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Jul 2018

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Extraction of RNA from Recalcitrant Tree Species Paulownia elongata
从顽拗型树种泡桐中提取RNA   

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Abstract

Isolation of pure RNA is the basic requisite for most molecular biology work. Plants contain polyphenols and polysaccharides, which can interfere with isolation of pure RNA from them. Especially hardwood tree species like Paulownia elongata have surplus amount of RNA-binding alkaloids, proteins and secondary metabolites that can further complicate the process of RNA extraction. Paulownia elongata is a fast-growing tree species which is known for its role in environmental adaptability and biofuel research. Here we describe an economical, efficient and time-saving method (2 h) to extract RNA from leaf tissues of the tree Paulownia elongata. Lack of DNA contamination and good RNA integrity were confirmed using RNA Gel electrophoresis. The purity of RNA was confirmed using Nanodrop spectrophotometer that revealed an A260:A280 ratio of about 2.0. The purified RNA was successfully used in the downstream applications such as RT-PCR (Reverse Transcription PCR) and qPCR (quantitative PCR). This method could be used for RNA extraction from several other recalcitrant tree species.

Keywords: RNA (核糖核酸), Recalcitrant plant species (顽拗型植物品种), RNA extraction (RNA 提取), Paulownia elongata (兰考泡桐), TrizolTM (TrizolTM)

Background

Paulownia elongata is a widely distributed tree that belongs to the family of Paulowniaceae (Zhu et al., 1986). It is known for its high adaptability and rapid growth rate (Chaires et al., 2017). Paulownia woods are gaining demands from all over the world due to their high stability, low thermal conductivity, decay and rot resistance etc. (Ayrilmis and Kaymakci, 2013). Apart from this, P. elongata is also known to show tolerance to a variety of biotic and abiotic stresses (Chaires et al., 2017). However not many studies are done on understanding its stress tolerance mechanism, which requires extraction of high-quality RNA.

High-quality RNA refers to RNA that is devoid of any genomic DNA, phenols, polysaccharides, secondary metabolites, etc. that interfere with molecular biology techniques (Ouyang et al., 2014). Genomic DNA contamination will affect the detection and quantification of gene expression analysis such as RT-PCR, qPCR, northern blotting and RNA sequencing (Añez-Lingerfelt et al., 2009). This is because the reaction cannot differentiate between cDNA (complementary DNA) and genomic DNA, which will lead to overestimation of gene expression present (Añez-Lingerfelt et al., 2009). Phenols in RNA can oxidize to form quinones that will bind to nucleic acids irreversibly (Ouyang et al., 2014). Polysaccharides and secondary metabolites can co-precipitate and degrade the RNA in the sample thereby affecting the yield, quality and downstream applications of RNA (Ouyang et al., 2014; Ghawana et al., 2011). Since P. elongata leaves are known to have a high content of alkaloids and proteins (Kirov et al., 2014), isolation of pure RNA from them poses a challenge. Methods using spin columns did not yield a good amount of RNA (data not shown) from P. elongata leaves, and this could be due to the fact that spin columns efficiency decreases in the presence of alkaloids (Ouyang et al., 2014). CTAB (cetyltrimethyl ammonium bromide) based methods are spin-column free but are time-consuming (Ouyang et al., 2014). Thus, we combined the Trizol (InvitrogenTM) extraction method and spin-column based purification method in our protocol.

In our study we extracted high quantity RNA using a modified Trizol (InvitrogenTM) method. The quality of RNA was improved by using RNA Clean and Concentrator Kit (ZYMO RESEARCH, USA) with modifications. The RNA yield was measured using Nanodrop spectrophotometer (Figure 1). The Nanodrop measurement peaks can also analyze the presence of phenols or polysaccharides in the sample. In our study, the peak indicated that the RNA was free from phenol or polysaccharide contamination (Figure 1). The integrity of the RNA was further affirmed by running an RNA gel (Formaldehyde free RNA gel kit, Amresco, USA). The gel revealed that the RNA extracted using our method was free from genomic DNA contamination. Clear 28S and 18S rRNA bands were observed (Figure 2). The RNA was then successfully used in downstream applications like RT-PCR and qPCR which amplified the ubiquitin gene (Figures 3 and 4). In RT-PCR, we used cDNA synthesized from our P. elongata RNA as the template. The cDNA was amplified using ubiquitin qPCR primers. The ubiquitin primers used in the study are as follows:
Forward Primer- 5’ GTC AGG AGG AAC ACC TTC TTT 3’
Reverse Primer- 5’ CCT TGA CTG GGA AGA CCA TTAC 3’

