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3’ Rapid Amplification of cDNA Ends (3’ RACE) Using Arabidopsis Samples
对拟南芥样本的cDNA 3'末端进行快速扩增(3' RACE)   

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Abstract

Production of functional eukaryotic RNA is a very elaborate process that involves a complex interplay between transcription and various RNA processing activities, including splicing, 5’ capping, and 3’ cleavage and polyadenylation (Bentley, 2014). Accurate mapping of RNA ends provides a valuable tool to assess transcriptional and post-transcriptional events giving rise to different gene transcripts. The abundance of such transcripts most likely depends on exogenous and developmental cues, or mutations. In the reference plant Arabidopsis, perturbation of the HUA-PEP post-transcriptional regulatory factors (Rodríguez-Cazorla et al., 2015) leads to the accumulation of aberrant transcripts of the key floral homeotic gene AGAMOUS (AG) (Yanofsky et al., 1990) that retain intronic sequence. It was determined by 3’ RACE reactions that such erroneous transcripts correspond to premature processing and polyadenylation events taking place at the AG intron region. Here we describe a protocol that is suitable for analysis of relatively abundant transcripts and also for detecting aberrant RNA species that are likely prone to rapid turnover. Likewise, the method, here adapted to Arabidopsis reproductive tissues, can be applied to characterize RNA species from other organs (leaf, root) and/or other plant species. We provide a detailed protocol of our 3’ RACE procedure comprising four major parts: Total RNA extraction, RNA amount determination and quality control, the RACE procedure itself, and isolation of the resulting RACE products for cloning and sequencing.

Keywords: Arabidopsis(拟南芥), 3' RACE(3的比赛), RNA extraction(RNA的提取)

Materials and Reagents

  1. Disposable gloves
  2. Sterile disposable RNase-free pipette tips
  3. RNase-free microcentrifuge tubes
  4. Plant sample (Arabidopsis young flower buds, stages 1 through 9)
    Note: Arabidopsis flower stages according to Smyth et al., 1990. Other tissue/species can be tested as well.
  5. Liquid nitrogen
  6. GeneJET Plant RNA Purification Kit (Thermo Fisher Scientific, catalog number: K0801 )
  7. 1 M DTT (Sigma-Aldrich, catalog number: 43816 )
  8. Absolute ethanol (JT Baker 8006) and 96% Ethanol (as recommended by the RNA extraction kit manufacturer, see point 6 above)
  9. 4M LiCl (made in distilled water and autoclaved, not necessarily fresh)
  10. DNase I, RNase-free (Thermo Fisher Scientific, catalog number: EN0525 )
  11. RiboLock RNase Inhibitor (Thermo Fisher Scientific, catalog number: EO0382 )
  12. dNTPs mix 10 mM each (Thermo Fisher Scientific, catalog number: R0192 )
  13. OligodT-Anchor Primer (Roche 5’/3’ RACE Kit) (Roche Diagnostics, catalog number: 03 353 621 001 , version 10)
  14. Maxima Reverse Transcriptase + buffer 5x (Thermo Fisher Scientific, catalog number: EP0741 )
  15. RNase-free water
  16. High Fidelity PCR Enzyme Mix + buffer 10x (Thermo Fisher Scientific, catalog number: K0191 )
  17. PCR Anchor Primer (Roche 5’/3’ RACE Kit) (Roche Diagnostics, catalog number: 03 353 621 001 )
  18. Gene specific 5’ Primer/s (Table 1)
  19. GeneJET Gel Extraction Kit (Thermo Fisher Scientific, catalog number: K0691 )
  20. StrataClone PCR Cloning Kit (Agilent Technologies, catalog number: 240205 )
  21. Taq DNA Polymerase (EURx, catalog number: EK2500-04 )
  22. GeneRulerTM 100 bp Plus DNA Ladder (Thermo Fisher Scientific, catalog number: SM0321 ) (or any other suitable molecular marker for your convenience)

Equipment

  1. Mortar and pestle (beaked Haldenwanger mortar, 63 mm. diameter. Clean, autoclave and dry before use)
  2. Thermomixer (water bath or heat block can be used as well) (Eppendorf)
  3. Microcentrifuge (Heraeus, Biofuge Pico)
  4. Refrigerated centrifuge BR15 (B. Braun Melsungen AG) with rotor 12,148-H (Sigma-Aldrich) for 1.5 ml Eppendorf tubes
  5. BioPhotometer Plus (Eppendorf)
  6. Thermal Cycler T100 (Bio Rad) (or any other conventional PCR device)
  7. Electrophoresis system
  8. UV transilluminator

