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Circular RT-PCR Assay Using Arabidopsis Samples
拟南芥样本的循环RT-PCR实验   

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参见作者原研究论文

本实验方案简略版
Proceedings of the National Academy of Sciences of the United States of America
Nov 2014

Abstract

Post-transcriptional processing is critical for RNA biogenesis, in which conventional functional RNA transcripts are generated, such as messenger RNAs (mRNAs), transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) for translation as well as emerging non-coding RNAs with known or unknown regulatory functions. To determine the precise termini of an RNA molecule during or after processing, the primer extension and Rapid Amplification of cDNA Ends (RACE) methods have been routinely utilized for the precise mapping of 5’ or 3’ ends. Different from these assays, which are designed to detect only one end of a specific target RNA at a time, circular Reverse Transcription-Polymerase Chain Reaction (cRT-PCR) is able to simultaneously determine both the 5’ and 3’ ends of the target RNA. In Arabidopsis thaliana, cRT-PCR has been wildly applied to identify both the 5’ and 3’ extremities of the ribosomal RNA precursors, or to assess the length or post-transcriptional extensions at the 3’ end of a matured mRNA. In this protocol, we summarize and present a detailed procedure of the cRT-PCR assay in Arabidopsis thaliana, which is also successfully used in our previously published work (Hang et al., 2014).

Keywords: Circular RT-PCR (循环检测), Post-transcriptional processing (转录后加工), Pre-rRNA processing (rRNA前体的加工), Ribosome biogenesis (核糖体合成), PRMT3 (PRMT3)

Materials and Reagents

  1. Arabidopsis thaliana 14-day-old seedlings
  2. M13F: 5′-TGTAAAACGACGGCCAGT-3′ and M13R: 5’-CAGGAAACAGCTATGAC-3’
  3. TRNzol Reagent (TIANGEN®, catalog number: DP40502 )
  4. Dynabeads® mRNA Purification Kit (Life Technologies, catalog number: 61006 )
  5. FirstChoice® RLM-RACE Kit (Life Technologies, catalog number: AM1700 )
  6. RNA 5’ Polyphosphatase (Epicentre, catalog number: RP8092H )
  7. T4 RNA Ligase 1 (New England Biolabs, catalog number: M0204S )
  8. T4 RNA Ligase Reaction Buffer (New England Biolabs, catalog number: B0216L )
  9. Adenosine 5'-Triphosphate (New England Biolabs, catalog number: P0756S )
  10. RNasin® Plus RNase Inhibitor (Promega Corporation, catalog number: N2615 )
  11. Glycogen (RNA grade) (Thermo Fisher Scientific, catalog number: R0551 )
  12. TransScript® II First-Strand cDNA Synthesis SuperMix (TransGen Biotech, catalog number: AH301 )
  13. TaKaRa ExTaq® DNA Polymerase (Takara Bio Company, catalog number: RR001B )
  14. pEASY® -T1 Cloning Kit (TransGen Biotech, catalog number: CT10102 )
  15. Trans2K® Plus II DNA Marker (TransGen Biotech, catalog number: BM121-01 )
  16. Diethyl pyrocarbonate/DEPC (Sigma-Aldrich, catalog number: D5758 )
  17. TIANgel Midi Purification Kit (TransGen Biotech, catalog number: DP209 )
  18. UltraPureTM Agarose (Life Technologies, catalog number: 75510019 )
  19. DEPC-treated deionized water (see Recipes)
  20. 3 M sodium acetate (see Recipes)
  21. 70%, 100% ethanol (see Recipes)
  22. 50x TAE buffer (see Recipes)

Equipment

  1. MaxyClear Microtubes (1.5 ml) (Corning Incorporated, Axygen®, catalog number: MCT150C )
  2. NanoDropTM 1000 Spectrophotometer (Thermo Fisher Scientific, catalog number: ND-1000 )
  3. Thermomixer comfort (Eppendorf, catalog number: 5362.000.019 )
  4. Eppendorf Centrifuge (Eppendorf, catalog number: 5417R )
  5. PCR system (Eastwin Life Science, catalog number: EDC-810 )
  6. Other standard laboratory equipment

