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Polysomal-mRNA Extraction from Arabidopsis by Sucrose-gradient Separation
采用甘蔗梯度分离法提取拟南芥多聚核糖体-mRNA

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Abstract

mRNAs surrounded by polysomes are ready for translation into proteins (Warner et al., 1963); these mRNAs are defined as polysomal-mRNAs (Mustroph et al., 2009). The process is affected by various growth conditions or surrounding situations. Microarray analysis is a powerful tool for detecting genome-wide gene expression. Therefore, using polysomal-mRNAs for microarray analysis can reflect the gene translation information (the translatome) under different developmental stages or environmental conditions from eukaryotes. Polysomal-mRNAs can be collected from the polysomal fraction by sucrose-gradient separation for further quantitative PCR or microarray assay. We modified a protocol (Mustroph et al., 2009) for collecting polysomal-mRNAs via sucrose-gradient separation to eliminate monosomal-mRNA contamination from pLAT52:HF:RPL18 Arabidopsis. This transgenic Arabidopsis uses a pollen-specific promoter (ProLAT52) to generate epitope-tagged polysomal-RNA complexes that could be purified with a specific antigen (Lin et al., 2014). The polysomal-mRNAs we obtained via sucrose-gradient separation and antibody purification underwent in vivo translation in pollen tubes grown from self-pollinated gynoecia of Arabidopsis thaliana.

Keywords: Polysomal-mRNA(多聚核糖体的mRNA), Sucrose-gradient seperation(蔗糖密度梯度分离), In vivo translation of genes(基因的体内翻译)

Material and Reagents

  1. pLAT52: HF-RPL18 transgenic Arabidopsis
  2. RNase-free water
  3. Tris buffer (Sigma-Aldrich, catalog number: T1378 )
  4. KCl (Sigma-Aldrich, catalog number: P9541 )
  5. EGTA (Sigma-Aldrich, catalog number: E3889 )
  6. MgCl2 (Sigma-Aldrich, catalog number: M8266 )
  7. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  8. Cycloheximide (Sigma-Aldrich, catalog number: C7698 )
  9. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
  10. Polyoxyethylene 10 tridecyl ether (PTE) (Sigma-Aldrich, catalog number: P2393 )
  11. Sodium deoxycholate (DOC) (Sigma-Aldrich, catalog number: D6750 )
  12. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  13. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  14. Triton X-100 (Sigma-Aldrich, catalog number: X-100)
  15. Polyoxyethylene(23)lauryl ether (Brij -35) (Sigma-Aldrich, catalog number: P6938 )
  16. Tween-20 (Sigma-Aldrich, catalog number: P1379 )
  17. NP-40 (Sigma-Aldrich, catalog number: NP 40 )
  18. Polyoxyethylene (Sigma-Aldrich, catalog number: P2393)
  19. Deoxycholic acid (Sigma-Aldrich, catalog number: D2510 )
  20. ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich, catalog number: A2220 )
  21. RNAsin (Promega Corporation, catalog number: N2511 )
  22. 3x FLAG peptide (Sigma-Aldrich, catalog number: F4799 )
  23. Sucrose (Sigma-Aldrich, catalog number: 84097 )
  24. Polysomal extraction buffer preparation (see Recipes)
  25. 20% detergent mixture (see Recipes)
  26. 20% PTE and 10% DOC (see Recipes)
  27. Sucrose gradient layer preparation (see Recipes)
  28. 10x sucrose salts (see Recipes)
  29. Preparation of the FLAG M2 Agarose beads (see Recipes)

Equipment

  1. QIAGEN QIA shedder columns (QIAGEN, catalog number: 27115 )
  2. 15-ml tube (Labcon, catalog number: 9205-946CB-946C )
  3. Hitachi ultracentrifuge himac CP100WX (Hitachi, catalog number: CP100WX)
  4. Hitachi P40ST swing rotor (Hitachi, model: P40ST)
  5. 13PA tubes (Hitachi, catalog number: 332901A )
  6. Spectrophotometer (Beckman Coulter, catalog number: DU648B )
  7. UV monitor (GE Healthcare, catalog number: UVIS-920 )
  8. Pump (GE Healthcare, model: P-50 )
  9. Fraction collector (Gilson Products, catalog number: FC80 )
  10. UV detector system (GE Healthcare, catalog number: monitor UVIS-0912 )
  11. Recorder (GE Healthcare, catalog number: pharmaria LKB REC102 )