Procedure

  1. Extracting total RNA from flower buds
    1. Pour liquid nitrogen in the mortar, let it to evaporate and then carefully place plant material. Grind until a fine homogeneous powder is obtained. Store in a 1.5 ml Eppendorf tube with a small spatula.
      Note: Operate at room temperature. Plant material can be freshly collected or previously harvested and stored at -80 °C. In both cases, however, harvesting must be carried out under freezing conditions (it is recommended to keep the Eppendorf tube and spatula in liquid nitrogen prior to use).
    2. Extract total RNA by using GeneJET Plant RNA Purification Kit (see protocol).
      1. Add 20 μl of 1 M DTT to each 500 μl of RNA Lysis Solution (containing the chaotropic salt guanidine thiocyanate). Add this mixture to the tube containing the tissue powder (up to 100 mg) and vortex for 10-20 sec to mix thoroughly. This step should be performed quickly to avoid degradation.
      2. Incubate for 3 min at 56 °C in a thermomixer or heat block.
      3. Centrifuge for 5 min at maximum speed (≥20,000 x g, ≥13,000-14,000 rpm).
      4. Collect the supernatant (usually 450-550 μl) and transfer to a clean 1.5 ml tube.
      5. Add 250 μl of 96% ethanol. Mix by pipetting.
      6. Transfer the mixture to a purification column inserted in a 2 ml collection tube (both provided with the kit).
      7. Centrifuge the column for 1 min at 12,000 x g (~11,000 rpm). Discard the flow-through solution and reassemble column and collection tube.
      8. Add 700 μl of Wash Buffer WB 1 (provided with the kit) to the purification column.
      9. Centrifuge for 1 min at 12,000 x g (~11,000 rpm). Discard the flow-through and collection tube. Place the purification column into a clean 2 mL collection tube.
      10. Add 500 μl of Wash Buffer 2 (provided with the kit) to the purification column.
      11. Centrifuge for 1 min at 12,000 x g (~11,000 rpm). Discard the flow-through solution and reassemble column and collection tube.
      12. Repeat previous steps A2j-k and re-spin the column for 1 min at maximum speed to remove all traces of Wash Buffer 2.
      13. Elute the RNA with 50 μl of nuclease-free water and centrifuge for 1 min at 12,000 x g (~11,000 rpm). Repeat this step with the same volume of nuclease-free water to increase the yield of RNA.
      14. Use the purified RNA immediately or store until use. Storage at -80 °C is recommended.
    3. DNase treatment and concentration.
      1. Treat the sample with 5 µl (5 U) of DNase I (Thermo Fisher Scientific, according to manufacturer’s protocol with minor modifications) for 1 h at 37 °C to eliminate traces of genomic DNA. Include RiboLock RNase Inhibitor at 1 U/µl to prevent RNA degradation.
      2. Add 10 µl 50 mM EDTA and incubate at 65 °C for 10 min.
      3. Immediately, mix the RNA sample with two volumes of cold 100% ethanol and store at -20 °C for several hours or overnight.
      4. Add 1/10 volume of 4 M LiCl and centrifuge at 12,000 rpm and 4 °C for 10 min. Remove the supernatant carefully.
      5. Wash with 500 µl cold 70% ethanol and centrifuge as in step A3d.
      6. Remove as much supernatant as possible with the pipette tip. Put the microcentrifuge tube with the open lid in a box with ice, and let the pellet dry for 5 min at the bench or in a laminar flux cabin.
        Note: Drying the pellet at room temperature also works fine.
      7. Resuspend in 25 μl RNase-free water and store at -80 °C if not used immediately.
      8. Determine RNA concentration photometrically, and check integrity by visualizing 500 ng in a 2% agarose gel (Figure 1).
        Note: It is highly recommended to use photometer cuvettes and gel trays previously treated with 0.4 M NaOH (at least for 5 min) to minimize the presence of RNases.
      9. Control PCR. Despite DNase I treatment after RNA extraction, 0.5 µl of the sample was used as template for a conventional PCR reaction with deoxynucleotide primers to test absence of genomic DNA. Carry out PCR with a pair of primers of your choice to test the presence of DNA in your sample. Actin or other housekeeping genes might be suitable.
        PCR components:
        16 µl Distilled water
        2.5 µl 10x Buffer B (+15 mM MgCl2)
        1 µl dNTPs mix (5 mM each)
        1 µl ACT2-f (5 µM) (see oligonucleotide sequence below)
        1 µl ACT2-r (5 µM) (see oligonucleotide sequence below)
        0.25 µl (1.25 U) Taq DNA polymerase
        0.5 µl RNA simple
        PCR profile:
        1 cycle
        94 °C, 2 min
        35 cycles
        94 °C, 30 sec; 51 °C, 30 sec; 72 °C, 30 sec
        1 cycle
        72 °C, 10 min

