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Polyadenylated RNA Sampling
带有Ploy A 的RNA 的分离纯化   

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Abstract

Polyadenylation is a post-transcriptional modification of RNA occurring in prokaryotes, eukaryotes and organelles. However, the function and extent of bacterial polyadenylation are in marked contrast to those of eukaryotic poly(A) tails. In fact, the long poly(A) tails of eukaryotic mRNAs play an important role in their exportation to the cytoplasm and promote mRNA stability and translation, whereas the short bacterial tails facilitate RNA decay. One of the obstacles encountered by investigators studying bacterial polyadenylation is the scarcity of polyadenylated RNAs. The method described here allows reverse transcription and PCR amplification of the whole population of polyadenylated RNAs provided that the poly(A) tails are long enough to hybridize to oligo dT30 sequence. To this end utilization of exoribonucleases deficient strains may be useful.

Keywords: Polyadenylation(多聚腺苷酸化), Bacteria(细菌), Poly(A) tails(聚(A)尾), RNA degradation(RNA降解), Gene expression(基因的表达)

Materials and Reagents

  1. Kit SMART cDNA (BD Biosciences, catalog number: PT3041-1 )
  2. Sfi I restriction enzyme
  3. pDNR-LIB (BD Biosciences, catalog number: PT3508-5 )
  4. Jet sorb kit (Genomed)
  5. Escherichia coli XLI Blue cells
  6. Wild-type bacteria or bacteria deficient in 3’-->5’ exoribonucleases (see Reference 1)
  7. Phenol (MP) chloroform (Merck KGaA)
  8. T4 DNA ligase (New England Biolabs)
  9. ATP (Promega Corporation)
  10. Powerscript reverse transcriptase
  11. Ethanol
  12. Potassium chloride
  13. LB medium
  14. Buffer 1 (see Recipes)
  15. Buffer 2 (see Recipes)

Equipment

  1. Beckman centrifuge
  2. JA20 rotor

Procedure

  1. RNA extraction
    1. Bacteria were grown in LB medium.
    2. At OD650=0.4 10 ml aliquots were rapidly mixed with an equal volume of 100% ethanol preequilibrated at -70 °C.
    3. Cells were pelleted for 10 min at 4,300 x g and 4 °C in the JA20 rotor of a Beckman centrifuge.
    4. Cells were resuspended in 1.5 ml 4 °C preequilibrated buffer 1.
    5. Mix with 1.5 ml of buffer 2 lysis buffer prewarmed at 95 °C.
    6. Cells were lysed for 2 min at 95 °C.
    7. RNAs were extracted twice at 65 °C with 3 ml of phenol saturated with water.
    8. RNAs were extracted once with chloroform at room temperature.
    9. RNAs were then precipitated with 2.5 volumes ethanol in the presence of 0.3 M NaCl, washed with 70% ethanol, and dissolved in water.
    10. RNA concentration was determined using a Nanovue.

  2. RT-PCR
    1. 1 μg total RNA was incubated with 10 pmol CDSIII/3’PCR (5’-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30NN-3’) and 10 pmol d’oligo SMART IV (5’-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3’) oligos (kit SMART cDNA, BD Biosciences) dissolved in water.
    2. Incubate for 2 min at 72 °C then 2 min in ice.
    3. Add 10 units PowerScript reverse transcriptase in 2 mM DTT, 2 mM dNTP, 1x buffer from the supplier).
    4. Incubate for 1 h at 42 °C. 
    5. Amplify cDNA with 20 pmol 5’PCR Primer (5’-AAGCAGTGGTATCAACGCAGAGT-3’), 20 pmol CDSIII/3’PCR oligo, 0,2 mM dNTP, 2 μl Avantage 2 Polymerase Mix 1x. Incubate 1 min at 92 °C, then perform 22 cycles (15 sec at 95 °C, 6 min at 68 °C).
    6. PCR products were analysed on agarose 1% and purified according to their size if required.

  3. Cloning and sequencing
    1. Digest cDNAs with 200 units Sfi I for 3 h at 37 °C.
    2. Digest pDNR-LIB plasmid with 30 units Sfi I for 3 h at 37 °C.
    3. Purify the linear plasmid on agarose gel and elute the DNA by using the Jetsorb kit.
    4. Precipitate both cDNAs and plasmid and resuspend in water.
    5. Ligate cDNAs and linear plasmid with 600 units T4 DNA ligase, 1 mM ATP, in recommended buffer for the night at 16 °C for 24 h.
    6. 150 μl supercompetent Escherichia coli XLI Blue were transformed with 5 μl ligation mix.

Recipes

  1. Buffer 1
    10 mM Tris-HCl (pH 7.3)
    10 mM potassium chloride
    5 mM magnesium chloride buffer
  2. Buffer 2
    20 mM Tris-HCI (pH 7.9)
    200 mM NaCl
    40 mM EDTA
    1% SDS

Acknowledgments

This work was supported by the Centre National de la Recherche Scientifique (UPR9073; now FRE3630), University Paris-Diderot, and the “Initiative d’Excellence” program from the French State (Grant “DYNAMO,” ANR-11-LABX-0011). This protocol was originally published in Maes et al. (2012).

References

  1. BD SMART TM PCR cDNA Synthesis Kit User Manual.
  2. Maes, A., Gracia, C., Hajnsdorf, E. and Regnier, P. (2012). Search for poly(A) polymerase targets in E. coli reveals its implication in surveillance of Glu tRNA processing and degradation of stable RNAs. Mol Microbiol 83(2): 436-451.

