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Mitochondrial RNA Transcript Analysis Assay of Arabidopsis Leaf Tissues
拟南芥叶片组织中线粒体RNA转录水平的分析实验   

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Abstract

This qPCR-based assay provides an overview of the expression levels of all mitochondrial transcripts (mRNAs and rRNAs) as well as splicing efficiency in Arabidopsis. It was developed before RNAseq techniques were widely used (de Longevialle et al., 2007), but is nevertheless still useful as it is cheaper to run and the analysis is much easier and faster to perform if the aim is only to look at mitochondrial transcripts. For intron-containing mRNAs, the use of primer sets specifically amplifying spliced or unspliced forms allows the evaluation of the splicing efficiency.

Keywords: Mitochondrial transcriptome(线粒体转录组), Splicing assay(拼接法), Quantitative RT-PCR(定量RT-PCR), Mitochondria(线粒体), Arabidopsis thaliana(拟南芥)

Materials and Reagents

  1. Eppendorf-type microtubes (0.5 ml and 1.5 ml) (any brand)
  2. LightCycler® 480 Multiwell plate (white) (Roche Diagnostics, catalog number: 04729749001 )
  3. LightCycler® 480 Sealing Foil for qPCR run (Roche Diagnostics, catalog number: 04729757001 )
  4. Adhesive PCR film seals for storing plates containing primer mixes (Thermo Fisher Scientific, catalog number: AB0558 )
  5. Combitips 0.1 ml (Eppendorf, catalog number: 0030 089.405 or 0030 089.618 )
  6. Arabidopsis thaliana plants grown either in vitro or in soil
  7. RNeasy Plant Mini Kit (QIAGEN, catalog number: 74904 )
  8. Water, for molecular biology, DNAse, RNAse and Protease free (Thermo Fisher Scientific, ACROS OrganicsTM, catalog number: 327390010 )
  9. Ambion Turbo DNA-freeTM Kit (Life Technologies, catalog number: AM1907 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1907”.
  10. 3M Na acetate (pH 5.2)
  11. 100 % and 70 % ethanol
  12. Agarose LE, analytical grade (Promega Corporation, catalog number: V3125 )
  13. Primers for PCR and qPCR (see Table 1)

    Table 1. Primers used in this protocol
    Please click here for Table 1.

  14. Taq polymerase
    Note: any Taq polymerase can be used for this step, we use a home-purified enzyme.
  15. dNTP set (Life Technologies, catalog number: 10297-018 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 10297-018”.
  16. Superscript III Reverse transcriptase (Life Technologies, catalog number: 18080-044 )
    Notes: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-044”.
    This enzyme comes with 5x transcription buffer and a solution of 0.1 M DTT.
  17. Random Hexanucleotide Primers (Life Technologies, catalog number: 48190011 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 48190011”.
  18. RNaseOUT Recombinant Ribonuclease inhibitor (Life Technologies, catalog number:
    10777-019 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777-019”.
  19. LightCycler® 480 SYBR I Master mix (Roche Diagnostics, catalog number: 04887352001 )
    Note: This reagent is light sensitive.

Equipment

  1. Nanodrop UV-Vis Spectrophotometer (Thermo Fisher Scientific)
  2. ‘Multipette plus’ (Eppendorf, catalog number: 4981 000.019 )
  3. LightCycler® 480 System (Roche Diagnostics)

Software

  1. LightCycler® 480 Software version 1.5 (Roche Diagnostics, catalog number: 04994884001)

Procedure

  1. RNA extraction
    1. Total Arabidopsis RNA is extracted from young tissue (8 to 20 day-old leaves) with the RNeasy plant mini kit according to the manufacturer’s protocol.
    2. The RNA is eluted with 40 µl RNase free water and its concentration measured with a NanoDrop.