Procedure

Pre-treatment: All glass materials were heated in 180 °C for 12 h and all plastic materials were treated with non-sterile DEPC-H2O overnight, then autoclaved at 121 °C for 20 min. RNase-free water was used to prepare all buffers.

  1. Collect 0.5-1 mg plant tissue sample into a mortar and then add liquid nitrogen immediately.
  2. After grinding the plant tissue finely, add 1,250 μl polysome extraction buffer into the mortar.
  3. Transfer the crude extract in an Eppendorf tube and let it sit on ice for 10 min.
  4. Centrifuge the crude extract for 10 min at 13,000 rpm at 4 °C.
  5. Transfer the supernatant into a QIA shedder column (700 μl/column). The column is spun for 1 min at 13,000 rpm at 4 °C, then the flow-through is collected.
  6. Detect the OD260 unit for the flow-through by spectrophotometry.
  7. Prepare the four different sucrose gradient layers in the Hitachi centrifuge column.
  8. Carefully add 800 μl sample (20-25 OD260 unit; the nucleic acid concentration for 1 OD260 unit is equal to 40 µg/ml RNA) onto the top sucrose layer (the 20% layer). Balance tubes and buckets to 0.03 g by adding sample or polysome extraction buffer.
  9. Ultracentrifuge for 225 min at 39,000 rpm at 4 °C in an Hitachi P40AT swing rotor.
  10. Link the pump with a UV detector and the fraction collector. Link the UV detector with the recorder.
  11. Put the 260-nm filter into the UV detector system and turn it on at least 20 min before polysome profile analysis.
  12. Put the probe into a sample separated in sucrose gradient after ultracentrifugation, turn on the pump to suck the sample, and turn on the recorder to create the sucrose gradient profile.
  13. According to the sucrose gradient profile, combine the polysomal fractions together into a 15-ml tube.
  14. Add the washed FLAG beads to the 15-ml tube and incubate for 2 h at 4 °C on a rocking platform.
  15. Centrifuge the mixture for 3 min at 3,000 rpm at 4 °C. Remove the supernatant.
  16. Add 2 ml wash buffer to the tube and mix gently by inverting it for 5 min at 4 °C, then centrifuge the mixture for 3 min at 3,000 rpm at 4 °C. Remove the supernatant (first wash).
  17. Add 2 ml wash buffer to the tube and mix gently by inverting it for 5 min at 4 °C, then centrifuge the mixture for 3 min at 3,000 rpm at 4 °C. Remove the supernatant. Repeat 3 times.
  18. Transfer the mixture to a new Eppendorf tube.
  19. To elute the affinity-purified polysomes, add the 300 μl FLAG peptide (400 ng/μl) with RNAsin (20 U/ml) to the Eppendorf tube and incubate for 30 min at 4 °C on a rocking platform.
  20. Centrifuge for 1 min at 8,200 x g at 4 °C. Transfer the supernatant to a new Eppendorf tube. If the supernatant still contains the beads, centrifuge again for 5 min at 13,000 rpm at 4 °C.
  21. Add additional wash buffer to the eluent to reach 500 μl volume.
  22. Add phenol/chloroform (1:1) in an equal volume (500 μl) to the eluent and mix by inverting.
  23. Centrifuge for 10 min at 13,000 rpm at 4 °C.
  24. Transfer the supernatant to a new Eppendorf tube and add an equal volume of chloroform.
  25. Centrifuge for 10 min at 13,000 rpm at 4 °C.
  26. Transfer the supernatant to a new Eppendorf tube and add 0.1 volume 3 M NaOAC (pH 5.2) and 2.5 volume 100% EtOH to precipitate the RNA overnight at -20 °C.
  27. Centrifuge for 25 min at 13,000 rpm at 4 °C.
  28. Pour out the supernatant, then wash the pellet with 70% EtOH twice.
  29. Air-dry the RNA pellet and use 20 μl RNase-free water to suspend the RNA pellet.