Thus bands of about 250 bp were observed as a result of successful amplification of ubiquitin (Figure 3). Our method of RNA isolation not only has high and pure yield but is also time-saving. The entire procedure takes about 2 h.

Materials and Reagents

  1. Nuclease-free microfuge tubes
  2. Nuclease-free micropipette tips
  3. Nitrile powder free gloves
  4. Liquid nitrogen
  5. RNase (ribonuclease) Away (Thermo Fisher Scientific, catalog number: 7000TS1 )
  6. Trizol (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596026 )
  7. 24:1 Chloroform:Isoamyl alcohol (Acros Organics, catalog number: 327155000
  8. 75% and 100% ethanol
  9. Nuclease-free water (Not DEPC-treated) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 4387936 )
  10. Turbo DNase (deoxyribonuclease) (Thermo Fisher Scientific, AmbionTM, catalog number: AM2238 )
  11. RNA Clean and Concentrator kit (ZYMO RESEARCH, catalog number: R1015 )
  12. Chilled isopropanol

Equipment

  1. Pipettes (BioExpress, GeneMate, catalog number: P-3960-20 )
  2. Mortar and pestle (Harold Import, HIC, catalog number: 43717 )
  3. Vortexer (BioExpress, GeneMate, catalog number: S-3200-1 )
  4. Refrigerated microcentrifuge (Labnet International, catalog number: C2500-R )
  5. Water bath
  6. -80 °C freezer
  7. Fume hood (KEWAUNEE, model: H05_5460-00 )
  8. Nanodrop Spectrophotometer (Thermo Fisher Scientific, USA)

Procedure

  1. RNA Extraction
    Note: It is modified from standard TrizolTM procedure.
    1. Spray gloves, equipment and all materials using RNase (ribonuclease) AWAY.
    2. Snap freeze about 100 mg of leaves by immersing the leaves for 5 sec in liquid nitrogen.
      Tip: Snap freezing works better than crushing the leaves directly in liquid nitrogen.
    3. Grind the frozen leaves in the presence of 1 ml of Trizol using a mortar and pestle (not necessary to be cold) and transfer to a nuclease-free microfuge tube using a pipette.
      Note: This step should be performed inside a fume hood.
    4. Incubate the tube for 5 min at room temperature (RT).
    5. Then add 200 μl of 24:1 Chloroform:Isoamyl alcohol into the tube and vortex to mix thoroughly.
      Tip: The sample must be vortexed until it is homogeneously green. This vortexing step is critical for high yield of RNA.
    6. Incubate this mixture for 2 min at RT.
    7. Then centrifuge at 12,000 x g for 15 min at 4 °C.
    8. Transfer the supernatant (the aqueous phase containing RNA) to a new tube.
      Tip: Note the volume of phase transferred.
    9. Add chilled isopropanol into the new tube at a volume of 0.5 times the original volume of aqueous phase present.
      Tip: Isopropanol must be chilled, otherwise the yield of RNA might be low.
    10. Vortex the mixture vigorously for at least 10 sec and incubate for about 15 min at -20 °C.
    11. Centrifuge the mix for 10 min at 12,000 x g at 4 °C.
    12. RNA can be seen as a small white pellet at the bottom of the tube.
    13. Remove the supernatant isopropanol without disturbing the pellet. Wash the RNA pellet by adding 500 μl of 75-80% ethanol and discard the supernatant after spinning at 7,000 x g for 5 min (Potential stop point: can be stored at -20 °C for up to a week).
    14. Spin the tube again (without adding any reagents) and then remove any residual ethanol that sticks to the wall of the microcentrifuge tube.
    15. Air dry the pellet for 15 min by placing the inverted tube inside the fume hood.
    16. Then resuspend the pellet in 40 μl of nuclease-free water and incubate in a water bath with the tube open at 60 °C for 15 min to completely dissolve it. The cap of microfuge tube should be kept open for efficient evaporation of residual ethanol (Potential Stop point: samples can be stored at -80 °C). The samples stored at -80 °C must be thawed on ice and treated by the following steps.