Procedure

  1. Primer design
    1. In general, primers should be highly specific for the target sequences, able to form stable duplexes with their target sequences, and free of secondary structure.
    2. Three primers around 20~25 nucleotides in length should be synthesized. The GC content of these primers is designed as similar as possible.
      Note: We recommend the GC ratio to be between 40%~55%. e.g., GC ratios of primers listed in Figure 2A are in this range. However, this parameter is not fixed and it depends on your gene.
    3. The first gene-specific primer cRT (Figure 1) should be localized in the known region and used for reverse transcription (RT).
      Note: cRT is of reverse orientation of the gene. Its localization is recommended within 100~200 nt from the anticipated 5’ end of the RNA. e.g., 18c is within 204 nt from the anticipated 5’ end of mature 18S rRNA (Figure 2A).
    4. The other two primers, cF1 and cR1, localized in 5’ and 3’ sides of the cRT primer, respectively (Figure 1), are for primary PCR amplification of the 5’ and 3’ extremities of the target transcript. cF1 and cR1 anneal to sequences located 3’ and 5’ (with respect to cDNA not RNA) of cRT, respectively. Their localizations are recommended within ~100 nt from the anticipated 5’ and 3’ end of the RNA, respectively.
      Note: Primers cF2 or cR2, immediately down-stream of the cF1 or cR1 primer, respectively (Figure 1), are needed for further nested PCR to improve PCR specificity. Primers cF2 and cR2 for nested PCR could be designed in known region of mature RNA or predicted region only exist in precursor RNA. Compared to cF1/cR1, each of these three primer combinations (cF1/cR2, cF2/cR1 and cF2/cR2) could be used for nested PCR.


    Figure 1. Diagrammatic representation of circular RT-PCR (cRT-PCR). Total RNA or polyadenylated RNA are circularized with T4 RNA ligase and reverse transcribed with gene-specific primer cRT. Then 5’ and 3’ extremities are amplified and sequenced. A and D mark the known 5’ and 3’ extremities of a specific RNA. Blue box from A to B and black box from C to D represent loci of PCR primers cF1 and cR1, respectively. White box between B and C indicates the locus of cRT primer. Primers cF2 and cR2 adjacent to the two primary primers cF1 and cR1, respectively, are designed for nested PCR.

  2. RNA extraction (Linear RNA)
    1. Total RNA is extracted from 0.1 g of 14-day-old fresh Arabidopsis seedlings with TRNzol Reagent according to standard manufacturer’s instruction, dissolved in DEPC-treated deionized water and quantified with NanoDropTM 1000 Spectrophotometer.
      Note:At least 50 μg total RNA could be extracted from 0.1 g seedlings. Therefore in theory, 0.01 g seedlings may be sufficient for one RNA circularization reaction (10 μg RNA). In addition, the starting amount of plant tissue may vary depending on specific tissue type, since different tissues may need optimized protocols and reagents for better RNA extraction efficiency.
    2. For RNAs with polyadenylated tails, enrichment with a Dynabeads® mRNA Purification Kit is recommended.
      Note: In general, polyadenylations are often identified in 3’-end of most nucleus-encoded mRNAs in eukaryotes, and RNA molecules in prokaryotes and organelles (Slomovic et al., 2008). In addition, most of other non-coding RNAs are also found to be polyadenylated for processing or degradation, like rRNAs, snoRNA, snRNA and so on (Lykke-Andersen et al., 2009).
  3. RNA ligation (Circular RNA, cRNA)
    1. 10 μg of total RNA or 0.5 μg of poly(A) RNA was used for each ligation reaction (50 μl). RNA is pre-incubated in Thermomixer comfort at 70 °C for 5 min, and then immediately chilled on ice for 2 min.
      Note: RNA decapping step is recommended before RNA ligation when analyzing nucleus-encoded mRNAs (Slomovic et al., 2008), since RNAs with 5’-cap structure will inhibit the RNA ligation efficiency. Although this step was not applied in our previous work (Hang et al., 2014), we recommend performing this assay if necessary with these two kits according to their corresponding protocols. RNA 5´ Polyphosphatase from Epicentre
      (http://www.epibio.com/docs/default-source/protocols/rna-5-polyphosphatase.pdf?sfvrsn=12) or Tobacco Acid Pyrophosphatase supported in FirstChoice® RLM-RACE Kit (https://tools.lifetechnologies.com/content/sfs/manuals/cms_056070.pdf).
    2. Prepare the following reaction system:
      RNA 10 μg total/0.5 μg poly(A)
      T4 RNA Ligase 1 (10,000 U/ml) 3 μl (30 U)
      10x T4 RNA Ligase 1 buffer 5 μl
      Adenosine-5'-Triphosphate (10 mM ATP) 5 μl (1 mM)
      RNase inhibitor (40 U/μl) 1 μl (40 U)
      DEPC-treated deionized water to 50 μl
      Note: The ligation reaction system may be optimized in the dosage of T4 RNA ligase 1 or ATP. Be aware that the appropriate concentration of ATP should be added if it is not supplied in the T4 RNA ligase buffer. The appropriate concentration of ATP should be in accordance with the product information of the commercial T4 RNA ligase 1. We used T4 RNA Ligase 1 from New England Biolabs (see in materials and reagents). In its properties and usage section, 1 mM ATP is suggested to add into the reaction system, since there is no ATP in the 10 x T4 RNA ligase reaction buffer (https://www.neb.com/products/m0204-t4-rna-ligase-1-ssrna-ligase - tabselect0). In addition, make reference to the companion protocol optimized for RNA circularization (https://www.neb.com/protocols/1/01/01/rna-circularization-using-t4-rna-ligase-1). In this protocol, 20 μl reaction system supplemented with 20-50 μM ATP and 10% PEG8000 is set up and then incubated at 25 °C for 1~2 h, or overnight at 16 °C for better yield.
    3. Mix well and incubate in Thermomixer comfort at 37 °C for 1~2 h.
    4. Incubate at 65 °C for 15 min to inactivate the T4 RNA Ligase 1.
    5. Ethanol precipitate the RNA by adding 5 μl 3 M sodium acetate (pH 5.2), 2 μl of glycogen (20 mg/ml), and 171 μl of 100% ethanol. Keep the mixture on ice for 30 min (short time) or store at -20 °C overnight to precipitate the ligated RNA.
      Note: Longer precipitation at -20 °C overnight is recommended for better RNA recovery. The concentration of glycogen is 20 mg/ml. Its working concentration here is around 0.7 μg/μl when 2 μl glycogen is added into 50 μl reaction product plus 5 μl 3M sodium acetate. It is notable that a final 0.05-1 μg/μl concentration of glycogen is suggested according to its product instruction.
    6. Spin the sample at 12,000 rpm for 15 min at 4 °C.
      Note:The force for a given rpm varies depending on the rotor. When we used Eppendorf Centrifuge 5417R, the speed 12,000 rpm equals to 15,294 x g.
    7.  Discard the supernatant and wash the pellet with 500 μl of 70% ethanol, then spin at 12,000 rpm for 15 min at 4 °C.
    8.  Discard the supernatant and air-dry the pellet. Resuspend the RNA in DEPC-treated deionized water, 70 μl for total RNA or 7 μl for poly(A) RNA. RNA samples can be stored at -80 °C until used.
      Note: We have not compared the half-life of the cRNA and normal RNA strored in -80 °C yet, although we predict that the cRNA is stable and could be kept for a long time in -80 °C. We recommend to perform the reverse transcription as soon as possible.

