搜索

1 user has reported that he/she has successfully carried out the experiment using this protocol.
Visualization of RNA 3’ ends in Escherichia coli Using 3’ RACE Combined with Primer Extension
3'RACE联合引物延伸法检测大肠埃希氏菌RNA 3'末端   

评审
匿名评审
引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

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
Jun 2015

Abstract

In this assay, 3’ RACE (Rapid Amplification of cDNA 3’ Ends) followed by PE (primer extension), abbreviated as 3’ RACE-PE is used to identify the mRNA 3’ ends. The following protocol describes the amplification of the mRNA 3’ ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, the definite primer is 5’ end-labeled using [γ-(32) P] ATP and T4 polynucleotide kinase, which anneals to the specific DNA molecules within the PCR product of the 3’ RACE. The conventional PE can only be used to locate the 5’ end of an mRNA transcript since reverse transcriptase (RTase) polymerizes only in the 5’ → 3’ direction. Thus, Taq polymerase is used instead of RTase, PCR is performed. Therefore, we are able to locate the 3’ end of the mRNA using this assay. The relative amount of the 3’ end can be directly visualized and quantified by way of separating DNA products in a denaturing 8% urea-PAGE (Polyacrylamide Gel Electrophoresis) gel. The exact position of the 3’ ends can be sequenced by comparison of these final DNA products with the corresponding DNA sequencing ladder.

Keywords: 3’ RACE (3' RACE), Primer extension (引物延伸), RNA 3’ ends (RNA 3' 末端), E. coli (大肠杆菌), Galactose operon (半乳糖操纵子)

Background

The synthesis of the mRNA 3’ end is an important step in E. coli that produces a stable messenger RNA (mRNA). In eukaryotic cells, the mRNA 3’ end formation is through a cleavage from an internal phosphodiester bond, followed by the addition of a poly (A) tail; whereas in prokaryotic cells, the 3’ ends of mRNAs are generated by termination of transcription or by processing of the primary transcript (Altman and Robertson, 1973; Nudler and Gottesman, 2002; Zhao et al., 1999). Therefore, it is important to analyze the exact position and relative quantity of mRNA 3’ end to understand the mechanism of mRNA generation.

3’ RACE assay is a particular procedure to obtain the 3’ end sequence information of a defined RNA transcript (Sambrook and Russell, 2006). Generally, the experiment procedure starts with ligating the 3’ end of RNA to a synthetic RNA oligo, followed by the synthesis of cDNA using RTase and a complementary primer (3RP) to the RNA oligo. Subsequently, specific cDNA is amplified by PCR using the gene-specific primer and the primer, 3RP. Usually, RACE products are directly sequenced, however, based on our modified procedure, PCR products undergo another concluding step of primer extension (PE), which uses Taq polymerase instead of RTase. The labeled primer integrated into the PCR products are extended in a denaturing PAGE gel which makes us visualize and quantify each product. The scheme of 3’ RACE-PE is presented in Figure 1. The polycistronic gal operon encodes amphibolic enzymes for the amphibolism of the sugar D-galactose (Holden et al., 2003). Using this method, we have identified and quantitated the 3’ ends of the gal operon mRNAs in wild type and mutant strains (Lee et al., 2008; Wang et al., 2014 and 2015).