  2. RNA Purification
    Note: It is modified from ZYMO Clean and Concentrator KitTM protocol (ZYMO RESEARCH, USA).
    1. Add 80 μl of RNA binding buffer to the RNA (see above) and gently mix it using pipette tip.
    2. Then add 120 μl of 100% ethanol to the tube followed by gentle mixing.
    3. Transfer the RNA sample to a Zymospin column in a collection tube and spin at 12,000 x g for 30 sec at 4 °C.
    4. Wash the column with 400 μl of RNA wash buffer by spinning at 12,000 x g for 30 sec at 4 °C. The flow-through will be discarded.
    5. Prepare DNase cocktail by mixing 5 μl of Turbo DNase (Thermo Fisher Scientific, AmbionTM) and 35 μl of DNA digestion buffer (ZYMO RESEARCH, USA) gently in a microfuge tube.
    6. Add the cocktail to the RNA column and incubate for 20 min at RT.
      Tip: Longer incubation times do not improve purity.
    7. Add about 400 μl of RNA Prep buffer (or RNA Pre-Wash Buffer) to the column and spin at 12,000 x g for 30 sec at 4 °C. Then discard the flow through.
    8. Wash the column again twice using 400 μl of RNA Wash buffer like Step B4.
    9. Spin the column (without adding any reagents) for 2 min to remove the residual ethanol.
    10. Finally, transfer the column to a new nuclease-free microfuge tube.
    11. Add about 20 μl of nuclease-free water to the column and incubate for 2 min at RT. Collect the flow through after spinning for 2 min at 12,000 x g at 4 °C.
    12. Pipette out the flow through and add it to the column again.
    13. Incubate the column for 2 min at RT and collect the pure RNA after spinning for 2 min at 12,000 x g at 4 °C.

Data analysis

  1. RNA measurement: The concentration of extracted RNA was measured using a Nanodrop spectrophotometer. Four biological replicates were used. The spectrophotometric images (Figure 1) showed that the RNA we extracted was of high yield starting from 240 ng. Pure RNA is considered to have an A260:A280 ratio between 1.8 and 2.2 (Ouyang et al., 2014). Since our RNA had ratios of 2.06-2.11, it revealed that this extraction protocol yields pure RNA. The single clear peak at 260 nm shows that the RNA extraction is devoid of any contaminants.


    Figure 1. Nanodrop spectrophotometer images of RNA extracted from P. elongata using the modified method. The 260/280 ratios are 2.09 (A), 2.07 (B), 2.06 (C) and 2.11 (D). Four pictures represent four different biological samples.

    RNA integrity:
    The RNA integrity was assessed by running an RNA gel. The RNA samples extracted without following our purification procedure was compared to RNA samples extracted following our modified protocol. About 5 μl of each RNA sample was loaded onto each well. The RNA gel revealed clear, distinct 18S and 28S ribosomal RNA bands for samples obtained using our protocol (Figure 2). The RNA samples obtained with other methods had smears indicating that there is genomic DNA contamination.


    Figure 2. RNA gel pictures. A1, A2, A3: P. elongata RNA without our purification procedure. B1, B2, B3, B4: P. elongata RNA extracted using modified method 1-4 indicates the different biological controls (The picture’s color has been altered to grayscale).