  2. DNase treatment
    Genomic DNA needs to be removed prior to the reverse transcription step.
    This is achieved using the Ambion Turbo DNA-free kit.
    1. Add in a 0.5 ml tube
      A maximum of 10 µg RNA (in 45 µl RNase-free water)
      4.7µl 10x Turbo DNase buffer
      1 µl DNase
    2. Mix by pipetting and leave tube at 37 °C for 1 h.
    3. The kit contains a DNase inactivator, which leads to a great loss of material and possible residual resin. Instead, precipitate the reaction with 1/10 vol 3 M Na acetate and 2.5 to 3 volumes of 100% ethanol (30 min to 1h at -20 °C). Spin down at max speed (20,800 x g) for 30 min at 4 °C. Carefully discard the supernatant.
    4. Wash the pellet with 70% ethanol (carefully add 300 µl of 70% ethanol without disrupting the pellet and spin down for 15 min. This step removes salts, and this will be achieved better if the tube is kept in a -20 °C freezer for 1 h or overnight.
    5. Dry pellets and resuspend with 30 µl RNase free water.
    6. Measure concentration with NanoDrop and check RNA quality by running about 100 ng on a 2% agarose-TBE gel. This is a regular DNA gel, just use clean buffer and wash the tray, comb and tank with 0.1% SDS to get rid of RNases. Figure 1A shows intact and degraded leaf RNA samples.
    7. The absence of contaminating DNA is confirmed by PCR on diluted RNA (1/100) using primers specific for plastid and mitochondrial transcripts.
      For example, the pairs Chloro44F and Chloro44R (amplifying rpoB) as well as Mito41F and Mito41R (amplifying cox2) are suitable for this test.
      Chloro44F
      GCTCCGGAGATAGTTCCCTT
      rpoB
      Chloro44R
      TTCTAGTTCTATCATCAGCTATGGG



      Mito41F
      CTCCAGCCGCTCACTGTAAT
      cox2
      Mito41R
      TCCGATGAGCAGTCACTCAC
      The great abundance of plastid DNA in plant cells justifies the use of plastid primers to check the efficiency of the DNase treatment.
      Use 1 µl of a 1/100 RNA dilution as template per reaction. Perform the following program: 2 min at 94 °C, 35 cycles (20 sec at 94 °C, 30 sec at 55 °C, 1 min at 72 °C) and finally 5 min at 72 °C.
      Do not forget positive (genomic DNA) and negative controls, as ideally this PCR should result in no amplification. When you are satisfied that there is no genomic DNA left in the RNA (see Figure 1B), proceed to cDNA synthesis. Otherwise repeat the DNase treatment.


      Figure 1. A. TBE gel showing intact (first lane) and degraded (second lane) RNA samples. B. Result of the PCR: the lack of amplification shows that the contaminating DNA has been efficiently removed by the DNase treatment.

  3. cDNA synthesis
    1. Use 1-3 µg of DNA-free RNA (0.5 µg is enough in the case of difficult samples), just make sure that the initial quantities of RNA are similar in all samples to be compared and process all samples at the same time.
    2. It is crucial to use hexanucleotide primers (a mixture of primers comprising six random nucleotides) rather than oligo dT for this step as plastid and plant mitochondrial transcripts do not have polyA tails (unless destined to be degraded). Use the Superscript III First Strand Synthesis System.
    3. Add in a 0.5 ml tube
      0.5 - 3 µg RNA in 11 µl
      1 µl 10 mM dNTPs
      1 µl random primers at 100 mg/ml
      Incubate for 5 min at 65 °C, then incubate on ice for 1-2 min.
    4. Add
      4 µl 5x transcription buffer
      1 µl 0.1 M DTT
      1 µl RNaseOUT
      1 µl Superscipt III Reverse Transcriptase
      Incubate 5 min at 25 °C
      Incubate 50 min at 50 °C
      Inactivate the reaction for 15 min at 70 °C and keep the samples on ice or at -20 °C until you are ready to proceed to the qPCR.

  4. Quantitative RT-PCR-general considerations
    1. The cDNAs can be tested by a first round of qPCR using one pair of nuclear primers and one pair of mitochondrial primers from the assay (see Table 1). This will allow you to adjust the cDNA concentrations (by comparing the crossing points (Cp) for all samples) before setting up 3 or 6 x 384-well plates (Figure 2).


      Figure 2. Amplification of rpl2. A. Amplification curves of the standard dilutions of the control sample (brown curves) and 14 other samples (red curves). In green, the negative control. B. Standard curve showing a primer efficiency close to 2.

