20.500.12556/RUL-151221 Optimizacija postopkov za oceno velikosti genoma s pretočno citometrijo pri repatih dvoživkah Optimisation of flow cytometry for genome size estimates of salamanders Genomi repatih dvoživk (Urodela) sodijo med največje genome tetrapodov - sama velikost genoma ima tudi pomemben vpliv na morfologijo, metabolizem, ekologijo in evolucijo repatcev. Tukaj predstavljamo optimiziran postopek za oceno velikosti genoma pri repatih dvoživkah s pretočno citometrijo. Metodo smo optimizirali na krvnih vzorcih španskega pupka (Pleurodeles waltl) in aksolotla (Ambystoma mexicanum), ter jo preizkusili na močerilu (Proteus anguinus). Pri optimizaciji metode pretočne citometrije smo primerjali različne pufre za liziranje celic in sproščanje jeder, primernost različnih pogosto uporabljenih fluorokromov za obarvanje jeder ter vpliv encima RNaza A na kvaliteto vzorcev pretočne citometrije. Kot ustrezen postopek priprave vzorcev za pretočno citometrijo smo določili 1) liziranje celic in sproščanje jeder v Galbraithovem pufru, 2) barvanje jeder s propidijevim jodidom v koncentraciji 100 µg/mL, brez potrebe po uporabi encima RNaze. Potrdili smo, da je A. mexicanum primeren interni standard za oceno velikosti genoma repatcev s pretočno citometrijo. Z našim postopkom pridobljene ocene velikosti genoma P. waltl so bile skladne z obstoječimi ocenami velikosti genoma te vrste, ter z meritvami velikosti genoma s slikovno citometrijo. Primerjali smo tudi štiri načine fiksacije krvnih vzorcev repatcev s časom fiksacije 7 in 14 dni. Za pretočno citometrijo so ustrezni vzorci, fiksirani v 3:1 v/v mešanici metanola in ocetne kisline. Postavljena metoda je nedestruktivna in zato primerna tudi za uporabo pri ogroženih vrstah dvoživk. Salamanders (Urodela) possess some of the largest genomes among tetrapods - and the size of these gargantuan genomes profoundly shapes the morphology, metabolism, ecology, and evolution of this group of amphibians. We present an optimized method for estimating genome size using flow cytometry. The method was optimized on blood samples of the Iberian ribbed newt (Pleurodeles waltl) and the Mexican axolotl (Ambystoma mexicanum) and tested on the Olm (Proteus anguinus). We demonstrate that A. mexicanum is a suitable internal reference standard for genome size estimation of salamanders by flow cytometry. We evaluated the necessity of using the enzyme RNase A in sample preparation and compared the suitability of different lysis buffers and various fluorochromes commonly used in flow cytometry. The optimized protocol was set as follows: 1) cell lysis and nuclear isolation in Galbraith’s buffer, 2) nuclei staining with 100 µg/mL propidium iodide. The genome size of P. waltl estimated by this method was consistent with existing genome size estimates for this species and with our estimates using image cytometry of Feulge-stained nuclei. We compared four methods for fixation of salamander blood samples, with fixation times of 7 and 14 days. Samples fixed in a 3:1 v/v mixture of methanol and acetic acid gave acceptable results when analyzed by flow cytometry. The method presented here is non-destructive and thus suitable for use with endangered salamander species. velikost genoma Urodela pretočna citometrija vrednost C genome size Urodela flow cytometry C-value true false false [B. Vencelj Merc] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-10-01 07:16:41 2023-10-01 07:16:42 2023-10-20 09:10:49 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 577:597.6/.9(043.2) 169212675 232231 169204995 36932.pdf 36932.pdf 1 E734E5DA2CB8AF63E3E043D7B5FAAE11 7548dd020fb1d5e3c5aa386b3a0916cfd7acfbf842b5944edffbf66c725bd013 b5e3d4a9-6019-11ee-930c-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=176132 Biotehniška fakulteta 0 0 0