Representative data



Figure 1. Polysomal profiles and validation of the specificity of immunopurification of mRNAs associated with pollen from pollinated floral buds, in vivo-pollinated pollen tubes, and in vitro-cultured pollen tubes of LAT52:HF-RPL18 transgenic plants. (A) Typical sucrose-gradient absorbance (A 260) profiles of ribosome complexes obtained from Bud stage, in vivo stage, and in vitro-cultured pollen. Positions of peaks corresponding to polysomes (line), 40S ribosomal subunits and 60S ribosomal subunit/80S monosomes (arrowheads) are indicated. The arrow for the sedimentation reflects the sucrose gradient from 20% to 60% (top to bottom). (B) Quantitative RT-PCR with primer sets targeting the FLAG-RPL18 transgene and organ-specific genes to confirm that the RNA extracted from purified polysomal mRNAs examined by sucrose-gradient separation and further purified by FLAG-agarose beads was all male gametophyte-specific. Primers span both the His6-FLAG tag and the RPL18 sequence (FLAG-PRL18); pollen-specific PLIM2 and VGD1, female-specific SHP1 and SR, and petal/sepal-specific AP1. ACT2 was an internal control (Lin et al., 2014).

Table1. Primer pairs for Q-PCR in Figure 1 (Lin et al., 2014)

Notes

  1. Prepare enough samples for sucrose gradient profile presentation.

    Table 2. Number of flowers used for the polysomal-RNA extraction for LAT52:HF-RPL18 Arabidopsis (Lin et al., 2014)
    Sample ID
    No. of flowers
    RNA level (ng/μl)
    aRNA level (ng/μl)
    Bud
    800-900
    8.761
    198.1
    in vivo
    500-700
    0.455
    1,159.1
    in vitro
    7,200
    2.897
    1,036.4
    aRNA: antisense RNA synthesized

Recipes

  1. Polysomal extraction buffer preparation

    2 M Tris-HCl (pH 9.0)
    1 ml
    2 M KCl
    1 ml
    0.5 M EGTA (pH 8.3)
    0.5 ml
    1 M MgCl2
    0.36 ml
    dd H2O
    To 8 ml
    Mix well

    β-mercaptoethanol
    80 μl
    50 mg/ml cycloheximide
    10 μl
    50 mg/ml chroramphenicol
    10 μl
    20% detergent mixture
    0.5 ml
    2% PTE, 10% DOC
    1 ml
    0.5 M DTT
    20 μl
    0.5 M PMSF
    20 μl
    ddH2O
    To 10 ml

  2. 20% detergent mixture

    Triton X-100
    10 ml
    Brij 35
    10 g
    Tween-20
    10 ml
    NP-40
    10 ml
    ddH2O
    To 50 ml

  3. 20% PTE and 10% DOC

    Polyoxyethylene 10 trydecyl ether
    4 ml
    Deoxychollic acid
    2 g
    ddH2O
    To 20 ml

  4. Sucrose-gradient layer preparation
    Add 0.1 μl per 1 ml volume of cyclohexamide (50 mg/ml) and chloramphenicol (50 mg/ml) to each layer

    % sucrose
    2 M (68.5%) sucrose
    10x sucrose salt
    ddH2O
    Vol/gradient
    60
    44 ml
    5 ml
    1 ml
    1.5 ml
    45
    66 ml
    10 ml
    24 ml
    3.0 ml
    30
    44 ml
    10 ml
    46 ml
    3.0 ml
    20
    14.5 ml
    5 ml
    30.5 ml
    1.5 ml