    2. Quantitative RT-PCR is conducted in 384-well plates (LightCycler 480 Multiwell plate) with a LightCycler 480. A reaction volume of 5 µl is sufficient as mitochondrial transcripts are highly expressed. For the qPCR run, the plates are sealed with the dedicated LightCycler 480 Seal Foil. Keep plates on ice and in the dark until the run.
    3. Each 5-µl reaction contains 0.5 µl of cDNA dilution, 2.5 µl of LightCycler 480 SYBR I Master mix (comprising DNA polymerase, buffer and DNA double-strand specific SYBR Green I dye) and 2 µl of 2.5 µM primer mix (1.25 µM each). The details of the serial dilutions and reactions mixes are given in Table 3. The plate setup shown in Table 2 allows the analysis of 6 samples (3x 384-well plates for each assay), one sample being used as a control for quantitation (3 biological repeats of wild-type and 3 of mutants for example). Three technical repeats per point (1/200 dilution) are necessary.
    4. A standard curve is performed for each primer pair, using one of the samples as a control. This cDNA is diluted (1/20, 1/200, 1/1,000, 1/5,000) and the remaining samples at 1/200 only). The primer efficiency is calculated for each pair of primers by the software when doing the standard curve.
    5. Primer pairs for the mitochondrial transcriptome assay are described in Table 1, as well as the primers for reference genes that will be used for normalisation of the results. It is very difficult to find reference genes whose expression will not vary between samples; it is a matter of trying several, picking the most suitable ones within an experiment and combining them. Alternatively, normalisation can be made to the median of mitochondrial transcripts.
    6. The primers used for the splicing assay (i.e. for quantifying the splicing of mitochondrial mRNAs) were designed to amplify 100-200 bp regions spanning intron-exon junctions (unspliced forms) or spanning splice junctions (spliced forms) of each gene (Table 1).

  5. Quantitative RT-PCR-time course for pipetting
    1. Prepare the primer plates
      The primers (2 µl at 1.25 µM each) are dispensed into the wells with an automatic dispenser ‘Multipette plus’, one primer pair per horizontal line (see Table 2). All plate set up stages must be done on ice, or in the cold room in case of multiple plate set up, to limit evaporation of the reagents.
      This step can be done in advance and the plates (sealed with Adhesive PCR film seals from Thermo Scientific) can be kept at -20 °C.

      Table 2. 384-well plate set up for mitochondrial transcriptome and splicing assay




    2. The reaction mixes are prepared according to Table 3 and kept on ice.
      Prepare serial cDNA dilutions and add the exact volume of SYBR Master mix for each dilution.
    3. Add 3 µl of this mix (SYBR Master mix + cDNA) to each well already containing 2 µl of primer mixes at 1.25 µM each.
    4. Seal and briefly spin the plates, keep them on ice and in the dark.

      Table 3. Serial cDNA dilutions and qPCR mixes
      Volume
      52 µl
      76 µl
      52 µl
      26 µl
      26 µl
      76 µl
      Dilution
      1/20
      1/200
      1/1,000
      1/5,000
      0
      Mutant/200
      Water
      57.8 µl
      78.8 µl
      45.8 µl
      20.8 µl
      26
      75.6 µl
      cDNA
      3.04 µl




      0.38 µl
      1/20

      8.75 µl




      1/200


      11.44 µl



      1/1,000



      5.2 µl




      1/20
      1/200
      1/1,000
      1/5,000
      0
      Mutant/200
      Master mix
      260 µl
      380 µl
      260 µl
      130 µl
      130 µl
      380 µl
      cDNA
      52 µl
      76 µl
      52 µl
      26 µl
      26 µl
      76 µl

    5. Perform qPCR run as soon as possible. The thermal cycling program is: 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 sec, 60 °C for 10 sec and 72 °C for 20 sec.
    6. The plate set-up should be entered in the LC480 software before starting the run, but this can be done later if necessary. Under ‘absolute quantitation’, fill in the ’Subset editor’ with the transcript names and positions in the plate and finally the ‘Sample editor’.

