Veterinary Research Forum
Vet Res Forum

The challenge of getting a high quality of RNA from oocyte for gene expression study

Document Type : Short Communication

Authors

Yusoff Maisarah 1
Noor Hashida Hashim 2
Mohd-Yusuf Yusmin 2, 3

1 Institute of Advanced Studies, University of Malaya, Kuala Lumpur, Malaysia

2 Biology Division, Centre for Foundation Studies in Science, University of Malaya, Kuala Lumpur, Malaysia

3 Centre for Research in Biotechnology for Agriculture, University of Malaya, Kuala Lumpur, Malaysia

https://doi.org/10.30466/vrf.2018.92133.2230
Abstract
The extraction of intact RNA from oocyte is quite challenging and time-consuming. A standard protocol using commercial RNA extraction kit, yields a low quantity of RNA in oocytes. In the past, several attempts in getting RNA for gene expression study ended up with a few different modified methods. Extraction of high-quality RNA from oocyte is important before further downstream analyses such as reverse transcription-polymerase chain reaction, quantitative polymerase chain reaction, or northern blot analysis. In this review, the efficiency of RNA extraction methods from all species oocytes was compared between published articles and our research to gather all possible methods of RNA extraction. Two different methods of RNA extraction that were proposed from various experiments were reviewed to determine the best method of RNA extraction from the oocyte. Modified TRIzol method can be concluded as an efficient RNA extraction method especially for good RNA from oocytes. Meanwhile, comparing RNA extraction kits to extract the RNA from oocytes or pre-implantation embryos, the micro RNA extraction kit type is the best. Therefore, an appropriate RNA extraction method is important to obtain high quality of total RNA for gene expression profiling analysis.

Keywords

Gamete
Pre-implantation embryos
RNA extraction
RT-PCR
TRIzol

  1.  

    1. Virant-Klun I, Knez K, Tomazevic T, et al. Gene expression profiling of human oocyte developed and matured in vivo or in vitro. BioMed Res Int 2013; 2013: 1-20.
    2. Hafidh S, Capková V, Honys D. Safe keeping the message: mRNP complexes tweaking after transcription. Adv Exp Med Biol 2011; 722: 118-136.
    3. Manes C, Byers MJ, Carver AS. Cellular and molecular aspects of implantation (pp 113-124). In: Glasser SR, Bullock DW (Eds). Mobilization of genetic information in the early rabbit trophoblast. New York, USA: Plenum Press 1981; 113-124.
    4. Pikó L, Clegg KB. Quantitative changes in total RNA, total poly(A) and ribosomes in early mouse embryos. Dev Biol 1982; 89(2): 362-378.
    5. Rascado TS, Martins LR, Watanabe Minto B, et al. Parthenogenetic development of domestic cat oocyte treated with ionomycin, cycloheximide, roscovitine and strontium. Theriogenology 2010; (4): 596-601.
    6. Zhao X, Ma W, Das SK, et al. Blastocyst H (2) receptor is the target for uterine histamine in implantation in the mouse. Development 2000; 127(12): 2643-2651.
    7. Veselá J, Rehák P, Mihalik J, et al. Expression of serotonin receptors in mouse oocyte and pre-implantation embryos. Physiol Res 2003; 52(2): 223-228.
    8. Cikos S, Rehak P, Czikkova S, et al. Expression of adrenergic receptors in mouse preimplantation embryos and ovulated oocyte. Reproduction 2007; 133(6): 1139-1147.
    9. Pavani KC, Baron EE, Faheem M, et al. Optimisation of total RNA extraction from bovine oocyte and embryos for gene expression studies and effects of cryo-protectants on total RNA extraction. Tsitol Genet 2015; 49 (4): 25-34.
    10. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 1987; 162(1): 156-159.
    11. Kimble K, Dickinson S, Biase F. Extraction of total RNA from single-oocytes and single-cell mRNA sequencing of swine oocytes. BMS Res Notes 2018; 11: 155.
    12. Cikos S, Burkus J, Bukovska A, et al. Expression of adiponectin receptors and effects of adiponectin isoforms in mouse preimplantation embryos. Hum Reprod 2010; 9: 2247-2255.
    13. Jeong YJ, Choi HW, Shin HS, et al. Optimization of real time RT-PCR methods for the analysis of gene expression in mouse eggs and preimplantation embryos. Mol Reprod Dev 2005; 71(3): 284-289.
    14. Steuerwald N, Cohen J, Herrera RJ, et al. Analysis of gene expression in single oocyte and embryos by real time rapid cycle fluorescence monitored RT-PCR. Mol Hum Reprod 1999; 11: 1034-1039.
    15. Zhu GY, Feng ST, Li JT, et al. Comparison of gene expression patterns between porcine cumulus-oocyte complexes and naked oocytes. S Afr J Anim Sci 2007; 37(1): 57-63.
    16. Luz JV, Alcântara-Neto AS, Batista, RITP, et al. Expression of CD44 in sheep oocyte and pre-implantation embryos. Genet Mol Res 2012; 2: 799-809.
    17. Biase FH, Martelli L, Puga R. Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos. Fertil Steril 2010; 93: 2507-2512.
    18. Mishra A, Reddy IJ, Gupta PSP et al. Total RNA content in sheep oocytes and developing embryos produced in vitro, a comparative study between spectrophoto-metric and fluorometric assay. Cytology and Genetics 2018; 52: 62-74.
    19. Brązert M, Kranc W, Nawrocki MJ, et al. New markers for regulation of transcription and macromolecule metabolic process in porcine oocytes during in vitro maturation. Molecular Medicine Reports 2020; 21: 1537-1551.
    20. Poong SW, Lim PE, Jeannette WSL, et al. Optimization of high quality total RNA isolation from the microalga, Chlorella sp. (Trebouxiophyceae, Chlorophyta) for next-generation sequencing. Phycological Research 2017; 65(2): 12165. doi: 10.1111/pre.12165.
    21. Lisandra CC, Cristiana LMF, Luciene AB, et al. Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation. Anim Reprod 2019; 16 (2): 290-296.

     

Veterinary Research Forum
Volume 11, Issue 2
Spring 2020
Pages 179-184

  • Receive Date 18 August 2018
  • Accept Date 29 December 2018

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