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  • 1.
    Etezadi Amoli, Mahmood Reza
    University of Skövde, School of Life Sciences.
    Evaluation of different modifications of TRIzol Reagent method for total RNA isolation from bull spermatozoa2013Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Transcriptional profile of spermatozoa can be used to assess male fertility; differences in spermatozoa attributes require adjustment of protocols for RNA isolation. Ten RNA precipitation modifications of heated TRIzol protocol were applied to total RNA extraction form bull spermatozoa. Motile spermatozoa were isolated from cryopreserved semen straws prepared from an ejaculate of a fertile bull. To evaluate total RNA purity and quantity, Ribogreen RNA detection system and Nanodrop 2000 were used. In RiboGreen assay, a consistent standard curve was not obtained, thus the measurements were ignored. Total RNA purity and concentrations also were assessed using Nanodrop. The A260/280 ratio obtained from Nanodrop showed a good range in the isolated total RNA samples (1.9±0.4). However, all RNA samples presented very low A260/230 ratios (≥1.26). It might be the effect of low concentration of the isolated total RNA. Statistical analysis of total RNA concentrations showed using manufacturer standard protocol had the highest concentration yield. Using either ethanol or Isopropanol as RNA precipitant and changing the incubation temperature had not significant effect on RNA concentration yields. However incubating samples in freezer overnight lowered the total RNA concentration. Using glycogen as a RNA carrier increased the total RNA precipitation yield. In other similar studies the highest total RNA concentration was obtained by using RNA carrier. Validating these findings by qRT-PCR and Bioanalyser might be helpful to enhance the total RNA extraction yield from bull sperm as well as for subsequent genomic studies of bull fertility.

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