Data analysis

The data are analysed using the inbuilt LightCycler 480 software version 1.5. The Second Derivative Maximum Method, which determines the Cp value at the beginning of the log-linear phase of the real-time fluorescence (Luu-The et al., 2005) is fast and easy, but the fit point method can be used too. All transcripts are quantified relative to an internal control sample. For each primer pair, a standard curve is done with the internal control sample. The efficiency of the PCR reaction is checked (optimal efficiency is 2, but is acceptable between 1.6 and 2.4). For the error to be minimal, standard errors should be less than 10 %, but for some primer pairs it is difficult to achieve.
The figures are then copied into a spreadsheet and calculations are done as follows:

  1. A normalisation step is necessary: For this, use either the geometric mean of 2 to 3 suitable reference genes or normalise to the median of all mitochondrial transcripts. It is always best to try several ways of doing it.
  2. Normalise each sample to its control, i.e., calculate ratio of expression of mutant M1 to WT1, M2/WT2, M3/WT3 or whatever comparison you need to do.
  3. Draw a first graph of transcript accumulation in mutant/WT. Showing the results with a log2 scale can be easier in some cases (see Figure 3).
  4. Calculate an average and a standard deviation (SD) between biological repeats. If the interval between the average log2 ratio + SD and average log2 ratio-SD does not contain zero, then the change is considered significant.
  5. For the splicing assay, the results are best presented as the log2 ratio of (spliced mRNA in the mutant to spliced mRNA in the WT) to (unspliced mRNA in the mutant to the unspliced mRNA in the WT) (Figure 3A). Nevertheless, to be sure what you see is the effect of a true splicing defect, you must check that the accumulation of spliced mRNA is down in the mutant and that of the unspliced mRNA is up in the mutant.
  6. Results can be confirmed by northern blots if necessary.

Representative data


Figure 3. Representative data. A. Panel A presents the qRT-PCR mitochondrial splicing assay for the mutant wtf9 (Francs-Small et al., 2012), which has a defect in rpl2 and ccmFc transcript splicing. The defects are very obvious when the data is represented as a log2 value of the relative quantities of spliced to unspliced forms of each transcript. B. Panel B shows the transcriptome data, where mRNA levels are expressed as a ratio of transcript levels in mutants compared with levels in WT Col-0 plants.

Notes

These assays are generally very reproducible and reliable due to the abundance of mitochondrial transcripts in plant tissues. Of course, in some mutants, some transcripts are expressed at significantly different levels compared to WT but this is what the essay is designed to detect.

Acknowledgments

This protocol was originally developed by Etiennne Delannoy and Andéol Falcon de Longevialle for work supported by the Australian Research Council Centre of Excellence grant CE0561495 (de Longevialle et al., 2007; Koprivova et al., 2010; Kühn et al., 2009). Some primer pairs have been modified or added by C. Colas des Francs-Small, in particular for the study of nad5 splicing (Colas des Francs-Small et al., 2014).

References

  1. Colas des Francs-Small, C., Falcon de Longevialle, A., Li, Y., Lowe, E., Tanz, S. K., Smith, C., Bevan, M. W. and Small, I. (2014). The pentatricopeptide repeat proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 are involved in the splicing of the multipartite nad5 transcript encoding a subunit of mitochondrial complex I. Plant Physiol 165(4): 1409-1416.
  2. de Longevialle, A. F., Meyer, E. H., Andres, C., Taylor, N. L., Lurin, C., Millar, A. H. and Small, I. D. (2007). The pentatricopeptide repeat gene OTP43 is required for trans-splicing of the mitochondrial nad1 Intron 1 in Arabidopsis thaliana. Plant Cell 19(10): 3256-3265.
  3. Francs-Small, C. C., Kroeger, T., Zmudjak, M., Ostersetzer-Biran, O., Rahimi, N., Small, I. and Barkan, A. (2012). A PORR domain protein required for rpl2 and ccmF(C) intron splicing and for the biogenesis of c-type cytochromes in Arabidopsis mitochondria. Plant J 69(6): 996-1005.
  4. Koprivova, A., des Francs-Small, C. C., Calder, G., Mugford, S. T., Tanz, S., Lee, B. R., Zechmann, B., Small, I. and Kopriva, S. (2010). Identification of a pentatricopeptide repeat protein implicated in splicing of intron 1 of mitochondrial nad7 transcripts. J Biol Chem 285(42): 32192-32199.
  5. Kühn, K., Richter, U., Meyer, E. H., Delannoy, E., Falcon de Longevialle, A. , O'Toole, N., Börner, T., Millar, A. H., Small, I. D. and Whelan, J. (2009). Phage-type RNA polymerase RPOTmp performs gene-specific transcription in mitochondria of Arabidopsis thaliana. Plant Cell 21(9): 2762-2779.
  6. Luu-The, V., Paquet, N., Calvo, E. and Cumps, J. (2005). Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction. Biotechniques 38(2): 287-293.