Figure 1. An illustration of the procedure 3’ RACE-PE in E. coli

Materials and Reagents

  1. Pipettes tips (DNase/RNase-free, Sorenson Bioscience)
  2. 1.5 ml centrifuge tube (SARSTEDT, catalog number: 72.690.001 )
  3. Cell culture flasks (Corning, catalog number: 3056 )
  4. 20 x 150 mm Test tube (Karter Scientific Labware Manufacturing, catalog number: 212W5 )
  5. Sephadex G-50 column (GE Healthcare, catalog number: 27-5330-01 )
  6. Whatman 3MM paper (GE Healthcare, catalog number: 3017-915 )
  7. Kodak CL-XPosure Film (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34090 )
  8. Gloves (Ultra TEX Glove, Taeshin Bio Science, catalog number: UT-11 )
  9. E. coli strains (CGSC, the coli genetic stock center) (an example)
  10. Lysozyme (Roche Diagnostics, catalog number: 10837059001 )
  11. TRIZOL (Molecular Research Center, catalog number: TR 118 )
  12. Chloroform (Merck, Sigma-Aldrich, catalog number: C2432 )
  13. Isopropanol (Merck, Sigma-Aldrich, catalog number: I9516 )
  14. Ethanol (Merck, Sigma-Aldrich, catalog number: E7023 )
  15. RNA storage solution (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM7001 )
  16. Alkaline phosphatase (Takara Bio, catalog number: 2250A )
  17. DNase I (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2222 )
  18. RNasin® Ribonuclease Inhibitors (Promega, catalog number: N2111 )
  19. PCI (Phenol:Chloroform:Isoamyl Alcohol) Solution 25:24:1 (Merck, Sigma-Aldrich, catalog number: 77617 )
  20. Sodium acetate (Merck, Sigma-Aldrich, catalog number: S2889 )
  21. RNase and DNase-free water (Bioneer, catalog number: C-9011 )
  22. T4 RNA ligase (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2141 )
  23. The synthesized 3’ RACE RNA oligo: 3’-inverted deoxythymidine (3’-idT) RNA (Dharmacon)
  24. 3RP primer (5’AGCATGCGGCCGCTAAGAAC3’)
  25. dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0191 )
  26. HotStarTaq Plus DNA Polymerase (QIAGEN, catalog number: 203603 )
  27. Gal-specific primers (Table 1)

    Table 1. Sequence of each galactose operon gene specific primers are listed


  28. T4 Polynucleotide Kinase (New England Biolabs, catalog number: M0201L )
  29. ATP, [γ-32P]- 6,000 Ci/mmol (PerkinElmer, catalog number: NEG002Z250UC )
  30. 5’ end labeled PE primer (5’ GAGAGTAGGGAACTGCCA 3’)
  31. 5x Developer (Vivid X-RAY DEVELOPER, Duksan (DS) Lab, catalog number: 0514_00004 )
  32. 5x Fixer (Vivid X-RAY RAPID FIXER, Duksan (DS) Lab, catalog number: 0514_00003 )
  33. Tryptone (BD, DifcoTM, catalog number: 211705 )
  34. Yeast extract (BD, DifcoTM, catalog number: 212750 )
  35. Sodium chloride (NaCl) (Merck, Sigma-Aldrich, catalog number: 746398 )
  36. Galactose (Merck, Sigma-Aldrich, catalog number: G5388 )
  37. Acrylamide (Merck, Sigma-Aldrich, catalog number: V900845 )
  38. Bis-acrylamide (Merck, Sigma-Aldrich, catalog number: V900301 )
  39. 5x TBE (Bioneer, catalog number: C-9002 )
  40. Urea (Merck, Sigma-Aldrich, catalog number: U5128 )
  41. Ammonium persulfate (Merck, Sigma-Aldrich, catalog number: 248614 )
  42. TEMED (Merck, Sigma-Aldrich, catalog number: T9281 )
  43. Formamide (Merck, Sigma-Aldrich, catalog number: F9037 )
  44. 0.5 M EDTA, pH 8.0 (Bioneer, catalog number: C-9007 )
  45. Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
  46. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  47. 1 M Tris-HCl, pH 8.0 (Bioneer, catalog number: C-9006 )
  48. Sucrose (Merck, Sigma-Aldrich, catalog number: S7903 )
  49. Omniscript RT Kit (QIAGEN, catalog number: 205111 )
  50. Sigmacote (Sigma-Aldrich, catalog number: SL2 )
  51. LB + 0.5% galactose (see Recipes)
  52. 8% sequencing solution (see Recipes)
  53. 8% Urea-PAGE gel (see Recipes)
  54. 2x gel loading mix (see Recipes)
  55. Protoplasting buffer (see Recipes)

Equipment

  1. Automatic pipette aid (Thermo Fisher Scientific, Thermo ScientificTM, model: S1 Pipette Filler )
  2. Pipettes (Gilson Company, USA)
  3. 37 °C shaking incubator (Hanbaek Scientific Co., Korea (S))
  4. 37 °C and 42 °C heat block (FINEPCR, Korea (S))
  5. Spectrophotometer (BECKMAN COULTER, USA)
  6. Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 )
  7. 4 °C micro-centrifuge (Beckman Coulter, USA)
  8. NanoDrop 1000 (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 1000 )
  9. PCR machine (Bio-Rad Laboratories, USA)
  10. Shallow fixer tray (Bio-Rad Laboratories, USA)
  11. Sequencing gel electrophoresis apparatus (LABREPCO, USA)
  12. X-ray cassette (Duksan (DS) Lab, Korea (S))
  13. Power supply (Bio-Rad Laboratories, USA)
  14. Autoclave (Dong Won Scientific Corp, Korea (S))