  4. Reverse transcription with gene-specific primer (Circular cDNA, ccDNA)
    1. Prepare the following reaction system and pre-incubate the mixture at 70 °C for 5 min, then chill it on ice for 2 min. 1 μg of total cRNA or 0.5 μg of poly(A) cRNA is used for each reverse transcription.
      cRNA 7 μl
      RT-primer (cRT in Figure 1) 2 pmol
      DEPC-treated deionized water to 9 μl
    2. Spin the mixture down slightly and add the following reaction mixture to each sample.
      2x TS Reaction Mix 10 μl
      TransScript® II RT/RI Enzyme Mix 1 μl
      Mix well and incubate at 50 °C for 1 h.
      Note:The 2x TS Reaction Mix and TransScript® II RT/RI Enzyme Mix are components supported in TransScript® II First-Strand cDNA Synthesis SuperMix. They have no catalog numbers and therefore not listed in the materials and reagents section.
    3. Incubate at 85 °C for 5 min to inactivate the TransScript® II RT enzyme.
    4. Add 180 μl of DEPC-treated deionized water to dilute the product to the final volume of 200 μl.

  5. PCR amplification
    1. Prepare the general reaction mixture for PCR (50 μl in total):
      cDNA 5 μl
      ExTaq 10x buffer 5 μl
      dNTPs mix (2.5 mM each dNTP) 4 μl
      Forward primer (cF1, 10 μM) 1 μl (10 pmol)
      Reverse primer (cR1, 10 μM) 1 μl (10 pmol)
      TaKaRa ExTaq® DNA Polymerase (5 U/μl) 0.12 μl (0.6 U)
      Deionized water 33.88 μl
    2. Run the PCR amplification according to the program described below:
      a. 3 min at 94 °C;
      b. 30 sec at 94 °C;
      c. 30 sec at 55 °C;
      d. 1 min at 72 °C; Repeat steps b-d for 30 cycles
      e. 10 min at 72 °C;
      f. Hold at 20 °C.
      Note: Annealing temperature and elongation time could be optimized case by case. We recommend to try the routine annealing temperature between 55~60 °C first. As we know, higher annealing temperature may increase the product specificity. Therefore, we could compare the PCR products in 55 °C, 58 °C and 60 °C to choose the best one. For the elongation time, we prefer to choose 1 min for the first try to have a look at the PCR product length (we suppose it isn’t longer than 1,000 bp). Then, we will adjust the elongation time accordingly. In summary, the elongation time depends on the potential length of PCR products and elongation rate of DNA polymerase.
    3. Load 8 μl of the PCR products on 1% agarose gel (with Ethidium Bromide, EtBr) and run until the bromophenol blue dye is 2/3rds of the way down the gel. Image the gel and determine whether this batch of circular RT-PCR is successful (sharp and strong bands) and which candidate band should be cut for further analysis.
      Note: If it does not work well, PCR system should be optimized in annealing temperature, amplification cycles, PCR enzyme, and so on. In addition, on the basis of the primary PCR with cF1/cR1, a further nested PCR with cF1/cR2, cF2/cR1 or cF2/cR2 could be helpful to improve the product specificity as described in Figure 1. For the nested PCR, the PCR mix components and PCR program are the same as the first round PCR, except for the primer combination and DNA template.
    4. Load all the remaining PCR products on a new 1% agarose gel. Cut the candidate bands and purify the DNA from the gel with TIANgel Midi Purification Kit.