简介

这种基于qPCR的测定提供了所有线粒体转录物(mRNA和rRNA)的表达水平以及拟南芥中的剪接效率的概述。 它是在RNAseq技术被广泛使用之前开发的(de Longevialle等人,2007),但是仍然有用,因为它运行更便宜,并且分析更容易和更快地执行,如果目标 只是看看线粒体转录物。 对于含内含子的mRNA,使用特异性扩增剪接或未剪接形式的引物组允许评价剪接效率。

关键字:线粒体转录组, 拼接法, 定量RT-PCR, 线粒体, 拟南芥

材料和试剂

  1. Eppendorf型微管(0.5ml和1.5ml)(任何品牌)
  2. LightCycler 480多孔板(白色)(Roche Diagnostics,目录号:04729749001)
  3. 用于qPCR运行的LightCycler 480密封箔(Roche Diagnostics,目录号:04729757001)
  4. 用于存储含有引物混合物的板的粘合剂PCR膜密封件(Thermo Fisher Scientific,目录号:AB0558)
  5. Combitips 0.1ml(Eppendorf,目录号:0030 089.405或0030 089.618)
  6. 在体外或土壤中生长的拟南芥植物
  7. RNeasy Plant Mini Kit(QIAGEN,目录号:74904)
  8. 水,用于分子生物学,DNAse,RNAse和无蛋白酶(Thermo Fisher Scientific,ACROS Organics TM ,目录号:327390010)
  9. Ambion Turbo无DNA的 TM Kit(Life Technologies,目录号:AM1907)
    />
  10. 3M乙酸钠(pH 5.2)
  11. 100%和70%乙醇
  12. 琼脂糖LE,分析级(Promega Corporation,目录号:V3125)
  13. PCR和qPCR引物(见表1)

    表1.本协议中使用的引物
    请点击表1的此处

  14. Taq聚合酶
    注意:任何Taq聚合酶都可用于此步骤,我们使用家用纯化的酶。
  15. dNTP组(Life Technologies,目录号:10297-018)
    注意:目前,它是"Thermo Fisher Scientific,Invitrogen TM ,目录号:10297-018"。
  16. Superscript III逆转录酶(Life Technologies,目录号:18080-044)
    注意:目前, 是"Thermo Fisher Scientific,Invitrogen TM ,目录号:18080-044" 此酶含有5x转录缓冲液和0.1 M DTT溶液。
  17. 随机六核苷酸引物(Life Technologies,目录号:48190011)
    注意:目前,它是"Thermo Fisher Scientific,Invitrogen TM ,c
  18. RNaseOUT重组核糖核酸酶抑制剂(Life Technologies,目录号:
    10777-019)
    注意:目前,它是"Thermo Fisher Scientific,Invitrogen TM ,目录号:10777-019"。
  19. LightCycler 480 SYBR I Master mix(Roche Diagnostics,目录号:04887352001)
    注意:此试剂对光敏感。

设备

  1. Nanodrop紫外可见分光光度计(Thermo Fisher Scientific)
  2. 'Multipette plus'(Eppendorf,目录号:4981 000.019)
  3. LightCycler ? 480 System(Roche Diagnostics)