    RNA downstream applications: To test the efficiency of the extracted RNA, the samples were used in RT-PCR and qPCR as templates. PCR products were successfully amplified using ubiquitin primers from the cDNA reverse-transcribed from the extracted RNA. The PCR conditions used were–initial denaturation at 95 °C for 5 min, 30 PCR cycles with denaturation at 95 °C for 30 sec, annealing at 50 °C for 30 sec and extension at 72 °C for 45 sec, with a final extension at 72 °C for 7 min. Four biological replicates were used for RT-PCR. Clear bands of ubiquitin were observed at about 250 bp (Figure 3). The integrity of mRNA (messenger RNA) was tested using qPCR. The qPCR conditions were: initial denaturation at 95 °C for 30 sec, 39 cycles with denaturation at 95 °C for 5 sec, annealing at 50 °C for 30 sec followed by melting-curve analysis. Four biological replicates and 3 technical replicates were used for qPCR.
    The amplification curve obtained from qPCR (Figure 4) showed that the mRNA was not degraded in the total RNA sample and that the ubiquitin gene was present in all 4 biological samples. The melt curve picture reveals that the ubiquitin primers were highly specific and that the results are not due to false positives (Figure 4). These results showed that the quality of RNA we extracted is suitable for basic experiments like PCR and qPCR.


    Figure 3. RT-PCR gel for ubiquitin gene using RNA extracted by this method (Ubiquitin bands of 250 bp size are observed in all four lanes from 3-6). Lane 1: Ladder; Lane 2: Negative control (water); Lanes 3-6: P. elongata cDNA amplified by ubiquitin primers using RNA extracted from 4 different biological controls.


    Figure 4. Amplification curve (top) and melt curve (bottom) observed in qPCR of ubiquitin gene from P. elongata cDNA

  2. Conclusion
    In our study, we developed a protocol for efficient isolation of high-quality RNA from the leaves of a recalcitrant tree species P. elongata. Traditional methods that use CTAB or spin columns were not able to produce this quality of RNA with high yield (data not shown), due to the presence of high amount of alkaloids and proteins in the leaves of P. elongata. The method we developed is rapid (2 h), universal and high yielding. We believe that this method could be used for RNA isolation from various recalcitrant plant species.

Acknowledgments

The authors declare no conflict of interests. The authors want to acknowledge the following two awards/scholarships awarded to NR at the California State University, Northridge: Thesis/Project /Dissertation/Support Award and Richard Duenckel Gardens Club Scholarship. This protocol has been used in our recently published research paper (Ramadoss et al., 2018).

References

  1. Añez-Lingerfelt, M., Fox, G. E. and Willson, R. C. (2009). Reduction of DNA contamination in RNA samples for reverse transcription-polymerase chain reaction using selective precipitation by compaction agents. Anal Biochem 384(1): 79-85.
  2. Ayrilmis, N. and Kaymakci, A. (2013). Fast growing biomass as reinforcing filler in thermoplastic composites: Paulownia elongata wood. Ind Crops Prod 43: 457-464.
  3. Chaires, M., Gupta, D., Joshee, N., Cooper, K. K. and Basu C. (2017) RNA-seq analysis of the salt stress-induced transcripts in fast-growing bioenergy tree, Paulownia elongata. Journal of Plant Interactions. 12(1):128-136.
  4. Ghawana, S., Paul, A., Kumar, H., Kumar, A., Singh, H., Bhardwaj, P. K., Rani, A., Singh, R. S., Raizada, J., Singh, K. and Kumar, S. (2011). An RNA isolation system for plant tissues rich in secondary metabolites. BMC Res Notes 4: 85.
  5. Kirov, V., Shindarska, Z., Kostadinova, G., Gencheva, A., Hadgiev, S., Penev, T. and Baykov, B. (2014). Comparative study of new energy crops for the production of biogas. Int J Curr Microbiol App Sci 3(11): 181-188.
  6. Ouyang, K., Li, J., Huang, H., Que, Q., Li, P. and Chen, X. (2014). A simple method for RNA isolation from various tissues of the tree Neolamarckia cadamba. Biotechnol Biotechnol Equip 28(6): 1008-1013.
  7. Ramadoss, N., Gupta, D., Vaidya, B. N., Joshee, N. and Basu, C. (2018). Functional characterization of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Arabidopsis thaliana and its potential in providing flood tolerance. Biochem Biophys Res Commun. doi: 10.1016/j.bbrc.2018.06.036.
  8. Zhu, Z. H., Chao, C. J., Lu, X. Y. and Xiong Y. G. (1986). Paulownia in China: cultivation and utilization by Chinese academy of forestry staff. Asian Network for Biological Sciences and International Development Research Centre.

