CAJ | 학술논문

RNA 提取是分子生物学研究的基础实验,由于海洋无脊椎动物组织结构以及体成分的特殊性,导致现有的 RNA 提取方法不能完全胜任此工作。作者以刺参(Apostichopus japonicus)、栉孔扇贝(Chlamys farreri)和凡纳滨对虾(Litopenaeus vannamei)为研究对象,对异硫氰酸胍RNA提取方法进行了改进。栉孔扇贝和凡纳滨对虾的肝胰腺富含RNase,刺参体壁结构致密、富含胶原蛋白,而成熟期精巢富含多糖,因此在这些动物组织中提取RNA时,在原有方法基础上增加了瞬时匀浆、增加氯仿及酸性酚的抽提次数,或添加高盐溶液等方法。实验结果显示,改进方法后获得的RNA具有完整的28S、18S和5S rRNA条带, A260nm/A280nm和A260nm/A230nm比值分别为1.89~2.0和1.98~2.11,β-actin基因扩增条带清晰,所获得的组织RNA的纯度和质量符合cDNA合成和基因功能研究的要求。
RNA 제취시분자생물학연구적기출실험,유우해양무척추동물조직결구이급체성분적특수성,도치현유적 RNA 제취방법불능완전성임차공작。작자이자삼(Apostichopus japonicus)、즐공선패(Chlamys farreri)화범납빈대하(Litopenaeus vannamei)위연구대상,대이류청산고RNA제취방법진행료개진。즐공선패화범납빈대하적간이선부함RNase,자삼체벽결구치밀、부함효원단백,이성숙기정소부함다당,인차재저사동물조직중제취RNA시,재원유방법기출상증가료순시균장、증가록방급산성분적추제차수,혹첨가고염용액등방법。실험결과현시,개진방법후획득적RNA구유완정적28S、18S화5S rRNA조대, A260nm/A280nm화A260nm/A230nm비치분별위1.89~2.0화1.98~2.11,β-actin기인확증조대청석,소획득적조직RNA적순도화질량부합cDNA합성화기인공능연구적요구。
RNA extraction is the basic experiment of the molecular biology study. The particularity of marine in-vertebrate in tissue structure and component results in some disadvantage to obtaining pure RNA using general RNA extraction methods. In this study, sea cucumber, Apostichopus japonicus, scallop, Chlamys farreri and the white shrimp, Litopenaeus vannamei were used as the experimental materials. The method was modified from the method of Guandine Throcyanate Reagent. Hepatopancreas of scallop and white shrimp were rich in endogenous RNase. The sea cucumber body wall was compact and rich in collagen and its testis was rich in polysaccharides. During the process of RNA extraction from these tissues, extraction time of chloroform and phenol-chloroform was increased, and brief homogenate and high concentrated solution of NaCl were used. The results show that A260nm/A280nm and A260nm/A230nm ratio of RNA were 1.89~2.0 and 1.98~2.11, respectively. The bands of 28S, 18S and 5S were integrity, and the amplified band of β-actin gene was clear. The data demonstrate that the RNA obtained was suitable for cDNA synthesis and future functional genomic studies.