  2. 3’ RACE
    1. Designing specific primers (forward primers).
      Primers were designed using the free online software OligoCalc (http://www.basic.northwestern.edu/biotools/oligocalc.html). Best suited primers usually are 20 to 25 nt in length, and 50-60% GC content (as suggested by the Roche 5’/3’ RACE Kit protocol). Melting temperature (Tm) must be close to that of the PCR Anchor primer (see below), not exceeding a 5 °C difference.
    2. Retrotranscription.
      According to the instructions provided by the Maxima Reverse Transcriptase protocol with minor modifications.
      1. Mix the following components for the annealing reaction mixture:
        5 µg of previously isolated total RNA
        1 µl of Oligod(T)-Anchor Primer (37.5 µM) from Roche 5’/3’ RACE Kit (A polydT stretch followed by an adapter sequence, see primer sequence below)
        1 µl dNTPs mix (10 mM each)
        14.5 µl Distilled water (nuclease-free)
      2. Incubate at 65 °C for 5 min. Chill on ice, centrifuge for a few seconds (to place the whole reaction at the bottom) and place on ice again.
      3. Add the following reaction components to the annealing mixture in the indicated order
        4 µl 5x RT Buffer (provided with the enzyme batch)
        0.5 µl (20 U) RiboLock
        1 µl (200 U) Maxima Reverse Transcriptase
      4. Incubate at 60 °C for 30 min.
        Note: Retrotranscription of GC-rich RNAs can be performed at 65 °C. Indeed, temperature may vary from 50 to 65 °C. Pilot experiments might be necessary to find optimal temperature.

      5. Terminate the reaction by heating at 85 °C for 5 min.
    3. Retrotranscription verification.
      This is an optional step. Carry out a conventional PCR procedure with a pair of primers of your choice to corroborate the presence of cDNA in your sample. Actin or other housekeeping genes might be suitable.
    4. First PCR (PCR amplification of cDNA). According to the High Fidelity PCR Enzyme Mix protocol with some modifications.
      1. Prepare the following reaction mixture
        30 µl Distilled water
        5 µl 10x High Fidelity PCR buffer (+ 15 mM MgCl2)
        1 µl dNTPs mix, 10 mM each
        1 µl specific primer (forward primer), 12.5 µM
        5 µl Template DNA
        0.4 µl (2 U) High Fidelity PCR Enzyme Mix
        8 µl PCR Anchor Primer (reverse primer) 12.5 µM (from Roche 5’/3’ RACE Kit, corresponding to the adapter sequence. See primer sequence below)
      2. PCR profile (check manufacturer’s indications, parameters may vary according to the length of the expected products)
        1 cycle 94 °C, 2 min
        35 cycles 94 °C, 30 sec; 57 °C, 30 sec; 68 °C, 4 min 30 sec
        1 cycle 68 °C, 10 min
        Note: It might be convenient to perform simultaneous reactions with distinct forward primers (2-4) hybridizing at close locations in order to select that one yielding optimal results.
      3. Check 10 µl of the reaction in an agarose gel (Figure 2)
        Note: Agarose concentration and running time may depend on the size of the expected products.
    5. Second PCR. Same as in step B4, except template (an aliquot of the first strand PCR), and a new forward primer (hybridizing downstream to the one used in the first reaction to endorse specificity).
      1. Reaction mixture
        34 µl Distilled water
        5 µl 10x High Fidelity PCR buffer (+ 15 mM MgCl2)
        1 µl dNTPs mix, 10 mM each
        1 µl specific primer (forward primer), 12.5 µM
        8 µl PCR Anchor Primer (reverse primer) 12.5 µM
        1 µl First PCR reaction
        0.4 µl (2 U) High Fidelity PCR Enzyme Mix
      2. Use same PCR profile as in step B4b.
        Note: Eventually the forward primer might be one of those tested for the first strand reaction provided that it lies downstream to the one used during that step.
    6. Isolate PCR products.
      1. Load the whole reaction in an agarose gel and carry out electrophoresis until the bands are separated as much as possible (Figure 3).
        Note: Agarose concentration may depend on the length of the expected products.
      2. Under a UV transilluminator, excise PCR DNA bands and store separately in Eppendorf tubes.
      3. Purify DNA using GeneJET Gel Extraction Kit (see protocol) or any other equivalent system. Finally, DNA is eluted from columns in a total volume of 30 µl.
    7. Cloning and sequencing.
      Eluted dsDNA PCR fragments contain 3'-A overhangs that allow for easy cloning in any T-vector plasmid system. For example, see protocol for StrataClone PCR Cloning Kit. Finally, sequence and analyze the structure of inserts contained in positive clones.