简介

纯RNA的分离是大多数分子生物学工作的基本要求。植物含有多酚和多糖,它们可以干扰从它们中分离纯RNA。特别是像泡桐(Paulownia elongata)这样的硬木树种具有过量的RNA结合生物碱,蛋白质和次级代谢物,这些可以进一步使RNA提取过程复杂化。 泡桐(Paulownia elongata)是一种快速生长的树种,以其在环境适应性和生物燃料研究中的作用而闻名。在这里,我们描述了一种经济,有效和省时的方法(2小时)从树的叶组织中提取RNA 泡桐(Paulownia elongata)。使用RNA凝胶电泳证实缺乏DNA污染和良好的RNA完整性。使用Nanodrop分光光度计确认RNA的纯度,其显示A 260 :A 280 比率为约2.0。纯化的RNA成功用于下游应用,例如RT-PCR(逆转录PCR)和qPCR(定量PCR)。该方法可用于从其他几种顽拗型树种中提取RNA。

【背景】泡桐是一种广泛分布的树,属于泡桐科(Zhu et al。,1986)。它以其高适应性和快速增长率而闻名(Chaires et al。,2017)。由于其高稳定性,低导热性,腐烂和腐烂抗性等,泡桐木材正在受到来自世界各地的需求。(Ayrilmis和Kaymakci,2013)。除此之外, P.还已知细菌对各种生物和非生物胁迫具有耐受性(Chaires et al。,2017)。然而,在理解其胁迫耐受机制方面没有进行太多研究,这需要提取高质量的RNA。

高质量的RNA是指缺乏任何干扰分子生物学技术的基因组DNA,酚类,多糖类,次级代谢产物等的RNA(欧阳等。, 2014)。基因组DNA污染将影响基因表达分析的检测和定量,例如RT-PCR,qPCR,northern印迹和RNA测序(Añez-Lingerfelt et al。,2009)。这是因为反应不能区分cDNA(互补DNA)和基因组DNA,这将导致对基因表达的过高估计(Añez-Lingerfelt et al。,2009)。 RNA中的酚可以氧化形成醌,这种醌将不可逆地与核酸结合(Ouyang et al。,2014)。多糖和次级代谢产物可共沉淀并降解样品中的RNA,从而影响RNA的产量,质量和下游应用(Ouyang et al。,2014; Ghawana et al。,2011)。自 P.已知长叶叶具有高含量的生物碱和蛋白质(Kirov et al。,2014),从它们中分离纯RNA提出了挑战。使用旋转柱的方法没有从 P产生大量RNA(数据未显示)。长叶叶,这可能是由于在生物碱存在下旋转柱效率降低的事实(Ouyang et al。,2014)。基于CTAB(十六烷基三甲基溴化铵)的方法不含自旋柱,但耗时(Ouyang 等,,2014)。因此,我们在我们的方案中结合了Trizol(Invitrogen TM )提取方法和基于旋转柱的纯化方法。

在我们的研究中,我们使用改良的Trizol(Invitrogen TM )方法提取高量RNA。通过使用RNA Clean and Concentrator Kit(ZYMO RESEARCH,USA)进行修改,提高了RNA的质量。使用Nanodrop分光光度计测量RNA产量(图1)。 Nanodrop测量峰还可以分析示例。在我们的研究中,峰值表明RNA不含苯酚或多糖污染(图1)。通过运行RNA凝胶(无甲醛游离RNA凝胶试剂盒,Amresco,USA)进一步确认RNA的完整性。凝胶显示使用我们的方法提取的RNA没有基因组DNA污染。观察到清晰的28S和18S rRNA条带(图2)。然后将RNA成功用于下游应用,如RT-PCR和扩增泛素基因的qPCR(图3和4)。在RT-PCR中,我们使用从我们的 P合成的cDNA。细长RNA RNA作为模板。使用遍在蛋白qPCR引物扩增cDNA。研究中使用的泛素引物如下:
Forward Primer-5'GTC AGG AGG AAC ACC TTC TTT 3'
反向引物-5'CCT TGA CTG GGA AGA CCA TTAC 3'