  6. DNA sequencing and analysis
    1. Purified DNA products are cloned into the pEasy-T vector.
    2. Make a master reaction mix for colony PCR, except the DNA template (20 μl in total):
      ExTaq 10x buffer 2 μl
      dNTPs mix (2.5 mM each dNTP) 1 μl
      M13F primer (10 μM) 0.5 μl (5 pmol)
      M13R primer (10 μM) 0.5 μl (5 pmol)
      TaKaRa ExTaq® DNA Polymerase (5 U/μl) 0.12 μl (0.6 U)
      Deionized water 15.88 μl
      PCR reaction mix is prepared first and aliquoted into 0.2 ml PCR tubes.
    3. Positive clones are chosen by white-blue colony selection. Take a 20 μl pipet tip and touch a positive colony very lightly and dip the tip a couple of times into the 20 μl of PCR mix.
    4. Run the PCR amplification according to the program described in E.2. The elongation time depends on the length of PCR product. For TaKaRa ExTaq® DNA Polymerase, the elongation time is 1 min for 1 kbp PCR products.
    5.  Analyze the PCR products using a 1% agarose gel with EtBr staining. Excise gel slices containing sharp target bands for DNA sequencing with M13F sequencing primer. We recommend to sequence at least 15 samples for a reliable result.
      Note: DNA sequences with both M13F and M13R primers are necessary to determine the 5’ and 3’ extremities of the target RNAs, especially for highly polyadenylated RNAs at the 3’ tail.
    6.  DNA sequencing data are analyzed with Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI). Choose the organism: Arabidopsis thaliana. Search database: Reference genomic sequences (refseq_genomic) using Megablast (Optimized for highly similar sequences).


    Figure 2. Circular RT-PCR to identify the pre-18S rRNAs in atprmt3-2. Total RNA from 14-day-old seedlings of both Col-0 and atprmt3-2 mutants are used for cRT-PCR assay. (A) Oligonucleotides used in this assay. 18c is the cRT primer for reverse transcription of pre-18S rRNAs. Their lengths, GC content and Tm value are listed. (B-C) Agarose gel analysis of PCR products amplified with primers r5/r6 (B) and r5/r8 (C) after reverse transcription with 18c. Reaction products were analyzed by gel electrophoresis in a 1.5% agarose gels and stained with 0.5 μg/ml ethidium bromide. DNA markers are indicated on the left. Excise candidate bands marked on the right and extract DNAs to clone into the pEasy-T vector. (D) Perform colony PCR with M13F and M13R primers for 30 cycles. Excise target bands with the right molecular weight (red arrow) for DNA sequencing with M13F sequencing primer. Avoid the PCR products with abnormal molecular weight (blue arrow), which are supposed to be contaminations in the gel-excising step. (E) The diagram illustrates the primers’ localizations, the amplified PCR fragments and the DNA sequencing results. For each fragment, the number of clones obtained from wild-type and atprmt3-2 samples is indicated on the right. 5’ ETS, external transcribed sequence. ITS1, internal transcribed sequence. P, P1 and P’ are known cleavage sites in 5’ ETS. D, A2 and A3 are known cleavage sites in ITS1.

Recipes

  1. DEPC-treated deionized water
    Add 1 ml DEPC to 1 L deionized water, mix and put at room temperature overnight
    Autoclave at 120 °C for 30 min
  2. 3 M sodium acetate (pH 5.2)
    Dissolve 40.8 g sodium acetate in 40 ml DEPC-treated deionized water, adjust to pH 5.2 with acetic acid.
    Add deionized water to the final volume of 100 ml
    Autoclave at 120 °C for 15 min
  3. 70%, 100% ethanol
    Diluted with DEPC-treated deionized water
  4. 50x TAE buffer
    Dissolve 242 g Tris base and 37.2 g Na2EDTA.2H2O in 800 ml deionized water, add 57.1 ml acetic acid and adjust to pH 8.0 with NaOH.
    Add deionized water to the final volume of 1,000 ml

Acknowledgments

This protocol was adapted from the previously published studies (Barkan, 2011; Slomovic et al., 2008) and Dr. Alice Barkan’s lab homepage (http://pml.uoregon.edu/protocols.html), and it was performed in (Hang et al., 2014). This work was supported by National Natural Science Foundation of China Grants 31330020 and 31210103901 (to X.C.), 31370770 and 31171184 (to C.L.), and 31200900 (to X.D.), and State Key Laboratory of Plant Genomics Grant SKLPG2011B0101.