  5. 10x sucrose salts

    Tris-base
    2.43 g
    KCl
    0.75 g
    MgCl2
    1.02 g
    ddH2O
    To 50 ml
    Adjust pH value with HCl
    8.4
    Autoclave 20 min, 121 °C
    Stored at -20 °C

  6. Preparation of the FLAG M2 Agarose beads
    1. Use cut pipette to transfer 100 μl FLAG M2 from a bottle into an Eppendorf tube, then centrifuge for 1 min at 8,200 x g at 4 °C.
    2. Remove the supernatant and add 500 μl wash buffer to resuspend the beads. Centrifuge for 1 min at 8,200 x g at 4 °C.
    3. Repeat the above step once.
    4. Wash buffer.

      2 M Tris-HCL (pH 9.0)
      1 ml
      2 M KCl
      1 ml
      0.5 M EGTA (pH 8.3)
      0.5 ml
      1 M MgCl2
      0.36 ml
      50 mg/ml cycloheximide
      10 μl
      50 mg/ml chroramphenicol
      10 μl
      0.5 M DTT
      20 μl
      0.5 M PMSF
      20 μl
      RNAsin
      20 U/ml
      ddH2O
      To 10 ml

Acknowledgments

We thank Dr. Julia Bailley-Serres (Department of Botany and Plant Sciences, University of California, Riverside) for the pLAT52-HF-RPL18 Arabidopsis and for sharing the polysomal-mRNA extraction protocol. This work was funded by Academia Sinica (Taiwan), the Taiwan National Science and Technology Program for Agricultural Biotechnology (Lin et al 31; NSTP/AB, 098S0030055-AA) and the Taiwan National Science Council (99-2321-B-001-036-MY3 and 102-2321-B-001-040-MY3) to G.-Y. Jauh.

References

  1. Lin, S. Y., Chen, P. W., Chuang, M. H., Juntawong, P., Bailey-Serres, J. and Jauh, G. Y. (2014). Profiling of translatomes of in vivo-grown pollen tubes reveals genes with roles in micropylar guidance during pollination in Arabidopsis. Plant Cell 26(2): 602-618.
  2. Mustroph, A., Juntawong, P. and Bailey-Serres, J. (2009). Isolation of plant polysomal mRNA by differential centrifugation and ribosome immunopurification methods. Methods Mol Biol 553: 109-126.
  3. Warner, J. R., Knopf, P. M. and Rich, A. (1963). A multiple ribosomal structure in protein synthesis. Proc Natl Acad Sci U S A 49: 122-129.

简介

由多核糖体包围的mRNA准备好翻译成蛋白质(Warner等人,1963);这些mRNA被定义为多核糖体mRNA(Mustroph等人,2009)。该过程受到各种生长条件或周围环境的影响。微阵列分析是检测全基因组基因表达的有力工具。因此,使用多核糖体mRNAs微阵列分析可以反映在不同发育阶段或环境条件下从真核生物的基因翻译信息(translatome)。可以通过蔗糖梯度分离从多聚糖部分收集多聚体mRNA,用于进一步的定量PCR或微阵列测定。我们修改了通过蔗糖梯度分离来收集多核糖体mRNA以从pLAT52:HF:RPL18拟南芥中消除单体mRNA污染的方案(Mustroph等人,,2009)。该转基因拟南芥使用花粉特异性启动子(ProLAT52 )产生可以用特异性抗原纯化的表位标记的多核糖体-RNA复合物(Lin等人,2014 )。我们通过蔗糖梯度分离和抗体纯化获得的多核糖体mRNA在从拟南芥的自花授粉的芽孢杆菌生长的花粉管中经历了体内翻译。