因此,由于遍在蛋白的成功扩增,观察到约250bp的条带(图3)。我们的RNA分离方法不仅具有高产量和纯产量,而且还节省时间。整个过程大约需要2小时。

关键字:核糖核酸, 顽拗型植物品种, RNA 提取, 兰考泡桐, TrizolTM

材料和试剂

  1. 无核酸酶微量离心管
  2. 无核酸酶微量吸头提示
  3. 丁腈粉手套
  4. 液氮
  5. RNase(ribonuclease)Away(赛默飞世尔科技,目录号:7000TS1)
  6. Trizol(Thermo Fisher Scientific,Invitrogen TM ,目录号:15596026)
  7. 24:1氯仿:异戊醇(Acros Organics,目录号:327155000) 
  8. 75%和100%乙醇
  9. 无核酸酶水(未经DEPC处理)(Thermo Fisher Scientific,Invitrogen TM ,目录号:4387936)
  10. Turbo DNase(脱氧核糖核酸酶)(Thermo Fisher Scientific,Ambion TM ,目录号:AM2238)
  11. RNA Clean and Concentrator kit(ZYMO RESEARCH,目录号:R1015)
  12. 冷冻异丙醇

设备

  1. 移液器(BioExpress,GeneMate,目录号:P-3960-20)
  2. 砂浆和杵(Harold Import,HIC,目录号:43717)
  3. Vortexer(BioExpress,GeneMate,目录号:S-3200-1)
  4. 冷冻微量离心机(Labnet International,目录号:C2500-R)
  5. 水浴
  6. -80°C冰柜
  7. 通风柜(KEWAUNEE,型号:H05_5460-00)
  8. Nanodrop分光光度计(Thermo Fisher Scientific,USA)

程序

  1. RNA提取
    注意:它是从标准的Trizol TM 程序修改的。
    1. 使用RNase(核糖核酸酶)AWAY喷涂手套,设备和所有材料。
    2. 通过将叶子在液氮中浸泡5秒来快速冷冻约100mg叶子。
      提示:快速冷冻比直接用液氮压碎叶子更好。
    3. 使用研钵和研杵(不需要冷却)在1ml Trizol存在下研磨冷冻叶子,然后用移液管转移到无核酸酶的微量离心管中。
      注意:此步骤应在通风橱内进行。
    4. 在室温(RT)下孵育管5分钟。
    5. 然后在管中加入200μl24:1氯仿:异戊醇,涡旋混匀。
      提示:样品必须涡旋直至其均匀绿色。这种涡旋步骤对RNA的高产量至关重要。
    6. 在室温下孵育该混合物2分钟。
    7. 然后在4,000℃下以12,000 x g 离心15分钟。
    8. 将上清液(含有RNA的水相)转移到新管中。
      提示:注意转移的相量。
    9. 将冷却的异丙醇加入新管中,体积为原始体积水相的0.5倍。
      提示:异丙醇必须冷却,否则RNA的产量可能会低。
    10. 将混合物剧烈涡旋至少10秒并在-20℃下孵育约15分钟。
    11. 将混合物在12,000 x g 4℃下离心10分钟。
    12. RNA可以看作是管底部的一个小白色颗粒。
    13. 加入500μl75-80%乙醇洗涤RNA沉淀,并在7,000 xg 旋转5分钟后丢弃上清液(潜在停止点可以存储在 - 20°C,最长一周)。
    14. 再次旋转试管(不添加任何试剂),然后除去粘在微量离心管壁上的残留乙醇。
    15. 将倒置管放入通风橱内,将颗粒风干15分钟。
    16. 然后将沉淀重悬于40μl不含核酸酶的水中,并在水浴中孵育,将管在60℃下打开15分钟以使其完全溶解。微量离心管的盖子应保持开放,以便有效蒸发残留的乙醇(潜在停止点:样品可以储存在-80°C)。储存在-80°C的样品必须在冰上解冻并按以下步骤处理。