Representative data

Table 1. Oligonucleotides used in this study

Name
Sequence (5’ – 3’)
References
Oligo d(T)-Anchor Primer
GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV
Roche 5’/3’ RACE Kit
PCR Anchor Primer
GACCACGCGTATCGATGTCGAC Roche
5’/3’ RACE Kit
ACT2-f
CTCTTAACCGTAAAGCTAACAG
An et al., 1996
ACT2-r
AGTGAGAATCTTCATGAGTGAG
An et al., 1996
AGIa (specific forward primer)
CGGATCGAGAACACAACGAATCG
Rodríguez-Cazorla et al., 2015
AGIb (specific forward primer)
GGTTTGCTCAAGAAAGCTTACGAGC
Rodríguez-Cazorla et al., 2015
V = A, C or G


Figure 1. Relative quantitation and qualitative analysis of total RNA in a 2% agarose gel stained with ethidium bromide (EtBr). Lane 1: Molecular weight ladder. Lanes 2-5: 500 ng of total RNA from different samples.


Figure 2. Verification of the first PCR step in an EtBr-stained agarose gel (1%). Lane 1: Molecular weight ladder. Lane 2: 10 µl of the first PCR reaction.


Figure 3. Verification of the second PCR step in an EtBr-stained agarose gel (1.5%). Lane 1: Molecular weight ladder. Lane 2: Total PCR sample (50 µl). Four bands of different size can be distinguished (A to D) that could be excised and purified as indicated in steps B6b-c.


Figure 4. Schematic representation of examples of prematurely processed AG transcripts identified by RACE. DNA sequence corresponding to exon 2 appears as white upper-case letters boxed in black. Intron 2 sequence is shown as lower-case black letters. Cleavage site is indicated (A in red). Additional representative data could also refer to Figures 4 and S11 from Rodríguez-Cazorla et al. (2015).

Acknowledgments

This work was supported by grants from Ministerio de Economía y Competitividad of Spain (https://sede.micinn.gob.es) (grant BIO2014-56321-P to AV) and National Science Foundation of USA (http://www.nsf.gov/; grant IOS-1121055 to MFY) and Paul D. Saltman Endowed Chair in Science Education (http://biology.ucsd.edu/news/awards-and-honors/endowedchairs.html) to MFY.

References

  1. An, Y. Q., McDowell, J. M., Huang, S., McKinney, E. C., Chambliss, S. and Meagher, R. B. (1996). Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J 10(1): 107-121.
  2. Bentley, D. L. (2014). Coupling mRNA processing with transcription in time and space. Nat Rev Genet 15(3): 163-175.
  3. Nojima, T., Gomes, T., Grosso, A. R., Kimura, H., Dye, M. J., Dhir, S., Carmo-Fonseca, M. and Proudfoot, N. J. (2015). Mammalian NET-Seq reveals genome-wide nascent transcription coupled to RNA processing. Cell 161(3): 526-540.
  4. Rodriguez-Cazorla, E., Ripoll, J. J., Andujar, A., Bailey, L. J., Martinez-Laborda, A., Yanofsky, M. F. and Vera, A. (2015). K-homology nuclear ribonucleoproteins regulate floral organ identity and determinacy in arabidopsis. PLoS Genet 11(2): e1004983.
  5. Smyth, D. R., Bowman, J. L. and Meyerowitz, E. M. (1990). Early flower development in Arabidopsis. Plant Cell 2(8): 755-767.
  6. Yanofsky, M. F., Ma, H., Bowman, J. L., Drews, G. N., Feldmann, K. A. and Meyerowitz, E. M. (1990). The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346(6279): 35-39.