关键字:多聚核糖体的mRNA, 蔗糖密度梯度分离, 基因的体内翻译

材料和试剂

  1. pLAT52:HF-RPL18 转基因拟南芥
  2. 无RNase水
  3. Tris缓冲液(Sigma-Aldrich,目录号:T1378)
  4. KCl(Sigma-Aldrich,目录号:P9541)
  5. EGTA(Sigma-Aldrich,目录号:E3889)
  6. MgCl 2(Sigma-Aldrich,目录号:M8266)
  7. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  8. 环己酰亚胺(Sigma-Aldrich,目录号:C7698)
  9. 氯霉素(Sigma-Aldrich,目录号:C0378)
  10. 聚氧乙烯10十三烷基醚(PTE)(Sigma-Aldrich,目录号:P2393)
  11. 脱氧胆酸钠(DOC)(Sigma-Aldrich,目录号:D6750)
  12. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  13. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  14. Triton X-100(Sigma-Aldrich,目录号:X-100)
  15. 聚氧乙烯(23)月桂基醚(Brij-35)(Sigma-Aldrich,目录号:P6938)
  16. Tween-20(Sigma-Aldrich,目录号:P1379)
  17. NP-40(Sigma-Aldrich,目录号:NP40)
  18. 聚氧乙烯(Sigma-Aldrich,目录号:P2393)
  19. 脱氧胆酸(Sigma-Aldrich,目录号:D2510)
  20. ANTI-FLAG M2 Affinity Gel(Sigma-Aldrich,目录号:A2220)
  21. RNAsin(Promega Corporation,目录号:N2511)
  22. 3x FLAG肽(Sigma-Aldrich,目录号:F4799)
  23. 蔗糖(Sigma-Aldrich,目录号:84097)
  24. 多聚体提取缓冲液制备(参见配方)
  25. 20%洗涤剂混合物(见配方)
  26. 20%PTE和10%DOC(参见配方)
  27. 蔗糖梯度层准备(参见配方)
  28. 10x蔗糖盐(见配方)
  29. 制备FLAG M2琼脂糖珠(参见配方)

设备

  1. QIAGEN QIA脱落柱(QIAGEN,目录号:27115)
  2. 15-ml试管(Labcon,目录号:9205-946CB-946C)
  3. 日立超速离心机himac CP100WX(日立,目录号:CP100WX)
  4. 日立P40ST摆动转子(日立,型号:P40ST)
  5. 13PA管(日立,目录号:332901A)
  6. 分光光度计(Beckman Coulter,目录号:DU648B)
  7. UV监视器(GE Healthcare,目录号:UVIS-920)
  8. 泵(GE Healthcare,型号:P-50)
  9. 馏分收集器(Gilson Products,目录号:FC80)
  10. UV检测器系统(GE Healthcare,目录号:监测器UVIS-0912)
  11. Recorder(GE Healthcare,目录号:pharmaria LKB REC102)

程序

预处理:将所有玻璃材料在180℃下加热12小时,所有塑料材料用非无菌DEPC-H 2 O 0处理过夜,然后在121℃高压灭菌20分钟。 使用无RNA酶的水制备所有缓冲液。