  2. RNA纯化
    注意:它是从ZYMO Clean and Concentrator Kit TM 协议(ZYMO RESEARCH,USA)修改的。
    1. 向RNA中加入80μlRNA结合缓冲液(见上文)并用移液管尖轻轻混合。
    2. 然后向管中加入120μl100%乙醇,然后温和混合。
    3. 将RNA样品转移到收集管中的Zymospin柱上,在4℃下以12,000 x g 旋转30秒。
    4. 通过在4,000℃下以12,000 x g 旋转30秒,用400μlRNA洗涤缓冲液洗涤柱子。流程将被丢弃。
    5. 通过在微量离心管中轻轻混合5μlTurboDNA酶(Thermo Fisher Scientific,Ambion TM )和35μlDNA消化缓冲液(ZYMO RESEARCH,USA)来制备DNase混合物。
    6. 将鸡尾酒加入RNA柱中,在室温下孵育20分钟。
      提示:更长的孵育时间不会提高纯度。
    7. 向柱中加入约400μlRNAPrep缓冲液,并在4℃下以12,000 x g 旋转30秒。然后丢弃流量。
    8. 使用400μlRNA洗涤缓冲液(如步骤B4)再次洗涤柱两次。
    9. 旋转色谱柱(不添加任何试剂)2分钟以除去残留的乙醇。
    10. 最后,将色谱柱转移到新的无核酸酶微量离心管中。
    11. 向柱中加入约20μl不含核酸酶的水,并在室温下孵育2分钟。在4℃下以12,000 x g 旋转2分钟后收集流量。
    12. 移出流出物并再次将其添加到色谱柱中。
    13. 在室温下孵育柱2分钟,并在4℃下以12,000 x g 旋转2分钟后收集纯RNA。

数据分析

  1. RNA测量:使用Nanodrop分光光度计测量提取的RNA的浓度。使用四个生物学重复。分光光度图像(图1)显示我们提取的RNA从240 ng开始具有高产量。纯RNA被认为具有A 260 :A 280 比率在1.8和2.2之间(Ouyang et al。,2014)。由于我们的RNA具有2.06-2.11的比率,因此显示该提取方案产生纯RNA。在260nm处的单个清晰峰显示RNA提取没有任何污染物。


    图1.从 P中提取的RNA的Nanodrop分光光度计图像。使用改进的方法进行细长 260/280比率为2.09(A),2.07(B),2.06(C)和2.11(D)。四张图片代表四种不同的生物样本。

    RNA完整性:通过运行RNA凝胶评估RNA完整性。将不经我们的纯化程序提取的RNA样品与按照我们修改的方案提取的RNA样品进行比较。将约5μl的每种RNA样品加载到每个孔上。 RNA凝胶显示使用我们的方案获得的样品的清晰,不同的18S和28S核糖体RNA条带(图2)。用其他方法获得的RNA样本有涂片,表明存在基因组DNA污染。


    图2. RNA凝胶图片。 A1,A2,A3: P.没有我们的纯化程序的细长RNA。 B1,B2,B3,B4: P.使用改良方法1-4提取的细菌RNA表明不同的生物控制(图片的颜色已经改变为灰度)。

    RNA下游应用为了测试提取的RNA的效率,将样品用于RT-PCR和qPCR作为模板。使用来自从提取的RNA逆转录的cDNA的遍在蛋白引物成功扩增PCR产物。使用的PCR条件是 - 在95℃下初始变性5分钟,30个PCR循环,在95℃变性30秒,在50℃退火30秒,在72℃延伸45秒,最后在72°C下延伸7分钟。四个生物学重复用于RT-PCR。在约250bp处观察到清晰的遍在蛋白条带(图3)。使用qPCR测试mRNA(信使RNA)的完整性。 qPCR条件为:在95℃下初始变性30秒,39个循环,在95℃变性5秒,在50℃退火30秒,然后进行熔解曲线分析。将4个生物学重复和3个技术重复用于qPCR。
    从qPCR获得的扩增曲线(图4)显示mRNA在总RNA样品中未降解,并且遍在蛋白基因存在于所有4种生物样品中。熔解曲线图显示泛素引物具有高度特异性,结果不是由于假阳性(图4)。这些结果表明,我们提取的RNA质量适合PCR和qPCR等基础实验。


