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1 海洋无脊椎动物组织总 RNA 提取方法的探讨冯政夫, 王琳, 李文侠, 部凡, 胡彦江, 朱伟 (, ) 摘要 : 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, A 260nm /A 280nm 和 A 260nm /A 230nm 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关键词 : 无脊椎动物 ; RNA 提取 ; 刺参 (Apostichopus japonicus); 栉孔扇贝 (Chlamys farreri); 凡纳滨对虾 (Litopenaeus vannamei) 中图分类号 : S488 文献标识码 : A 文章编号 : (2014) doi: /hykx RNA, RNA, mrna, RT-PCR cdna [1-2], (Apostichopus japonicus) (Chlamys farreri) (Litopenaeus vannamei),,, RNA, ( RNase,, ), RNA [3-5] RNA, ( Trizol ),, RNA, RNase,, RNA, [6-8],, RNA, RNA, RNA 1 材料与方法 1.1 实验材料,, 160 L,, 2 d,, 30 mg, 80, RNA RNA Tris : ; : : ( ), (SDAIT ), (SYJK 12-14) : (1965-),,, ;,, zhuw001@163.com 24 / 2014 / 38 / 11

2 ; M-MLV, PCR r-taq, dntp ; EP, 1 (diethyl pyrocarbonate, DEPC) 24 h 3% H 2 O 2 3 h, h, RNA RNase 1.2 实验方法 RNase 3, 1 DEPC (1) EP 3% H 2 O 2 3 h, 2, min,, (2) EP 0.5% SDS ( ) 3 h, 2, min,, (3) EP 30% 3 h, 2, min,, RNA [9-10] (1) 3 ml D(4 mmol/l, 0.17 mmol/l, mmol/l ) 500 μl CHCl 3 15 μl β-, 5 min mg, (FSH-II ), 5~10 min,, RNA 5~8 s, 2~3 min, RNase 5~10 min, RNase RNase, 80 RNase, ( 20 mg 1 ml D), 3~5 s, 2~3 min, RNase (2), 2, (5 mol/l) 0.8 mol/l,, 10% (w/v, 2%),, DNA CHCl 3, r/min, 10 min, RNA, DNA,,, (3) RNA, 3 mol/l NaAc (ph 5.2) (.... NaAc = ),, 20 2 h, r/min, 4 30 min (4) RNA RNase, 200 μl D, 5 min 100 μl 100 μl ( D.... = ), r/min, 4 30 min,,, 1 ml 75%, 0.5 ml(5 mol/l )NaCl, EP, r/min, 4 5 min,,, 40 μl RNase DNA RNA 5 µl 10 DNaseⅠbuffer 2 µl DNase Ⅰ(10U) 0.5 μl RNasin(40U) RNase 100 μl, 37 40~50 min, 50 μl 50 μl CHCl 3, r/min, 4 10 min RNA 3 mol/l NaAc (ph 5.2)(.... NaAc= ), 20 1 h, r/min, 4 30 min Marine Sciences / Vol. 38, No. 11 /

, 0.1 mol/l 70% 1~2,,, 10 000 r/min, 4 15 min,, 5~10 min, 10~20 μl RNase RNA 80 1.2.3 RNA RNA 70 15 min, 0.1 μl RNA 3 μl TE, 1.

3 , 0.1 mol/l 70% 1~2,,, r/min, 4 15 min,, 5~10 min, 10~20 μl RNase RNA RNA RNA min, 0.1 μl RNA 3 μl TE, 1.0% RNA DNA RNA 3 rrna (28S, 18S 5S), DNA, 1 μl RNA 19 μl RNase, A 280nm A 260nm A 230nm, RNA M-MLV cdna, PCR β-actin, RNA β-actin 1 表 1 β-actin 基因引物序列 Tab.1 Primer of β-actin gene (5-3 ) F1: AAGGTTATGCTCTTCCTCACGCT R1: GATGTCACGGACGATTTCACG F2: AGTAGCCGCCCTGGTTGTAGAC R2: TTCTCCATGTCGTCCCAGT (bp) F3: GCTGGTGATGGTGTTTCACA R3: GCCAGACTCGTCGTATTCCT 结果 2.1 去除 RNase 方法的比较 RNase, 3% H 2 O 2 0.5% SDS EP RNase, RNA 28S 18S 5S rrna, 3%H 2 O 2 DEPC ( 1A) RNA A 260nm /A 280nm A 260nm /A 230nm 1.96~ ~1.97, SDS A 260nm /A 230nm , RNA, SDS, β-actin PCR, 3% H 2 O 2 0.5% SDS (242bp), ( 1B) 1 RNase RNA β-actin PCR Fig.1 Electrophoresis profiles of total RNA from different methods to eliminate extrinsic RNase and amplification band of β-actin PCR A. RNA ; B. β-actin 1. 3% H 2 O 2 3 h; % SDS 3 h; 3. 30% CHCl 3 3 h; 4. 1 DEPC 12 h(); M. DL2000 marker A. Electrophoresis profiles of total RNA from muscal of Litopenaeus vannamei; B. Amplification band of β-actin PCR from muscal of Litopenaeus vannamei 1. plastic inundated by 3% H 2 O 2 for 3 hours; 2. plastic inundated by 0.5% SDS for 3 hours; 3. plastic inundated by 30% CHCl 3 for 3 hours; 4. plastic inundated by 1 DEPC for 12 hours (control); M. DL2000 marker RNase RNA, D (.. D=20 mg.. 1mL), 1 D, RNA ( 2) 3 RNA 28S 18S ( 2A), 50 μl D 28S 18S D A 260nm / A 280nm , A 260nm /A 230nm , 100 μl D A 260nm / A 230nm 2.45, ( 2A) β-actin PCR, D 2 (634bp), 50 μl D 100 μl D ( 2B) 2.2 不同方法获取 RNA 质量分析 RNA,, RNA RNA 3 rrna ( 3A), A 260nm /A 280nm A 260nm /A 230nm / 2014 / 38 / 11