Software

  1. ImageJ

Procedure

  1. RNA extraction
    1. Streak out the E. coli strain to be used for RNA extraction on an LB plate and incubate overnight at 37 °C.
    2. Select a single colony from the streaked LB agar plate. Inoculate E. coli strain in 3 ml fresh LB medium supplemented with 0.5% galactose, and then grow for 16 h. The next day, assess the bacterial growth by measuring the OD600, usually it is around 4.
    3. Dilute the culture in 50 ml pre-warmed (37 °C) LB medium supplemented with 0.5% galactose to give an OD600 reading of about 0.05.
    4. Grow the bacterial culture at 37 °C in a shaking incubator. Assess the OD600 till it reaches 0.6, usually it takes 1 h 50 min for the wild type E. coli cell. At this time, the number of cells in 1 ml culture is about 2 x 108.
    5. Collect 1 ml cells in a microcentrifuge tube, pellet the cells by centrifugation (12,000 x g, 1 min, 20 °C).
    6. Discard the supernatant by pipetting, resuspend the cell pellet in 50 µl protoplasting buffer, and add 5 µl lysozyme (50 mg/ml), mix thoroughly, incubate for 5 min at room temperature.
    7. Add 500 µl Trizol, mix thoroughly by vortexing for 15 sec. incubate e the lysed sample for 5 min at room temperature.
    8. Add 100 µl chloroform to the lysate, shake vigorously for 15 sec. incubate the resulting mixture at room temperature for 5 min, and centrifuge at 12,000 x g for 15 min at 4 °C.
    9. Following the centrifugation process, the mixture separates into 3 layers: a clear upper aqueous layer (containing RNA), an interphase and a red lower organic layer, carefully transfer 250 µl of the upper aqueous phase to a fresh tube.
    10. Precipitate RNA from the aqueous phase by mixing 250 µl (equal volume) of isopropanol.
    11. Store samples at room temperature for 10 min and centrifuge at 12,000 x g for 15 min at 4 °C.
    12. Decant the supernatant and wash the RNA pellet by vortexing in 1 ml 75% ethanol.
    13. Centrifuge the RNA suspension at 12,000 x g for 5 min at 4 °C.
    14. Remove the remaining ethanol by repeating the centrifugation process and briefly air-dry the RNA pellet for 5 min (air-dry can be carried on at room temperature).
    15. Dissolve RNA in 30 µl RNA storage solution.
    16. Measure the concentration of RNA by NanoDrop. Typically, 800-1,000 ng/µl RNA can be isolated from 1 ml of E. coli culture.

  2. DNase I and Alkaline Phosphatase treatment
    1. Resuspend 25 µg RNA in 1x AP Reaction Buffer to a final volume of 50 µl.
    2. Add 3 U of DNase I, and 45 U of Alkaline Phosphatase, 20 U of RNasin® Ribonuclease Inhibitors, mix thoroughly and incubate at 37 °C for 30 min.
    3. Add 50 µl PCI, mix thoroughly by vortexing for 10 sec.
    4. Centrifuge at 12,000 x g for 5 min.
    5. Collect 30 µl of supernatant, and transfer to a Sephadex G-50 column.
    6. Centrifuge at 2,000 x g for 2 min, collect the pass through.
    7. Measure the RNA concentration by NanoDrop. Typically, 300-400 ng/µl RNA can be collected.