  1. 收集0.5-1毫克植物组织样品在研钵中,然后立即加入液氮
  2. 研磨植物组织后,向研钵中加入1250μl多核糖体提取缓冲液
  3. 在Eppendorf管中转移粗提取物,让其置于冰上10分钟
  4. 在4℃下以13,000rpm离心粗提取物10分钟
  5. 将上清液转移到QIA脱落柱(700μl/柱)中。 将柱在4℃下以13,000rpm旋转1分钟,然后收集流出液。
  6. 用分光光度法检测流过物的OD <260>单位。
  7. 在Hitachi离心机柱中准备四个不同的蔗糖梯度层
  8. 在顶部蔗糖层上小心地加入800μl样品(20-25OD OD 260单位; 1OD小于260单位的核酸浓度等于40μg/ml RNA) (20%层)。 通过加入样品或多核糖体提取缓冲液将天平管和桶平衡至0.03g。
  9. 在Hitachi P40AT摇摆转子中在4℃下以39,000rpm超速离心225分钟
  10. 将泵与UV检测器和馏分收集器连接。 将UV检测器与记录仪相连。
  11. 将260-nm过滤器放入紫外检测器系统,并在多核糖结构分析前至少打开20分钟
  12. 将探针放入在超速离心后以蔗糖梯度分离的样品中,打开泵吸取样品,并打开记录仪以产生蔗糖梯度曲线。
  13. 根据蔗糖梯度曲线,将多糖部分合并成15ml管。
  14. 将洗涤过的FLAG珠加入15-ml试管中,并在4℃下在摇摆平台上孵育2小时。
  15. 在4℃下以3,000rpm离心混合物3分钟。 除去上清液。
  16. 向管中加入2ml洗涤缓冲液,轻轻混合,在4℃下翻转5分钟,然后在4℃下以3000rpm离心混合物3分钟。 取出上清液(第一次洗涤)。
  17. 向管中加入2ml洗涤缓冲液,轻轻混合,在4℃下翻转5分钟,然后在4℃下以3000rpm离心混合物3分钟。 除去上清液。 重复3次。
  18. 将混合物转移到新的Eppendorf管中
  19. 为了洗脱亲和纯化的多核糖体,将300μl带有RNAsin(20U/ml)的FLAG肽(400ng /μl)加入到Eppendorf管中,并在4℃下在摇摆平台上孵育30分钟。
  20. 在4℃下以8,200×g离心1分钟。 转移上清液到一个新的Eppendorf管。 如果上清液仍然含有珠子,在4℃下以13,000rpm再次离心5分钟
  21. 向洗脱液中加入另外的洗涤缓冲液以达到500μl体积
  22. 将等体积(500μl)的苯酚/氯仿(1:1)加入洗脱液中,倒转混合。
  23. 在4℃下以13,000rpm离心10分钟
  24. 将上清液转移到新的Eppendorf管中,加入等体积的氯仿
  25. 在4℃下以13,000rpm离心10分钟
  26. 将上清液转移到新的Eppendorf管中,加入0.1体积的3M NaOAC(pH 5.2)和2.5体积100%EtOH,使RNA在-20℃下沉淀过夜。
  27. 在4℃下以13,000rpm离心25分钟
  28. 倒出上清液,然后用70%EtOH洗涤沉淀两次
  29. 空气干燥RNA沉淀,并使用20μl无RNA酶的水悬浮RNA沉淀

代表数据



图1.多形体谱和对来自授粉花芽的花粉的mRNA的免疫纯化的特异性的验证 体内 花粉管和 体外 - 培养的花粉管 LAT52:HF-RPL18 转基因植物。(A)从芽期,体内阶段获得的核糖体复合物的典型蔗糖梯度吸光度>体外培养的花粉。指示对应于多核糖体(线),40S核糖体亚基和60S核糖体亚基/80S单体(箭头)的峰的位置。沉淀的箭头反映了蔗糖梯度从20%到60%(从上到下)。 (B)使用靶向FLAG-RPL18转基因和器官特异性基因的引物组的定量RT-PCR,以确认通过蔗糖梯度分离检查从纯化的多核糖体mRNA提取的RNA,并进一步通过FLAG-琼脂糖珠都是雄配子体特异性的。引物跨越His6-FLAG 标签和RPL18序列( FLAG-PRL18 );花粉特异性 PLIM2 和 VGD1 ,女性特异性 SHP1 和 > AP1 。 ACT2 是内部控制(Lin 等人,2014)。

Table1。图1中的Q-PCR引物对(Lin 等 ,2014)

笔记

  1. 准备足够的样品用于蔗糖梯度曲线呈现
    表2.用于对LAT52进行多聚体-RNA提取的花数:HF-RPL18 拟南芥(Lin 等/em> ,2014)
    样品编号
    花数
    RNA水平(ng /μl)
    aRNA水平(ng /μl)

    800-900
    8.761
    198.1
    体内
    500-700
    0.455
    1,159.1
    体外
    7,200
    2.897
    1,036.4
    aRNA:反义RNA合成

食谱

  1. 多聚体提取缓冲液制备

    2 M Tris-HCl(pH 9.0)
    1 ml
    2 M KCl
    1 ml
    0.5 M EGTA(pH 8.3)
    0.5 ml
    1 M MgCl 2
    0.36 ml
    dd H sub 2 O
    至8 ml
    混合良好