软件

  1. LightCycler 480软件版本1.5(Roche Diagnostics,目录号:04994884001)

程序

  1. RNA提取
    1. 总的拟南芥RNA从年轻组织(8至20日龄)提取 叶)与RNeasy植物迷你套件根据制造商的 协议。
    2. 用40μl无RNA酶的水洗脱RNA,并用NanoDrop测量其浓度。

  2. DNase治疗
    在逆转录步骤之前需要除去基因组DNA 这是使用Ambion Turbo无DNA试剂盒实现的
    1. 加入0.5 ml管
      最多10μgRNA(在45μl无RNA酶的水中)
      4.7μl10x Turbo DNase缓冲液
      1μlDNase
    2. 通过吸移混合,并在37℃下放置1小时
    3. 的 ?试剂盒含有DNase灭活剂,这导致了很大的损失 材料和可能的残余树脂。相反,沉淀反应 用1/10体积的3M乙酸钠和2.5至3体积的100%乙醇(30 min至-20℃下1小时)。在4℃下以最大速度(20,800×g )旋转30分钟 ?C。小心丢弃上清液。
    4. 用70% 乙醇(小心地加入300μl的70%乙醇而不破坏 沉淀并旋转15分钟。这一步除去盐,这将 如果管在-20℃的冷冻器中保持1小时或更好地实现 过夜
    5. 干燥颗粒,并用30μl无RNA酶的水重悬。
    6. 测量 ?浓度与NanoDrop和通过运行约100检查RNA质量 在2%琼脂糖-TBE凝胶上。这是一个常规的DNA凝胶,只是使用干净 缓冲液,并用0.1%SDS洗涤托盘,梳子和槽以除去 核糖核酸酶。图1A显示了完整和降解的叶RNA样品
    7. 的 通过对稀释的RNA(1/100)的PCR证实不存在污染性DNA 使用对质体和线粒体转录物特异的引物。
      对于 ?例如,对Chloro44F和Chloro44R(扩增 rpoB )以及 ?Mito41F和Mito41R(扩增 cox2 )适用于该测试。
      Chloro44F
      GCTCCGGAGATAGTTCCCTT
      rpoB
      Chloro44R
      TTCTAGTTCTATCATCAGCTATGGG



      Mito41F
      CTCCAGCCGCTCACTGTAAT
      cox2
      Mito41R
      TCCGATGAGCAGTCACTCAC
      的 ?植物细胞中大量的质体DNA证明了其使用 质体引物以检查DNase处理的效率 使用1 ?μl的1/100 RNA稀释作为模板。执行 程序:94℃2分钟,35个循环(94℃20秒,30秒,30秒) ?55℃,72℃1分钟),最后在72℃5分钟 不要忘记 阳性(基因组DNA)和阴性对照,理想情况下这个PCR应该 ?导致无扩增。当你确信没有 留在RNA中的基因组DNA(参见图1B),进行cDNA合成。 否则重复DNase处理

      图1。 A. TBE凝胶 显示完整(第一泳道)和降解(第二泳道)RNA样品。乙。 PCR结果:缺乏扩增显示 污染的DNA已经通过DNase处理有效去除

  3. cDNA合成
    1. 使用1-3微克无DNA的RNA(0.5微克足够在困难的情况下 样品),只是确保RNA的初始量是相似的 在所有样品中进行比较并同时处理所有样品。
    2. 它 ?使用六核苷酸引物(引物的混合物)是至关重要的 包括六个随机核苷酸),而不是该步骤的寡聚dT ?质体和植物线粒体转录物不具有polyA尾 (除非注定要退化)。使用Superscript III First Strand 合成系统
    3. 加入0.5 ml管
      在11μl中加入0.5-3μgRNA
      1μl10 mM dNTPs
      1微升随机引物,浓度为100 mg/ml
      在65℃孵育5分钟,然后在冰上孵育1-2分钟
    4. 添加
      4μl5×转录缓冲液
      1μl0.1M DTT
      1μlRNaseOUT
      1μlSuperscipt III Reverse Transcriptase
      在25°C孵育5分钟
      在50°C孵育50分钟
      在70°C使反应停止15分钟,将样品保存在冰上或-20°C,直到准备好进行qPCR。

  4. 定量RT-PCR-一般考虑
    1. cDNA可以通过第一轮qPCR使用一对来测试 核引物和一对线粒体引物 (参见表1)。这将允许您调整cDNA浓度(通过 ?在设置3之前比较所有样本的交叉点(C

      ) ?或6×384孔板(图2)。


      图2.扩增 的 rpl2 。 A。标准稀释的扩增曲线 对照样品(棕色曲线)和14个其他样品(红色曲线)。在 绿色,阴性对照。 B.显示引物的标准曲线 效率接近2.