简介

功能真核RNA的生产是非常复杂的过程,其涉及转录和各种RNA加工活性(包括剪接,5'加帽和3'剪切和聚腺苷酸化)之间的复杂相互作用(Bentley,2014)。 RNA末端的精确作图提供了评估产生不同基因转录物的转录和转录后事件的有价值的工具。这种转录物的丰度最可能取决于外源和发育线索或突变。在参考植物拟南芥中,HUA-PEP 转录后调节因子(Rodríguez-Cazorla等人,2015)的扰动导致保留内含子序列的关键花卉同源异源基因(AGO)( )(Yanofsky等人,1990)的异常转录物的积累。通过3'RACE反应测定,这样的错误转录物对应于在AG 内含子区发生的过早加工和聚腺苷酸化事件。在这里我们描述了一个适用于分析相对丰富的转录物以及用于检测可能易于快速更新的异常RNA种类的方案。同样地,此处适于拟南芥生殖组织的方法可用于表征来自其他器官(叶,根)和/或其它植物物种的RNA种类。我们提供我们的3'RACE程序的详细方案,包括四个主要部分:总RNA提取,RNA量测定和质量控制,RACE程序本身,和分离所得RACE产物用于克隆和测序。

关键字:拟南芥, 3的比赛, RNA的提取

材料和试剂

  1. 一次性手套
  2. 无菌一次性无RNase移液器吸头
  3. 无RNase微量离心管
  4. 植物样品(拟南芥幼芽,第1至9期)
    注意:根据Smyth等人,1990年的拟南芥花期。
  5. 液氮
  6. GeneJET Plant RNA纯化试剂盒(Thermo Fisher Scientific,目录号:K0801)
  7. 1 M DTT(Sigma-Aldrich,目录号:43816)
  8. 无水乙醇(JT Baker 8006)和96%乙醇(由RNA提取试剂盒制造商推荐,参见上面第6点)
  9. 4M LiCl(在蒸馏水中制备并高压灭菌,不一定是新鲜的)
  10. DNase I,无RNA酶(Thermo Fisher Scientific,目录号:EN0525)
  11. RiboLock核糖核酸酶抑制剂(Thermo Fisher Scientific,目录号:EO0382)
  12. dNTPs各自混合10mM(Thermo Fisher Scientific,目录号:R0192)
  13. OligodT-锚定引物(Roche 5'/3'RACE试剂盒)(Roche Diagnostics,目录号:03 353 621 001,版本10)
  14. Maxima Reverse Transcriptase + buffer 5x(Thermo Fisher Scientific,目录号:EP0741)
  15. 无RNase水
  16. 高保真PCR酶混合物+缓冲液10x(Thermo Fisher Scientific,目录号:K0191)
  17. PCR锚定引物(Roche 5'/3'RACE试剂盒)(Roche Diagnostics,目录号:03 353 621 001)
  18. 基因特异性5'引物(表1)
  19. GeneJET凝胶提取试剂盒(Thermo Fisher Scientific,目录号:K0691)
  20. Strata Clone PCR Cloning Kit(Agilent Technologies,目录号:240205)
  21. Taq DNA聚合酶(EURx,目录号:EK2500-04)
  22. GeneRuler TM 100bp Plus DNA Ladder(Thermo Fisher Scientific,目录号:SM0321)(或为了方便起见,任何其他合适的分子标记)

设备

  1. 砂浆和杵(冲洗Haldenwanger砂浆,直径63毫米,清洁,高压灭菌,使用前干燥)
  2. 热固机(水浴或加热块也可以使用)(Eppendorf)
  3. 微量离心机(Heraeus,Biofuge Pico)
  4. 带有转子12,148-H(Sigma-Aldrich)的冷冻离心机BR15(B.Braun Melsungen AG),用于1.5ml Eppendorf管
  5. BioPhotometer Plus(Eppendorf)
  6. 热循环仪T100(Bio Rad)(或任何其他常规PCR装置)
  7. 电泳系统
  8. 紫外透射仪