References

  1. Barkan, A. (2011). Studying the structure and processing of chloroplast transcripts. Methods Mol Biol 774: 183-197.
  2. Hang, R., Liu, C., Ahmad, A., Zhang, Y., Lu, F. and Cao, X. (2014). Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing. Proc Natl Acad Sci U S A 111(45): 16190-16195.
  3. Lykke-Andersen, S., Brodersen, D. E. and Jensen, T. H. (2009). Origins and activities of the eukaryotic exosome. J Cell Sci 122(Pt 10): 1487-1494.
  4. Slomovic, S., Portnoy, V. and Schuster, G. (2008). Detection and characterization of polyadenylated RNA in Eukarya, Bacteria, Archaea, and organelles. Methods Enzymol 447: 501-520.

简介

转录后加工对于RNA生物发生至关重要,其中产生常规功能性RNA转录物,例如用于翻译的信使RNA(mRNA),转移RNA(tRNA)和核糖体RNA(rRNA)以及已知的新编码RNA或未知的调节功能。为了在加工期间或之后确定RNA分子的精确末端,引物延伸和cDNA末端的快速扩增(RACE)方法已经常规地用于5'或3'末端的精确作图。与设计为一次仅检测特定靶RNA的一端的这些测定法不同,环状逆转录 - 聚合酶链式反应(cRT-PCR)能够同时测定靶标的5'和3'末端RNA。在拟南芥中,cRT-PCR已经被广泛应用于鉴定核糖体RNA前体的5'和3'末端,或者评估在3'末端的长度或转录后延伸成熟mRNA。在该方案中,我们总结并提出了在拟南芥中cRT-PCR测定的详细程序,其也成功地用于我们以前公开的工作中(Hang等人 ,2014)。

关键字:循环检测, 转录后加工, rRNA前体的加工, 核糖体合成, PRMT3

材料和试剂

  1. 拟南芥 14天的幼苗
  2. M13F:5'-TGTAAAACGACGGCCAGT-3'和M13R:5'-CAGGAAACAGCTATGAC-3'
  3. TRNzol试剂(TIANGEN ®,目录号:DP40502)
  4. Dynabeads mRNA纯化试剂盒(Life Technologies,目录号:61006)
  5. FirstChoice ® RLM-RACE Kit(Life Technologies,目录号:AM1700)
  6. RNA 5'多磷酸酶(Epicentre,目录号:RP8092H)
  7. T4 RNA连接酶1(New England Biolabs,目录号:M0204S)
  8. T4 RNA连接酶反应缓冲液(New England Biolabs,目录号:B0216L)
  9. 腺苷5'-三磷酸(New England Biolabs,目录号:P0756S)
  10. RNasin Plus RNA酶抑制剂(Promega Corporation,目录号:N2615)
  11. 糖原(RNA级)(Thermo Fisher Scientific,目录号:R0551)
  12. TransScript II第一链cDNA合成SuperMix(TransGen Biotech,目录号:AH301)
  13. TaKaRa ExTaq DNA聚合酶(Takara Bio Company,目录号:RR001B)
  14. pEASY -T1克隆试剂盒(TransGen Biotech,目录号:CT10102)
  15. Plus II DNA标记(TransGen Biotech,目录号:BM121-01)。
  16. 焦碳酸二乙酯/DEPC(Sigma-Aldrich,目录号:D5758)
  17. TIANgel Midi Purification Kit(TransGen Biotech,目录号:DP209)
  18. UltraPure TM 琼脂糖(Life Technologies,目录号:75510019)
  19. DEPC处理的去离子水(见配方)
  20. 3 M乙酸钠(见配方)
  21. 70%,100%乙醇(见配方)
  22. 50x TAE缓冲液(见配方)

设备

  1. MaxyClear Microtubes(1.5ml)(Corning Incorporated,Axygen ,目录号:MCT150C)
  2. NanoDrop 1000分光光度计(Thermo Fisher Scientific,目录号:ND-1000)
  3. Thermomixer comfort(Eppendorf,目录号:5362.000.019)
  4. Eppendorf离心机(Eppendorf,目录号:5417R)
  5. PCR系统(Eastwin Life Science,目录号:EDC-810)
  6. 其他标准实验室设备

程序

  1. 底漆设计
    1. 一般来说,引物应该对靶标具有高度特异性 序列,能够与其靶序列形成稳定的双链体,和   没有二级结构
    2. 三个引物约20〜25 长度的核苷酸应当被合成。 这些的GC含量 引物设计为尽可能相似。
      注意:我们建议 GC比例在40%〜55%之间。 例如,中列出的引物的GC比率 图2A在该范围内。 但是,此参数不是固定的,它   取决于你的基因。
    3. 第一个基因特异性引物cRT(图 1)应当位于已知区域中并用于反向 转录(RT)。
      注意:cRT是基因的反向。 其定位推荐在预期的100〜200 nt内 5'末端。 例如18c位于距预期5'末端204nt内   的成熟18S rRNA(图2A)。
    4. 其他两个引物,cF1和 cR1,分别位于cRT引物的5'和3'侧 (图1),用于初级PCR扩增5'和3' 目标转录物的末端。 cF1和cR1与序列退火 分别位于cRT的3'和5'(相对于cDNA非RNA)。 他们的本地化推荐距离预期约100 nt 5'和3'末端 注意:引物cF2或cR2, 紧接着在cF1或cR1引物的下游 1),进一步嵌套PCR以提高PCR特异性。 用于嵌套PCR的引物cF2和cR2可以在已知区域设计 成熟RNA或预测区域仅存在于前体RNA中。相比 cF1/cR1,这三种引物组合(cF1/cR2,cF2/cR1和 cF2/cR2)可用于嵌套PCR。