    2. 进行定量RT-PCR 384孔板(LightCycler 480多孔板) 5μl的反应体积足以作为线粒体 转录物高度表达。对于qPCR运行,板是 用专用的LightCycler 480密封箔密封。保持板在冰上 并在黑暗中直到跑步
    3. 每个5μl反应含有0.5μl 的cDNA稀释液,2.5μlLightCycler 480 SYBR I Master混合物 (包括DNA聚合酶,缓冲液和DNA双链特异性SYBR Green I染料)和2μl2.5μM引物混合物(各1.25μM)。细节 的连续稀释和反应混合物在表3中给出 表2所示的板设置允许分析6个样品(3x 384-孔板用于每个测定),一个样品用作对照 定量(3个生物学重复的野生型和3个突变体 例)。每点三次技术重复(1/200稀释) 必要。
    4. 对每个引物对进行标准曲线, 使用样品之一作为对照。将该cDNA稀释(1/20, 1/200,1/1,000,1/5,000),其余样品仅为1/200)。的 通过软件计算每对引物的引物效率 ?当做标准曲线时。
    5. 线粒体的引物对 转录组测定在表1中描述,以及引物 ?将用于结果的归一化的参考基因。它 很难找到其表达不会改变的参考基因 ?样品之间;这是一个尝试几个,挑选最多的问题 在实验中合适的和组合它们。或者, 可以对线粒体转录物的中值进行标准化。
    6. 的 ?用于剪接测定的引物(即用于定量剪接) 的线粒体mRNA)设计为扩增100-200 bp区域 跨越内含子 - 外显子连接(未剪接形式)或跨接剪接 每个基因的连接(剪接形式)(表1)。

  5. 移液的定量RT-PCR时间过程
    1. 准备底漆板
      引物(2μl,各1.25μM) 用自动分配器"Multipette plus"分配在孔中, 每个水平线一个引物对(参见表2)。所有板设置 阶段必须在冰上进行,或在多个情况下在冷室中进行 板设置,以限制试剂的蒸发 这一步可以 并用板(用粘合剂PCR膜密封件密封 ?Thermo Scientific)可以保持在-20℃。

      表2.用于线粒体转录组和剪接测定的384孔板




    2. 反应混合物根据表3制备并保存在冰上 制备连续cDNA稀释液,并为每个稀释液加入准确体积的SYBR Master混合物。
    3. 加入3微升此混合(SYBR主混合+ cDNA)到已经包含2微升引物混合物,每个1.25微米的每个孔。
    4. 密封并短暂旋转板,保持在冰上和在黑暗中。

      表3.系列cDNA稀释液和qPCR混合物
      Volum e
      52微升
      76 μl
      52微升
      26微升
      26微升
      76μl
      稀释
      1/20
      1/200
      1/1,000
      1/5,000
      0
      突变体/200

      57.8微升
      78.8微升
      45.8微升
      20.8μl
      26
      75.6μl
      cDNA
      3.04μl




      0.38微升
      1/20

      8.75微升




      1/200


      11.44微升



      1/1,000



      5.2微升




      1/20
      1/200
      1/1,00 0
      1/5,000
      0
      突变体/200
      主混音
      260微升
      380μl
      260微升
      130微升
      130微升
      380μl
      cDNA
      52微升
      76微升
      52微升
      26微升
      26微升
      76微升

    5. 执行 ?qPCR尽快运行。热循环程序为:95℃ ?10分钟,然后是95℃10秒,60℃10秒的45个循环 ?72℃20秒。
    6. 板设置应输入LC480 ?软件在开始运行之前,但这个可以在以后完成 必要。在"绝对定量"下,填写"子集编辑器" 与文本名称和位置在盘子和最后 '示例编辑器'。

数据分析

使用内置的LightCycler 480软件版本1.5分析数据。在实时荧光的对数线性阶段开始时确定Cp值的第二微分最大方法(Luu-The等人,2005)是快速和容易的,但是适合点法也可以使用。所有转录物相对于内部对照样品定量。对于每个引物对,用内部对照样品进行标准曲线。检查PCR反应的效率(最佳效率为2,但在1.6和2.4之间是可接受的)。为使误差最小,标准误差应小于10%,但对于某些引物对,很难实现。
然后将数字复制到电子表格中,并如下进行计算:

  1. 标准化步骤是必要的:为此,使用2至3个合适的参考基因的几何平均值或归一化到所有线粒体转录物的中值。总是最好尝试几种方法。
  2. 将每个样品标准化为其对照,即,计算突变体M1与WT1,M2/WT2,M3/WT3或任何需要进行的比较的表达的比率。
  3. 绘制突变体/WT中转录物积累的第一张图。在某些情况下,使用日志 2 缩放显示结果更容易(见图3)。
  4. 计算生物重复之间的平均值和标准偏差(SD)。如果平均log2比率+ SD和平均log2比率-SD之间的间隔不包含零,则认为该变化是显着的。
  5. 对于剪接测定,结果最好表示为(WT中突变体与剪接的mRNA的剪接的mRNA)与(WT中未剪接的mRNA的未剪接的mRNA)的log2比(图3A)。然而,要确保你看到的是真正的拼接缺陷的影响,你必须检查拼接的mRNA的积累在突变体中,并且未剪接的mRNA的积累在突变体中。
  6. 如果需要,可以通过Northern印迹确认结果。