DL2000 marker A. Electrophoresis profiles of total RNA from hepatopancreas of Chlamys farreri, B. Amplification band of β-actin PCR from hepatopancreas of Chlamys farreri 1.

4 2.03, β-actin cdna, (137bp, 3B), RNA,,, RNA ( 3B) 3 讨论 2 RNase RNA β-actin RT-PCR Fig.2 Electrophoresis profiles of total RNA from different methods to eliminate endogenous RNase and amplification band of β-actin PCR A. RNA ; B. β-actin 1. 1 D(); 2. 1 D 50 μl; 3. 1 D 100 μl; M. DL2000 marker A. Electrophoresis profiles of total RNA from hepatopancreas of Chlamys farreri, B. Amplification band of β-actin PCR from hepatopancreas of Chlamys farreri 1. first supernate without solution D (control); 2. first supernate with 50 μl solution D; 3. first supernate with 100 μl solution D; M. DL2000 marker 3 RNA Fig.3 Comparison of RNA quality from different extraction methods A. RNA B. β-actin 1. (); 2. (); 3.; 4.; M. DL2000 marker A. Electrophoresis profiles of total RNA, B. Amplification band of β-actin PCR 1. no repeat phenol and chloroform extraction in the body wall of sea cucumber two more times (control); 2. no high concentrated solution of NaCl used in the RNA extraction of testis for sea cucumber (control); 3. repeat phenol and chloroform extraction two more times in the body wall of sea cucumber; 4. high concentrated solution of NaCl used in the RNA extraction of testis for sea cucumber; M. DL2000 marker 2.2.2, 2, RNA( 3A) RNA 3 rrna,, A 260nm /A 280nm A 260nm /A 230nm , β-actin RNA RNA, [5-6] RNase,, RNA, RNA, [11] RNase EP RNA, 1 DEPC RNase, DEPC RNase, RNA, [11] 3% H 2 O 2 RNase,, DEPC RNase RNA, RNase, RNA, RNA [12] [6] RNase, RNase, RNase, D ( 4mmol/L ) 1 D,, RNase RNase, RNA D (.. D=12.. 1) RNase, RNA, ( 2A) RNA,,, RNA RNA [3-4] [4] 3 RNA RNA,, RNA, ( 3) [3] RNA, RNA, Marine Sciences / Vol. 38, No. 11 /

5 ,,,, RNA,, RNA,, 2,,, RNA : [1],,,. RNA [J]. ( ), 2012, 40(6): [2],,. RNA [J]., 2006, 2: [3],,. RNA [J]., 2007, 18(1): [4],,,. RNA [J]., 2010, 31(1): [5],,,. RNA [J]., 2011, 27(03): [6],,,. RNA RNA [J]., 2005, 13(6): [7],. RNA [J]., 2010, 37(1): [8],,,. [J]., 2013, 29(21): [9] Joseph S, David W R. Molecular Cloning: A Laboratory Manual(3 rd )[M]. USA: Cold Spring Harbor Laboratory Press, [10] Chan C X, Teo S S, Ho C L, et al. Optimisation of RNA extraction from Gracilaria changii[j]. Journal of Applied Phycology, 2004, 16: [11],. RNA [J]., 2010, 8: [12]. RNA [J]., 2012, 51(3): Extraction of total RNA from marine invertebrate tissue FENG Zheng-fu, WANG Lin, LI Wen-xia, BU Fan, HU Yan-jiang, ZHU Wei (College of Life Science, Qingdao Agricultural University, Qingdao , China) Received: Jan.,28,2014 Key words: marine invertebrate; RNA extraction; Apostichopus japonicus; Chlamys farreri; Litopenaeus vanna Abstract: RNA extraction is the basic experiment of the molecular biology study. The particularity of marine invertebrate 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 A 260nm /A 280nm and A 260nm /A 230nm 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. 28 / 2014 / 38 / 11 ( 本文编辑 : 梁德海 )

2004级研究生现代免疫学实验技术结业报告

2004级研究生现代免疫学实验技术结业报告 PCR PCR PCR RT-PCR PCR (Polymerase Chain Reaction,PCR) 80 DNA, DNA PCR PCR PCR DNA PCR -- -- DNA DNA 93 DNA PCR DNA DNA ( ) DNA 55 DNA DNA -- TaqDNA dntp DNA -- -- PCR PCR DNA DNA Y (1 X) n Y DNA X (Y)