    图1.循环RT-PCR(cRT-PCR)的图示。总RNA或聚腺苷酸化RNA用T4 RNA连接酶环化,并用基因特异性引物cRT反转录。然后扩增5'和3'末端并测序。 A和D标记特定RNA的已知5'和3'末端。蓝框从A到B和黑框从C到D分别表示PCR引物cF1和cR1的位点。 B和C之间的白色框表示cRT引物的基因座。与两个主要引物cF1和cR1相邻的引物cF2和cR2分别设计用于嵌套PCR。

  2. RNA提取(线性RNA)
    1. 紧接着在cF1或cR1引物的下游 1),进一步嵌套PCR以提高PCR特异性。 用于嵌套PCR的引物cF2和cR2可以在已知区域设计 成熟RNA或预测区域仅存在于前体RNA中。相比 cF1/cR1,这三种引物组合(cF1/cR2,cF2/cR1和 cF2/cR2)可用于嵌套PCR。


    图1.循环RT-PCR(cRT-PCR)的图示。总RNA或聚腺苷酸化RNA用T4 RNA连接酶环化,并用基因特异性引物cRT反转录。然后扩增5'和3'末端并测序。 A和D标记特定RNA的已知5'和3'末端。蓝框从A到B和黑框从C到D分别表示PCR引物cF1和cR1的位点。 B和C之间的白色框表示cRT引物的基因座。与两个主要引物cF1和cR1相邻的引物cF2和cR2分别设计用于嵌套PCR。

  3. RNA提取(线性RNA)
    1. ...
  4. RNA ligation (Circular RNA, cRNA)
    1. 10 μg of total RNA or 0.5 μg of poly(A) RNA was used for each ligation reaction (50 μl). RNA is pre-incubated in Thermomixer comfort at 70 °C for 5 min, and then immediately chilled on ice for 2 min.
      Note: RNA decapping step is recommended before RNA ligation when analyzing nucleus-encoded mRNAs (Slomovic et al., 2008), since RNAs with 5’-cap structure will inhibit the RNA ligation efficiency. Although this step was not applied in our previous work (Hang et al., 2014), we recommend performing this assay if necessary with these two kits according to their corresponding protocols. RNA 5´ Polyphosphatase from Epicentre
      (http://www.epibio.com/docs/default-source/protocols/rna-5-polyphosphatase.pdf?sfvrsn=12) or Tobacco Acid Pyrophosphatase supported in FirstChoice® RLM-RACE Kit (https://tools.lifetechnologies.com/content/sfs/manuals/cms_056070.pdf).
    2. Prepare the following reaction system:
      RNA 10 μg total/0.5 μg poly(A)
      T4 RNA Ligase 1 (10,000 U/ml) 3 μl (30 U)
      10x T4 RNA Ligase 1 buffer 5 μl
      Adenosine-5'-Triphosphate (10 mM ATP) 5 μl (1 mM)
      RNase inhibitor (40 U/μl) 1 μl (40 U)
      DEPC-treated deionized water to 50 μl
      Note: The ligation reaction system may be optimized in the dosage of T4 RNA ligase 1 or ATP. Be aware that the appropriate concentration of ATP should be added if it is not supplied in the T4 RNA ligase buffer. The appropriate concentration of ATP should be in accordance with the product information of the commercial T4 RNA ligase 1. We used T4 RNA Ligase 1 from New England Biolabs (see in materials and reagents). In its properties and usage section, 1 mM ATP is suggested to add into the reaction system, since there is no ATP in the 10 x T4 RNA ligase 反应缓冲液 ( https://www.neb.com/产物/m0204-t4-rna连接酶-1-ssrna连接酶 - tabselect0 )。此外,参考伴随协议 优化RNA环化 ( https://www。 neb.com/protocols/1/01/01/rna-circularization-using-t4-rna-ligase-1 )。  在此协议,20μl反应系统补充20-50μMATP 和10%PEG8000,然后在25℃下孵育1〜2小时 在16℃过夜以获得更好的产率。
    3. 充分混合并在Thermomixer中37°C温育1〜2小时
    4. 在65℃孵育15分钟以失活T4 RNA连接酶1.
    5. 乙醇通过加入5μl3M乙酸钠(pH 5.2)沉淀RNA,  2μl糖原(20mg/ml)和171μl100%乙醇。保持 混合物在冰上30分钟(短时间)或在-20℃下储存过夜 沉淀连接的RNA 注意:-20°C下的沉淀时间更长 建议过夜更好的RNA回收。 浓度 糖原为20mg/ml。 其工作浓度约为0.7μg/μl   当将2μl糖原加入50μl反应产物加上5μl3M 乙酸钠。 值得注意的是,最终0.05-1μg/μl的浓度   根据其产品说明建议糖原。
    6. 在4℃下以12,000rpm旋转样品15分钟。
      注意:   对于给定rpm的力根据转子而变化。 当我们使用 Eppendorf离心机5417R,速度12,000rpm等于15,294×g。
    7. 弃去上清液,用500μl70%乙醇洗涤沉淀,然后在4℃以12,000 rpm旋转15分钟。
    8.  丢弃上清液并风干沉淀。 重悬RNA中 DEPC处理的去离子水,70μl总RNA或7μl聚(A) RNA。 RNA样品可储存于-80℃直至使用 注意:我们有 没有比较cRNA和正常RNA在-80℃下的半衰期 然而,虽然我们预测cRNA是稳定的并且可以保持   长时间在-80°C。 我们建议进行逆转录 尽快。