代表数据


图3.代表数据。 A。图A显示了突变wtf9(Francs-Small等人,2012)的qRT-PCR线粒体剪接测定,其在rpl2中具有缺陷 em>和 ccmFc 转录本拼接。当数据表示为每个转录物的拼接至未剪接形式的相对量的log 2值时,缺陷是非常明显的。图B显示转录组数据,其中mRNA水平表示为与WT Col-0植物中的水平相比的突变体中转录物水平的比率。

笔记

由于植物组织中线粒体转录物的丰度,这些测定通常是非常可重复的和可靠的。当然,在一些突变体中,一些转录物以与WT相比显着不同的水平表达,但这是文章旨在检测的。

致谢

该协议最初由Etiennne Delannoy和AndéolFalcon de Longevial??le开发,由澳大利亚研究委员会卓越中心(Australian Research Council Center of Excellence)授予CE0561495(de Longevial??le等人,2007; Koprivova等人,/em,2010;Kühn等人,2009)。一些引物对已经被C.Colas des Francs-Small修饰或添加,特别是用于nad5剪接的研究(Colas des Francs-Small等人,2014年, )。

参考文献

  1. Colas des Francs-Small,C.,Falcon de Longevial??le,A.,Li,Y.,Lowe,E.,Tanz,S.K.,Smith,C.,Bevan,M.W.and Small,I.(2014)。 五肽复制蛋白TANG2和ORGANELLE TRANSCRIPT PROCESSING439参与多分体nad5转录物的剪接,编码亚基的线粒体复合物I. 植物生理学165(4):1409-1416。
  2. de Longevial??le,A.F.,Meyer,E.H.,Andres,C.,Taylor,N.L.,Lurin,C.,Millar,A.H。和Small,I.D。(2007)。 五子多肽重复基因OTP43是线粒体nad1内含子1的反式剪接所必需的,拟南芥。 植物细胞 19(10):3256-3265。
  3. Francis-Small,C.C.,Kroeger,T.,Zmudjak,M.,Ostersetzer-Biran,O.,Rahimi,N.,Small,I.and Barkan,A。(2012)。 rpl2和ccmF(C)内含子剪接所需的PORR结构域蛋白和c-在拟南芥线粒体中的类型细胞色素。 植物J 69(6):996-1005。
  4. Koprivova,A.,des Francs-Small,C.C.,Calder,G.,Mugford,S.T.,Tanz,S.,Lee,B.R.,Zechmann,B.,Small,I.and Kopriva, 鉴定与线粒体nad7转录本的内含子1的剪接有关的pentatricopeptide重复蛋白。 Biol Chem 285(42):32192-32199。
  5. Kühn,K.,Richter,U.,Meyer,EH,Delannoy,E.,Falcon de Longevial??le,A.,O'Toole,N.,B?rner,T.,Millar,AH,Small,ID和Whelan, (2009)。 噬菌体型RNA聚合酶RPOTmp在 Arabidop 的线粒体中进行基因特异性转录> 。 21(9):2762-2779。
  6. Luang-The,V.,Paquet,N.,Calvo,E。和Cumps,J.(2005)。 改进的实时RT-PCR方法用于使用二阶导数计算和双重校正的高通量测量。 em> Biotechniques 38(2):287-293。
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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. Delannoy, E., Falcon de Longevialle, A. and Francs-Small, C. C. (2015). Mitochondrial RNA Transcript Analysis Assay of Arabidopsis Leaf Tissues. Bio-protocol 5(20): e1620. DOI: 10.21769/BioProtoc.1620.
  2. Colas des Francs-Small, C., Falcon de Longevialle, A., Li, Y., Lowe, E., Tanz, S. K., Smith, C., Bevan, M. W. and Small, I. (2014). The pentatricopeptide repeat proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 are involved in the splicing of the multipartite nad5 transcript encoding a subunit of mitochondrial complex I. Plant Physiol 165(4): 1409-1416.
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