程序

  1. 从花芽中提取总RNA
    1. 在砂浆中倒入液氮,让其蒸发,然后 仔细放置植物材料。研磨直到细的均匀粉末 ?获得。用小刮刀存放在1.5 ml Eppendorf管中。
      注意:在室温下操作。植物材料可以是新鲜的 收集或预先收获并储存在-80℃。在这两种情况下, 然而,收获必须在冷冻条件下进行(它是 ?建议保持Eppendorf tub e和铲液体nitr ogen 使用前)。
    2. 使用GeneJET Plant RNA纯化试剂盒提取总RNA(参见方案)。
      1. 添加20微升的1 M DTT到每500微升的RNA裂解溶液 (含有离液盐的硫氰酸胍)。添加此混合物 ?到含有组织粉末(高达100mg)的管中并涡旋 10-20秒彻底混合。这一步应该快速执行 避免退化。
      2. 在56℃下在热混合器或加热块中孵育3分钟
      3. 以最大速度(≥20,000x g,<13,000-14,000 rpm)离心5分钟。
      4. 收集上清液(通常450-550微升),并转移到干净的1.5毫升管
      5. 加入250μl的96%乙醇。通过移液混合。
      6. 将混合物转移到插入2ml收集管(两者随试剂盒提供)中的纯化柱
      7. 以12,000×g(约11,000rpm)离心该柱1分钟。丢弃 ?流通溶液,并重新组装色谱柱和收集管
      8. 向纯化柱中加入700μl洗涤缓冲液WB 1(随试剂盒提供)
      9. 在12,000×g(约11,000rpm)离心1分钟。丢弃 流通和收集管。将纯化柱放入 清洁2 mL收集管
      10. 向纯化柱中加入500μl洗涤缓冲液2(随试剂盒提供)
      11. 在12,000×g(约11,000rpm)离心1分钟。丢弃 流过溶液并重新装配色谱柱和收集管
      12. 重复前面的步骤A2j-k,并以最大速度重新旋转色谱柱1分钟,以清除所有痕量的洗涤缓冲液2.
      13. 用50μl无核酸酶的水洗脱RNA,离心1分钟 ?min,在12,000×g(?11,000rpm)。以相同的音量重复此步骤 的无核酸酶水,以提高RNA的产量
      14. 立即使用纯化的RNA或存储直到使用。建议储存在-80°C。

    3. DNase处理和浓缩
      1. 用5μl(5 U)DNase I(Thermo Fisher Scientific, 根据制造商的方案,略有修改)1小时 在37℃消除痕量的基因组DNA。包括RiboLock核糖核酸酶 抑制剂1 U /μl,以防止RNA降解
      2. 加入10μl50mM EDTA,并在65℃孵育10分钟
      3. 立即,将RNA样品与两倍体积的冷的100%乙醇混合,并在-20℃下储存数小时或过夜。
      4. 加入1/10体积的4M LiCl,并在12,000rpm和4℃下离心10分钟。小心地清除上清液。
      5. 用500μl冷70%乙醇洗涤,并如步骤A3d离心
      6. 用移液管吸头尽可能多的上清液。放 微量离心管与开盖在一个带冰的盒子里,让 将颗粒在工作台上或在层流通风室中干燥5分钟 注意:在室温下干燥沉淀也很好。
      7. 重悬于25μl无RNA酶的水中,如果不立即使用,存放在-80°C
      8. 通过光度法测定RNA浓度,并通过在2%琼脂糖凝胶中显现500ng来检查完整性(图1)。
        注意:强烈建议使用光度计比色杯和凝胶盘 ?预先用0.4M NaOH处理(至少5分钟)以最小化 ?存在核糖核酸酶。
      9. 对照PCR。尽管DNase I治疗 在RNA提取后,将0.5μl样品用作a的模板 用脱氧核苷酸引物进行常规PCR反应以测试缺失 的基因组DNA。用您选择的一对引物进行PCR 测试样品中DNA的存在。肌动蛋白或其他家务 基因可能是合适的 PCR组件:
        16μl蒸馏水
        2.5μl10x缓冲液B(+ 15mM MgCl 2)
        1μldNTPs混合物(各5mM) 1μlACT2-f(5μM)(参见下面的寡核苷酸序列) 1μlACT2-r(5μM)(参见下面的寡核苷酸序列) 0.25μl(1.25U)Taq DNA聚合酶 0.5μlRNA简单
        PCR谱:
        1个周期
        94℃,2分钟
        35个周期
        94℃,30秒; 51℃,30秒; 72℃,30秒
        1个周期
        72℃,10分钟