  5. 用基因特异性引物(Circular cDNA,ccDNA)进行逆转录
    1. 准备以下反应系统并在0℃预孵育混合物   70℃5分钟,然后在冰上冷却2分钟。 1μg总cRNA或 对于每个逆转录使用0.5μg的poly(A)cRNA。
      cRNA 7μl
      RT引物(图1中的cRT)2 pmol
      DEPC处理的去离子水至9μl
    2. 轻轻旋转混合物,向每个样品中加入以下反应混合物 2x TS反应混合10μl
      TransScript II RT/RI Enzyme Mix 1μl
      充分混合,在50℃孵育1小时 注意:   2x TS Reaction Mix和TransScript 支持TransScript®II第一链cDNA合成SuperMix。 他们 没有目录号,因此没有列在材料和 试剂部分。
    3. 在85℃孵育5分钟以灭活TransScript II RT酶。
    4. 加入180μlDEPC处理的去离子水稀释产物至终体积200μl。

  6. PCR扩增
    1. 准备一般反应混合物用于PCR(总共50μl):
      cDNA5μl
      ExTaq 10x缓冲液5μl
      dNTPs混合物(2.5mM每种dNTP)4μl
      正向引物(cF1,10μM)1μl(10 pmol)
      反向引物(cR1,10μM)1μl(10pmol)
      TaKaRa ExTaq DNA聚合酶(5U /μl)0.12μl(0.6U)
      去离子水33.88μl
    2. 根据下述程序运行PCR扩增:
      一个。 94℃3分钟;
      b。 94℃30秒;
      C。 55℃下30秒;
      d。 72℃1分钟; 重复步骤b-d 30个周期
      e。 72℃10分钟;
      F。 保持在20°C。
      注意:   退火温度和延伸时间可以通过优化 案件。 我们建议尝试之间的常规退火温度 55〜60℃。 我们知道,较高的退火温度可能增加 产品特异性。 因此,我们可以比较PCR产物   55°C,58°C和60°C选择最好的一个。 对于伸长时间,   我们宁愿选择1分钟,第一次尝试看看PCR 产品长度(我们假设它不超过1000 bp)。 然后,我们会   相应地调节伸长时间。 总之,伸长时间   取决于PCR产物的潜在长度和延伸率 DNA聚合酶。
    3. 加载8μl的PCR产物在1%琼脂糖凝胶上 (用溴化乙锭,EtBr),并运行直到溴酚蓝染料 2/3下来的凝胶。图像凝胶,并确定是否这  批次的循环RT-PCR成功(锋利和强带)和 该候选频带应该被切割用于进一步分析。
      注意:如果它  不能很好地工作,PCR系统应该在退火中优化 温度,扩增循环,PCR酶等。此外, 基于用cF1/cR1的初级PCR,进一步嵌套PCR cF1/cR2,cF2/cR1或cF2/cR2可有助于改善产品 特异性,如图1所示。对于嵌套PCR,PCR混合物 组件和PCR程序与第一轮PCR相同,除外 对于引物组合和DNA模板。
    4. 加载所有 剩余的PCR产物在新的1%琼脂糖凝胶上。剪切候选条带 并用TIANgel Midi Purification Kit纯化凝胶中的DNA

