Design and optimization of primers for gene transcript and methylation analysis on bovine embryos

Batlle Pérez, Mar
The cattle industry is constantly evolving due to an increasing demand for livestock products. The need to breed more cattle and the increase of the temperatures due to climate change have motivated different studies with the aim to link heat stress and bovine reproduction problems. This is the case of the ongoing project in Ghent University entitled “Effects of sperm stressors on sperm methylation and subsequent embryo development in cattle”. My role in this project was: (I) to test and optimize primers for embryo transcript expression analysis and (II) to design, test and optimize primers for bisulfite treated embryos. In order to achieve the first objective, RNA extracted from frozen tissue was reverse transcribed, and the resulting cDNA was used as a template in RT-qPCRs to assess the specificity and the efficiency of the studied primers. The second objective was achieved by first, testing and optimizing the oxidative bisulfite sequencing protocol on frozen tissue and then on embryos. Afterwards, specific primers were designed and optimized in frozen tissue and then tested on in vitro produced embryos. The specificity of the primer pairs was tested by performing melt curves during the RT-qPCR program and then, characterizing the resulting amplicons. All the studied primers showed a single distinct peak, indicating high specificity. These peaks were characterized by assessing the uniqueness and length of the amplicons via agarose gel and then, sequencing. PCR efficiency was analyzed and all the assays obtained valid efficiency values backed up by optimal R2 values. On the other hand, the bisulfite conversion protocol showed trustworthy results both on testicle tissue and on embryos. However, the oxidative step results were not satisfactory. The designed primers for the original and the bisulfite treated DNMT1 gene showed solid results after analyzing them via PCR amplification, agarose gel electrophoresis and Sanger sequencing, therefore, the bisulfite sequencing protocol was verified. In conclusion, different sets of primers were successfully optimized for gene transcript analysis. The bisulfite conversion protocol was optimized, and the DNTM1 based primers for original and bisulfite treated DNA showed satisfactory results ​
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