  2. 3'RACE
    1. 设计特异性引物(正向引物) 设计引物 使用免费在线软件OligoCalc ( http://www.basic.northwestern.edu/biotools/oligocalc.html)。最适合 ?引物长度通常为20至25nt,GC含量为50-60%(as 建议由Roche 5'/3'RACE试剂盒协议)。熔点 (Tm)必须接近PCR锚定引物(见下文),而不是 超过5°C的差异
    2. 重转录。
      根据Maxima Reverse Transcriptase 协议提供的说明进行了微小的修改。
      1. 混合以下组分用于退火反应混合物:
        5μg以前分离的总RNA 将来自Roche 5'/3'RACE试剂盒(A)的1μlOligod(T)-Anchor Primer(37.5μM) polydT延伸,接着是接头序列,参见引物序列 下面)
        1μldNTPs混合物(每种10mM)
        14.5μl蒸馏水(不含核酸酶)
      2. 在65℃孵育5分钟。在冰上冷却,离心几个 秒(将整个反应置于底部)并置于冰上 再次。
      3. 将以下反应组分以指定的顺序
        加入到退火混合物中 4μl5x RT缓冲液(随酶批次提供)
        0.5μl(20 U)RiboLock
        1μl(200 U)Maxima Reverse Transcriptase
      4. 在60℃孵育30分钟。
        注意:富含GC的RNA的反转录可以在65℃进行。 实际上,温度可以在50至65℃之间变化。试点实验可能 必须找到最佳温度。
      5. 通过在85℃加热5分钟终止反应。
    3. 重转帐验证。
      这是一个可选步骤。进行常规PCR程序 ?您选择的一对引物来证实cDNA的存在 您的样品。肌动蛋白或其他管家基因可能是合适的
    4. 第一PCR(cDNA的PCR扩增)。根据高保真PCR酶混合物协议,有一些修改。
      1. 制备以下反应混合物
        30μl蒸馏水
        5μl10x高保真PCR缓冲液(+ 15mM MgCl 2)
        1μldNTPs混合物,每种10mM
        1μl特异性引物(正向引物),12.5μM
        5μl模板DNA
        0.4μl(2 U)高保真PCR酶混合物
        8μlPCR锚定引物(反向引物)12.5μM(来自Roche 5'/3'RACE Kit,对应于接头序列。参见下面的引物序列)
      2. PCR配置文件(检查制造商的说明,参数可能会根据预期产品的长度而变化)
        1个循环94℃,2分钟
        35个循环94℃C,30秒; 57℃,30秒; 68℃,4分30秒
        1个循环68℃,10分钟
        注意:执行同时反应可能很方便 不同的正向引物(2-4)在接近位置按顺序杂交 以选择一个产生最佳结果。
      3. 在琼脂糖凝胶(图2)中检查10μl反应物。
        注意:琼脂糖浓度和运行时间可能取决于预期产品的大小。
    5. 第二PCR。与步骤B4相同,除了模板(a 第一链PCR)和新的正向引物(杂交下游 在第一反应中使用的那一个来支持特异性)
      1. 反应混合物
        34μl蒸馏水
        5μl10x高保真PCR缓冲液(+ 15mM MgCl 2)
        1μldNTPs混合物,每种10mM
        1μl特异性引物(正向引物),12.5μM
        8μlPCR Anchor Primer(反向引物)12.5μM
        1μl第一次PCR反应
        0.4μl(2 U)高保真PCR酶混合物
      2. 使用与步骤B4b相同的PCR谱。
        注意:最终正向引物可能是测试的引物之一 第一链反应提供它位于一个下游 在该步骤期间使用。
    6. 分离PCR产物
      1. 加载 在琼脂糖凝胶中进行完全反应,并进行电泳直至 ?频带尽可能分开(图3)。
        注意:琼脂糖浓度可能取决于预期产品的长度。
      2. 在紫外透射仪下,切除PCR DNA条带,并单独储存在Eppendorf管中
      3. 使用GeneJET凝胶提取试剂盒纯化DNA(参见方案)或任何 其他等效系统。最后,DNA总共从柱上洗脱 体积30μl。
    7. 克隆和测序。
      洗脱dsDNA PCR 片段含有允许在任何中容易克隆的3'-A突出端 T-载体质粒系统。例如,请参阅用于StrataClone PCR的协议 克隆试剂盒。最后,序列和分析插入物的结构 包含在阳性克隆中。