  7. DNA测序和分析
    1. 将纯化的DNA产物克隆到pEasy-T载体中。
    2. 对于菌落PCR制备主反应混合物,除了DNA模板(总共20μl):
      ExTaq 10x缓冲液2μl
      dNTPs混合物(2.5mM每种dNTP)1μl
      M13F引物(10μM)0.5μl(5pmol) M13R引物(10μM)0.5μl(5pmol) TaKaRa ExTaq DNA聚合酶(5U /μl)0.12μl(0.6U)
      去离子水15.88μl
      首先制备PCR反应混合物,并等分到0.2ml PCR管中
    3. 通过白 - 蓝菌落选择选择阳性克隆。 取20μl   移液器吸头和触摸阳性菌落非常轻轻,浸泡的提示a 几次到20微升的PCR混合物
    4. 运行PCR 根据E.2中描述的程序进行扩增。 伸长率 时间取决于PCR产物的长度。 对于TaKaRa ExTaq ® DNA 聚合酶,1kbp PCR产物的延伸时间为1分钟
    5. 使用1%琼脂糖凝胶用EtBr染色分析PCR产物。 含有用于DNA测序的尖锐目标条带的消除凝胶切片 M13F测序引物。 我们建议至少排列15个样本   可靠的结果。
      注意:具有M13F和M13R的DNA序列 引物是确定5'和3'末端所必需的 靶RNA,特别是在3'尾的高聚腺苷酸化RNA。
    6.   DNA测序数据用基本局部比对搜索工具分析  (BLAST)从国家生物技术信息中心(NCBI)。 选择有机体: Arabidopsis thaliana 。搜索数据库:参考 基因组序列(refseq_genomic)使用Megablast(优化高  类似序列)。


    图2.用于鉴定atprmt3-2中的前-18S rRNA的循环RT-PCR 。来自Col-0和的14天龄幼苗的总RNA > atprmt3-2 突变体用于cRT-PCR测定。 (A)用于该测定中的寡核苷酸。 18c是用于pre-18S rRNA的逆转录的cRT引物。列出它们的长度,GC含量和Tm值。 (B-C)用18c反转录后用引物r5/r6(B)和r5/r8(C)扩增的PCR产物的琼脂糖凝胶分析。通过在1.5%琼脂糖凝胶中的凝胶电泳分析反应产物并用0.5μg/ml溴化乙锭染色。 DNA标记物显示在左边。将标记在右侧的切除候选条带并提取DNA克隆到pEasy-T载体中。 (D)用M13F和M13R引物进行菌落PCR 30个循环。用正确的分子量(红色箭头)切割目标条带用于使用M13F测序引物进行DNA测序。避免PCR产物具有异常的分子量(蓝色箭头),这是在凝胶切割步骤中的污染物。 (E)该图说明了引物的定位,扩增的PCR片段和DNA测序结果。对于每个片段,从野生型和atprmt3-2样品获得的克隆数目显示在右侧。 5'ETS,外部转录序列。 ITS1,内部转录序列。 P,P1和P'是5'ETS中的已知切割位点。 D,A2和A3是ITS1中的已知切割位点。

食谱

  1. DEPC处理的去离子水 将1ml DEPC加入1L去离子水中,混合并在室温下放置过夜
    在120℃高压灭菌30分钟
  2. 3 M乙酸钠(pH 5.2)
    将40.8g乙酸钠溶解在40ml DEPC处理的去离子水中,用乙酸调节至pH 5.2。
    加入去离子水至最终体积为100ml
    在120℃高压灭菌15分钟
  3. 70%,100%乙醇 用DEPC处理的去离子水稀释
  4. 50x TAE缓冲区
    在800ml去离子水中溶解242g Tris碱和37.2g Na 2 EDTA EDTA 2 H 2 O 2,加入57.1ml乙酸,并调节至 pH 8.0用NaOH。
    加入去离子水至最终体积为1000ml

致谢

该协议改编自以前发表的研究(Barkan,2011; Slomovic等人,2008)和Alice Barkan博士的实验室主页( http://pml.uoregon.edu/protocols.html ),并且在(Hang et al。 ,2014)。 这项工作得到中国国家自然科学基金31330020和31210103901(到X.C.),31370770和31171184(C.L.)和31200900(X.D.)和植物基因组学批准SKLPG2011B0101国家重点实验室的支持。

参考文献

  1. Barkan,A。(2011)。 研究叶绿体转录物的结构和加工。 方法 em> 774:183-197。
  2. Hang,R.,Liu,C.,Ahmad,A.,Zhang,Y.,Lu,F.and Cao,X.(2014)。 拟南芥蛋白精氨酸甲基转移酶3是核糖体生物发生所必需的,通过影响前体核糖体RNA processing。 Proc Natl Acad Sci USA 111(45):16190-16195。
  3. Lykke-Andersen,S.,Brodersen,D.E.and Jensen,T.H。(2009)。 真核生物外来体的起源和活性。细胞科学122(Pt 10):1487-1494。
  4. Slomovic,S.,Portnoy,V。和Schuster,G。(2008)。 检测和表征真核生物,细菌,古细菌和细胞器中的聚腺苷酸化RNA。方法Enzymol 447:501-520。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hang, R., Deng, X., Liu, C., Mo, B. and Cao, X. (2015). Circular RT-PCR Assay Using Arabidopsis Samples. Bio-protocol 5(14): e1533. DOI: 10.21769/BioProtoc.1533.
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