    图3.使用通过该方法提取的RNA的遍在蛋白基因的RT-PCR凝胶(在3-6的所有四个泳道中观察到250bp大小的遍在蛋白条带)。泳道1:梯形;泳道2:阴性对照(水);车道3-6: P.使用从4种不同生物对照中提取的RNA,通过遍在蛋白引物扩增细胞cDNA。


    图4.在来自 P的遍在蛋白基因的qPCR中观察到的扩增曲线(顶部)和解链曲线(底部)。细菌 cDNA

  2. 结论
    在我们的研究中,我们开发了一种方案,用于从顽拗型树种 P的叶子中有效分离高质量的RNA。泡桐。由于 P叶中存在大量生物碱和蛋白质,使用CTAB或旋转柱的传统方法无法以高产率生成这种质量的RNA(数据未显示)。泡桐。我们开发的方法是快速(2小时),通用和高产。我们相信这种方法可用于从各种顽拗型植物中分离RNA。

致谢

作者声明没有利益冲突。作者希望感谢以下两个奖项/奖学金颁发给加州州立大学北岭分校的NR:论文/项目/论文/支持奖和Richard Duenckel Gardens俱乐部奖学金。该协议已用于我们最近发表的研究论文(Ramadoss等,2018)。

参考

  1. Añez-Lingerfelt,M.,Fox,G。E.和Willson,R。C.(2009)。 使用压实剂选择性沉淀,减少RNA样品中的DNA污染,进行逆转录聚合酶链反应。 Anal Biochem 384(1):79-85。
  2. Ayrilmis,N。和Kaymakci,A。(2013年)。 快速生长的生物质作为热塑性复合材料中的增强填料:泡桐木木材。 Ind Crops Prod 43:457-464。
  3. Chaires,M.,Gupta,D.,Joshee,N.,Cooper,KK和Basu C.(2017)快速生长的生物能源树中的盐胁迫诱导转录本的RNA-seq分析,泡桐。 植物相互作用杂志。 12(1):128-136。
  4. Ghawana,S.,Paul,A.,Kumar,H.,Kumar,A.,Singh,H.,Bhardwaj,PK,Rani,A.,Singh,RS,Raizada,J.,Singh,K。和Kumar, S.(2011)。 用于富含次级代谢产物的植物组织的RNA分离系统。 BMC Res注释 4:85。
  5. Kirov,V.,Shindarska,Z.,Kostadinova,G.,Gencheva,A.,Hadgiev,S.,Penev,T。和Baykov,B。(2014)。 用于生产沼气的新能源作物的比较研究。 Int J Curr Microbiol App Sci 3(11):181-188。
  6. Ouyang,K.,Li,J.,Huang,H.,Que,Q.,Li,P。和Chen,X。(2014)。 从树木的各种组织中分离RNA的简单方法 Neolamarckia cadamba 。 Biotechnol Biotechnol Equip 28(6):1008-1013。
  7. Ramadoss,N.,Gupta,D.,Vaidya,B.N.,Joshee,N。和Basu,C。(2018)。 拟南芥中1-氨基环丙烷-1-羧酸氧化酶基因的功能特征及其提供的潜力洪水容忍。 Biochem Biophys Res Commun 。 doi:10.1016 / j.bbrc.2018.06.036。
  8. Zhu,Z。H.,Chao,C。J.,Lu,X。Y. and Xiong Y. G.(1986)。 中国的泡桐:中国林业工作人员的培养和利用。 亚洲网络生物科学与国际发展研究中心。
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引用:Ramadoss, N. and Basu, C. (2018). Extraction of RNA from Recalcitrant Tree Species Paulownia elongata. Bio-protocol 8(14): e2925. DOI: 10.21769/BioProtoc.2925.
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