猪精子RNA提取的初步研究

The Preliminary Study on Extraction of RNA in Swine Spermatozoa

实验采用Trizol一次法或试剂盒提取猪精子的总RNA,然后通过RT-PCR扩增的方法,检测精子和睾丸组织中鱼精蛋白(PRM-1)的mRNA;使用不同数量的精子,检测在本实验条件下能扩增出PRM-1带所需要的最小精子数量;通过OD260/280的测算,判断不同提取方法所得RNA的纯度;通过琼脂糖电泳得到精子RNA与睾丸、卵巢组织RNA的电泳图。实验结果表明:在使用RNA沉淀剂时,1.51×107精子可检测到PRM-1的mRNA,不使用沉淀剂则需要3.02×107精子才检测到;使用Trizol一次法提取的精子总RNA纯度较常规Mini试剂盒(W6221)高;本实验首次得到猪精子总RNA的普通琼脂糖凝胶电泳图。初步研究的结果表明,Trizol一次法可用来提取精子总RNA供进一步研究;猪精子RNA的28S和18S片段与睾丸、卵巢组织无明显差异。

In the present study, total RNA in swine spermatozoa, testis and ovarian tissue was extracted by Trizol and mini kit （W6221）. Concentration of total RNA recovered was evaluated by OD260/260 and agarose gel electrophoresis. transcript of protamine-1 （PRM-1）in total RNA were subsequently detected and by RT-PCR and PCR. The results revealed that the lowest sperm number required for RNA extraction and detection of PRM-1 was 1.5 1×10^7under the use of RNA carrier and 3.02×10^7 without using RNA carrier. The purity of total RNA extracted by Trizol was higher than those extracted by mini kit. Total RNA from swine spermatozoa was analyzed by gel electrophoresis. The results indicates that there was no significant difference between the bands of total RNAs from spermatozoa and tissues （testis and ovary）. It is concluded that extraction of total RNA from spermatozoa by Trizol was more efficient than by mini kit （W6221）,and the method by Trizol is feasible in swine spermatozoal RNA study.