代表数据

表1.本研究中使用的寡核苷酸

名称
Secuence(5' - 3')
参考资料
寡核苷酸(T)-Anchor Primer
GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTTV
罗氏5'/3'RACE试剂盒
PCR锚定引物
GACCACGCGTATCGATGTCGAC Roche
5'/3'RACE套件
ACT2-f
CTCTTAACCGTAAAGCTAACAG
An 等人,1996
ACT2-r
AGTGAGAATCTTCATGAGTGAG
An 等人,1996
AGIa(特异性正向引物)
CGGATCGAGAACACAACGAATCG
Rodríguez-Cazorla等人,2015年
AGIb(特异性正向引物)
GGTTTGCTCAAGAAAGCTTACGAGC
Rodríguez-Cazorla等人,2015年
V = A,C或G


图1.在用溴化乙锭(EtBr)染色的2%琼脂糖凝胶中的总RNA的相对定量和定性分析。泳道1:分子量梯度。泳道2-5:500ng来自不同样品的总RNA

图2.在EtBr染色的琼脂糖凝胶(1%)中第一PCR步骤的验证。泳道1:分子量梯度。泳道2:10μl的第一次PCR反应

图3.在EtBr染色的琼脂糖凝胶(1.5%)中第二PCR步骤的验证。泳道1:分子量梯度。泳道2:总PCR样品(50μl)。可以区分不同大小的四个条带(A至D),如步骤B6b-c所示,可以进行切割和纯化。

图4.由RACE识别的过早处理的 AG 成绩单的示例的示意图。 对应于外显子2的DNA序列显示为黑色大写字母。 Intron 2序列显示为小写黑色字母。显示切割位点(A为红色)。另外的代表性数据还可以参考来自Rodríguez-Cazorla等人的图4和S11。(2015)。

致谢

这项工作得到西班牙经济部长奖学金的支持( https://sede.micinn.gob.es )(授予BIO2014-56321-P至AV)和美国国家科学基金会( http://www。 nsf.gov/;授予IOS-1121055至MFY)和Paul D. Saltman授予科学教育主席(http://biology.ucsd.edu/news/awards-and-honors/endowedchairs.html)至MFY。

参考文献

  1. An,Y.Q.,McDowell,J.M.,Huang,S.,McKinney,E.C.,Chambliss,S。和Meagher,R.B。(1996)。 拟南芥ACT2/ACT8肌动蛋白亚类在营养中的强烈的组成型表达植物J 10(1):107-121。
  2. Bentley,D.L。(2014)。 将mRNA处理与时间和空间中的转录相结合。 Nat Rev Genet 15(3):163-175。
  3. Nojima,T.,Gomes,T.,Grosso,A.R.,Kimura,H.,Dye,M.J.,Dhir,S.,Carmo-Fonseca,M.and Proudfoot,N.J。 哺乳动物NET-Seq揭示了与RNA加工耦合的全基因组新生转录。 C 161(3):526-540
  4. Rodriguez-Cazorla,E.,Ripoll,J.J.,Andujar,A.,Bailey,L.J.,Martinez-Laborda,A.,Yanofsky,M.F.and Vera,A。(2015)。 K-同源核核糖核蛋白调节拟南芥中的花器官特性和确定性。 PLoS Genet 11(2):e1004983。
  5. Smyth,D.R.,Bowman,J.L.和Meyerowitz,E.M。(1990)。 拟南芥中的早期花卉发育 植物Cell 2(8):755-767。
  6. Yanofsky,M.F.,Ma,H.,Bowman,J.L.,Drews,G.N.,Feldmann,K.A。和Meyerowitz,E.M。 由拟南芥同源异型基因agamous编码的蛋白质类似于转录因子。/a> 346(6279):35-39
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rodríguez-Cazorla, E., Andújar, A., Ripoll, J. J., Bailey, L. J., Martínez-Laborda, A., Yanofsky, M. F. and Vera, A. (2015). 3’ Rapid Amplification of cDNA Ends (3’ RACE) Using Arabidopsis Samples. Bio-protocol 5(19): e1604. DOI: 10.21769/BioProtoc.1604.
  2. Rodriguez-Cazorla, E., Ripoll, J. J., Andujar, A., Bailey, L. J., Martinez-Laborda, A., Yanofsky, M. F. and Vera, A. (2015). K-homology nuclear ribonucleoproteins regulate floral organ identity and determinacy in arabidopsis. PLoS Genet 11(2): e1004983.
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