.N.A.®) that were commercially available were used. Each protocol yielded good quality total RNA and distinct 28S and 18S rRNA bands were observed in agarose gel electrophoreses. Amount of total RNA isolated was lower for EZ-RNA protocol. Column based protocols had RNA contaminated with great amount of genomic DNA, however, DNAse-I digestion was able to fully clean the DNA contamination from RNA in all the protocols used. Following cDNA synthesis and PCR, GAPDH, a housekeeping gene, bands were amplified from all the samples. In conclusion, all the protocols used extracted good quality but different amounts of total RNA and it is strongly recommended that RNA samples must undergo DNAse-I digestion before RT-PCR to eliminate gDNA contamination.
  • Küçük Resim
    Öğe
    Expression of epidermal growth factor (egf) and heparin-binding egf (hb-egf) mrna in mare endometrium
    (Veteriner Fakultesi Dergisi, 2012) Atli M.O.; Güzeloğlu A.; Kurar E.; Kayiş S.A.; Aslan S.; Semacan A.; Çelik S.
    EGF and HB-EGF play crucial roles in embryonic development and peri-implantation. In this study, aim was to characterize expression profiles of EGF and HB-EGF in mare endometrium during estrous cycle and early pregnancy. Endometrium biopsies were obtained from mares on day of ovulation (d0), late diestrus (LD) and after luteolysis (AL) in the estrus phase. In pregnant groups, biopsies were taken on days 14 (P14), 18 (P18), 22 (P22) and 60 (P60). Relative expression levels of genes were quantified using real-time RT-PCR. A mixed model was fitted on the normalized data and Least Significant Difference (LSD) test was employed to determine significantly different group(s). EGF mRNA expression was up-regulated at LD compared to d0 while HB-EGF expression was not changed throughout the cycle. EGF expression was also increased during early pregnancy with the highest expression level observed on P60. Similarly, HB-EGF mRNA level was increased on P60. Pregnancy induced EGF expression on P14 and P18 compared to LD and AL whereas expression of HB-EGF was only significantly higher on P18 than that of AL. These results indicate that EGF expression is up-regulated during the cycle at late diestrus when P4 is high and is increased by pregnancy. HB-EGF expression is induced later in the pregnancy. In conclusion, EGF and HB-EGF appear to involve in the events that happen in the mare endometrium during peri-implantation period.