    β-巯基乙醇 80μl
    50mg/ml放线菌酮 10微升
    50mg/ml氯霉素
    10微升
    20%洗涤剂混合物
    0.5 ml
    2%PTE,10%DOC
    1 ml
    0.5 M DTT
    20微升
    0.5 M PMSF
    20微升
    ddH sub 2 O
    至10ml

  2. 20%洗涤剂混合物

    Triton X-100
    10 ml
    Brij 35
    10克
    吐温-20
    10 ml
    NP-40
    10 ml
    ddH sub 2 O
    到50 ml

  3. 20%PTE和10%DOC

    聚氧乙烯10十三烷基醚 4 ml
    脱氧胆酸
    2克
    ddH sub 2 O
    至20ml

  4. 蔗糖梯度层准备
    向每层中加入0.1μl/ml体积的环己酰胺(50mg/ml)和氯霉素(50mg/ml)
    %蔗糖
    2 M(68.5%)蔗糖 10x蔗糖
    ddH sub 2 O
    音量/渐变
    60
    44 ml
    5 ml
    1 ml
    1.5 ml
    45
    66 ml
    10 ml
    24 ml
    3.0 ml
    30
    44 ml
    10 ml
    46 ml
    3.0 ml
    20
    14.5毫升
    5 ml
    30.5 ml
    1.5 ml

  5. 10x蔗糖

    Tris碱
    2.43克
    KCl
    0.75克
    MgCl 2
    1.02克
    ddH sub 2 O
    到50 ml
    用HCl调整pH值
    8.4
    高压灭菌器20分钟,121℃
    储存于-20°C

  6. 制备FLAG M2琼脂糖珠
    1. 使用切割移液管将100μlFLAG M2从瓶中转移到Eppendorf管中,然后在4℃下以8,200×g离心1分钟。
    2. 除去上清液,加入500μl洗涤缓冲液以重悬珠。 在4℃下以8,200×g离心1分钟。
    3. 重复上述步骤一次。
    4. 洗涤缓冲液。

      2 M Tris-HCL(pH 9.0)
      1 ml
      2 M KCl
      1 ml
      0.5 M EGTA(pH 8.3)
      0.5 ml
      1 M MgCl 2
      0.36 ml
      50mg/ml放线菌酮 10微升
      50mg/ml氯霉素
      10微升
      0.5 M DTT
      20微升
      0.5 M PMSF
      20微升
      RNAsin
      20 U/ml
      ddH sub 2 O
      至10ml

致谢

我们感谢Julia Bailley-Serres博士(加利福尼亚大学植物科学系Riverside)用于pLAT52-HF-RPL18 拟南芥和共享多核糖体mRNA提取方案。这项工作由中央研究院(台湾),台湾农业生物技术国家科学技术计划(Lin等人31; NSTP/AB,098S0030055-AA)和台湾国立科学委员会-2321-B-001-036-MY3和102-2321-B-001-040-MY3)。 Jauh。

参考文献

  1. Lin,S.Y.,Chen,P.W.,Chuang,M.H.,Juntawong,P.,Bailey-Serres,J.and Jauh,G.Y。 体内生长的花粉管的转录物的分析揭示了具有角色的基因在拟南芥授粉期间的微孔引导。 植物细胞26(2):602-618。
  2. Mustroph,A.,Juntawong,P。和Bailey-Serres,J。(2009)。 通过差速离心和核糖体免疫纯化方法分离植物多核糖体mRNA。方法Mol Biol 553:109-126。
  3. Warner,J.R.,Knopf,P.M.and Rich,A。(1963)。 蛋白质合成中的多核糖体结构。美国国家科学院院刊/em> 49:122-129。

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引用:Lin, S. and Jauh, G. (2014). Polysomal-mRNA Extraction from Arabidopsis by Sucrose-gradient Separation . Bio-protocol 4(24): e1364. DOI: 10.21769/BioProtoc.1364.
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