Veterinary Research Division, Department of Animal Reproduction and A.I, National Research Centre, Giza, Egypt.
Department of Biotechnology, Animal Production Research Institute, Giza, Egypt.
Andrologia. 2020 May;52(4):e13526. doi: 10.1111/and.13526. Epub 2020 Feb 26.
To date, there is no an established protocol for total RNA isolation in Egyptian buffalo spermatozoa. The present study aimed (I) to establish a defined protocol for total RNA isolation from fresh and frozen spermatozoa, (II) to evaluate RNA quality and quantity from different extraction methods and studying gene expression. Warm and standard room temperature modified QIAzol Lysis Reagents were used for total RNA extraction. The quality and quantity of extracted RNA were checked, and subsequently qRT-PCR was performed using androgen receptor-like and three reference gene primers (GAPDH, ACTB and 18S). The warm modified QIAzol Lysis Reagents resulted higher yield of good quality RNA from fresh (569.54 ± 18.83 ng/μl) and frozen spermatozoa (110.59 ± 4.43 ng/μl), compared to standard room temperature modified QIAzol (421.26 ± 7.18 ng/μl) and (29.07 ± 5.25 ng/μl), for fresh and frozen semen samples respectively. The 260/280 ratio was 1.90 and 1.89 for fresh and frozen isolated semen by warm method respectively. The integrity of RNA was good and appeared as a sharp band on 2% agarose gel. The most stable reference gene was 18S. Reliable extraction method of high quality RNA yield could be a step forward for understanding mechanisms of spermatogenesis for improving male fertility.
迄今为止，埃及水牛精子总 RNA 分离尚无既定方案。本研究旨在：(I) 建立新鲜和冷冻精子总 RNA 分离的明确方案；(II) 评估不同提取方法的 RNA 质量和数量，并研究基因表达。使用温热改良 QIAzol 裂解试剂提取总 RNA。检查提取 RNA 的质量和数量，然后使用雄激素受体样和三个参考基因引物（GAPDH、ACTB 和 18S）进行 qRT-PCR。与标准室温改良 QIAzol 相比，温热改良 QIAzol 裂解试剂从新鲜（569.54 ± 18.83ng/μl）和冷冻精子（110.59 ± 4.43ng/μl）中获得了更高产量的高质量 RNA，新鲜和冷冻精液样本分别为 421.26 ± 7.18ng/μl 和 29.07 ± 5.25ng/μl。温热法分离的新鲜和冷冻精液的 260/280 比值分别为 1.90 和 1.89。RNA 完整性良好，在 2%琼脂糖凝胶上呈清晰条带。最稳定的参考基因是 18S。高质量 RNA 产量的可靠提取方法可能是深入了解精子发生机制以提高男性生育力的重要一步。