简介

多腺苷酸化是在原核生物,真核生物和细胞器中发生的RNA的转录后修饰。 然而,细菌聚腺苷酸化的功能和程度与真核多聚腺苷酸尾部的功能和程度形成鲜明对比。 事实上,真核mRNA的长聚(A)尾部在它们输出到细胞质中起重要作用,并且促进mRNA稳定性和翻译,而短的细菌尾部促进RNA衰变。 研究细菌聚腺苷酸化的研究者遇到的障碍之一是聚腺苷酸化RNA的缺乏。 本文所述的方法允许对聚腺苷酸化的RNA的整个群体进行逆转录和PCR扩增,条件是聚(A)尾足够长以与oligo dT30序列杂交。 为此,使用核酸外切酶缺陷菌株可能是有用的。

关键字:多聚腺苷酸化, 细菌, 聚(A)尾, RNA降解, 基因的表达

材料和试剂

  1. Kit SMART cDNA(BD Biosciences,目录号:PT3041-1)
  2. Sfi I限制酶
  3. pDNR-LIB(BD Biosciences,目录号:PT3508-5)
  4. 喷气吸嘴套件(Genomed)
  5. 大肠杆菌 XLI蓝细胞
  6. 野生型细菌或缺乏3'→5'外核糖核酸酶的细菌(参见参考文献1)
  7. 苯酚(MP)氯仿(Merck KGaA)
  8. T4 DNA连接酶(New England Biolabs)
  9. ATP(Promega Corporation)
  10. Powerscript逆转录酶
  11. 乙醇
  12. 氯化钾
  13. LB培养基
  14. 缓冲区1(请参阅配方)
  15. 缓冲液2(见配方)

设备

  1. Beckman离心机
  2. JA20转子

程序

  1. RNA提取
    1. 细菌在LB培养基中生长。
    2. 在OD 650 = 0.4时,将10ml等分试样与等体积的在-70℃下预平衡的100%乙醇迅速混合。
    3. 在Beckman离心机的JA20转子中在4,300×g和4℃下将细胞沉淀10分钟。
    4. 将细胞重悬于1.5ml 4℃预平衡缓冲液1中。
    5. 与在95℃预热的1.5ml缓冲液2裂解缓冲液混合。
    6. 将细胞在95℃下裂解2分钟。
    7. RNA在65℃下用3ml用水饱和的苯酚提取两次。
    8. 在室温下用氯仿萃取RNA一次。
    9. 然后在0.3M NaCl存在下用2.5体积的乙醇沉淀RNA,用70%乙醇洗涤,并溶于水中。
    10. 使用Nanovue测定RNA浓度
  2. RT-PCR
    1. 1μg总RNA与10pmol CDSIII/3'PCR(5'-ATTCTAGA GGCCGAGGCGGCC GACATG-d(T)30NN-3')和10pmol的寡聚SMART IV(5'- AAGCAGTGGTATCAACGCAGAGT GGCCATTACGG GGG-3')寡核苷酸(试剂盒SMART cDNA,BD Biosciences)。
    2. 在72℃孵育2分钟,然后在冰中孵育2分钟。
    3. 在来自供应商的2mM DTT,2mM dNTP,1x缓冲液中加入10单位PowerScript逆转录酶)。
    4. 在42°C孵育1小时 
    5. 用20pmol 5'PCR引物(5'-AAGCAGTGGTATCAACGCAGAGT-3'),20pmol CDSIII/3'PCR寡核苷酸,0.2mM dNTP,2μlAvantage 2聚合酶混合物1x扩增cDNA。 在92℃孵育1分钟,然后进行22个循环(95℃15秒,68℃6分钟)。
    6. PCR产物在琼脂糖1%上分析,如果需要,根据它们的大小纯化
  3. 克隆和测序
    1. 用200单位Sfi I在37℃消化cDNA 3小时。
    2. 用30单位Sfi I在37℃消化pDNR-LIB质粒3小时。
    3. 在琼脂糖凝胶上纯化线性质粒,并使用Jetsorb试剂盒洗脱DNA。
    4. 沉淀cDNA和质粒,并重悬于水中。
    5. 用600单位T4 DNA连接酶,1mM ATP,在推荐的缓冲液中在16℃下24小时监测cDNA和线性质粒。
    6. 用5μl连接混合物转化150μl超感受态大肠杆菌 XLI Blue。

食谱

  1. 缓冲区1
    10mM Tris-HCl(pH7.3)
    10mM氯化钾 5mM氯化镁缓冲液
  2. 缓冲2
    20mM Tris-HCl(pH7.9) 200 mM NaCl
    40mM EDTA
    1%SDS

致谢

这项工作得到了巴黎狄德罗大学国家科学研究中心(UPR9073;现在的FRE3630)和来自法国国家的"卓越倡议"计划(Grant"DYNAMO",ANR-11-LABX-0011 )。 该协议最初出版于Maes等人(2012)。

参考文献

  1. BD SMART  TM PCR cDNA合成试剂盒用户手册。
  2. Maes,A.,Gracia,C.,Hajnsdorf,E。和Regnier,P。(2012)。 在 E中搜索poly(A)聚合酶靶标。 大肠杆菌揭示其在Glu tRNA加工和稳定RNA降解的监测中的意义。 83(2):436-451。
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  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hajnsdorf, E. (2012). Polyadenylated RNA Sampling. Bio-protocol 2(17): e254. DOI: 10.21769/BioProtoc.254.
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