  3. Ligation
    1. Deprotection of 3’-idT RNA oligo
      1. Centrifuge the tube briefly. Add 400 µl of deprotection buffer to the tube, vortex for 10 sec and centrifuge for 10 sec.
      2. Incubate at 60 °C for 30 min.
      3. Perform phenol extraction and ethanol precipitation as follows: add 400 µl of PCI solution to the tube, vortex for 5 sec.
      4. Centrifuge at 12,000 x g for 5 min at room temperature.
      5. Carefully remove the 300 µl upper aqueous phase to a new tube.
      6. Add 2.5 volume of ice cold 100% ethanol, 0.1 volume of 3 M sodium acetate, and vortex to mix thoroughly.
      7. Precipitate at -80 °C for 20 min.
      8. Centrifuge at 12,000 x g for 20 min at 4 °C.
      9. Decant the supernatant and carefully pipet off the remaining supernatant, wash pellet twice by adding 500 µl ice cold 70% ethanol.
      10. Air dry the pellet at room temperature and resuspend in 100 µl of RNase and DNase-free water.
    2. Set up a 20 μl reaction as follows:
      2 µl 10x RNA ligase buffer
      0.25 µl RNasin (40 U/µl)
      1 μl T4 RNA ligase (5 U/µl)
      2.5 µg RNA
      0.5 µl 3’ RACE RNA oligo (100 nM)
      RNase-free water
    3. Incubate at 37 °C for 3 h.
      The sequence of 3’ RACE RNA oligo is 5’ P. UUCACUGUUCUUAGCGGCCGCAUGCUC.idT
    4. Apply the ligation mix to a G-50 column for buffer exchange, centrifuge at 2,000 x g for 2 min and collect the pass through in a new micro centrifuge tube.

  4. Reverse transcription
    1. Assemble 3RP primer, dissolve the primer in RNase- and DNase-free water to make a 100 µM stock solution (= concentration). A working solution of 25 µM was made by diluting in RNase and DNase-free water from this stock.
      Sequence of 3RP primer (5’AGCATGCGGCCGCTAAGAAC3’)
    2. Set up a 20 μl reaction as follows:
      2 µl 10x RTase buffer
      0.25 µl RNasin (40 U/µl)
      1 μl RTase (4 U/µl)
      Ligation product 13.75 µl
      1 µl 3RP primer (25 µM)
      2 µl dNTP mix (5 mM)
    3. Incubate at 37 °C for 2 h.

  5. PCR
    1. Assemble primers, as in Table 1.
    2. Set up a 50 μl reaction as follows:
      5 µl 10x PCR buffer
      0.5 µl HotStarTaq plus DNA polymerase (5 U/µl)
      1 µl dNTP mix (10 mM)
      1 µl 3RP primer (25 µM)
      2.5 µl gal-specific primer (10 µM)
      2 µl cDNA
      38 µl RNase-free water
    3. Transfer PCR tubes to a PCR machine with the lid pre-heated to 105 °C and run the thermocycling process. Thermocycling conditions for PCR are shown in Table 2.

      Table 2. Thermocycling conditions for PCR


  6. Primer end labeling
    1. Set-up the following reaction to a final volume of 20 µl:
      2 µl 10x kinase buffer
      1 µl kinase (10 U/µl)
      2 µl primer (10 µM)
      2 µl [γ-32P] ATP
      13 µl RNase-free water
    2. Incubate at 37 °C for 30 min.
    3. Inactivate the enzyme at 65 °C for 30 min.
    4. Add 30 µl RNase and DNase-free water to the reaction mix, pass through the G-50 column by centrifuge at 2,000 x g for 2 min.
    5. Store the labeled primer at 4 °C.

  7. Primer extension
    1. Set-up the following reaction to a final volume of 20 µl:
      2 µl 10x PCR buffer
      0.2 µl HotStarTaq plus DNA polymerase (5 U/µl)
      0.3 µl dNTP mix (10 mM)
      0.3 µl 5’ end labeled PE primer
      2 µl DNA template
      15.2 µl RNase-free water
    2. Transfer PCR tubes to a PCR machine with the lid preheated to 105 °C and run the thermocycling process. Thermocycling conditions for PCR are shown in Table 3.

      Table 3. Thermocycling conditions for PE


  8. Gel electrophoresis
    1. Gel casting
      1. Assemble the gel according to manufacturer’s description and fix the gel in the gel-casting chamber. Use 0.4 mm thick spacers, glass plates (Inner plate x outer plate (W x H): 31 x 38.5 cm), 4 x 0.4 mm (14 cm) gel comb).
      2. Prepare 50 ml of the 8% urea-PAGE solution.
      3. Pour the gel immediately using an automatic pipette aid between the two glass plates. Avoid introducing air bubbles. Insert the comb and let the gel polymerize for 60 min. The assembled gel is indicated as in Figure 2.


        Figure 2. A picture of assembled gel in the gel-casting chamber with the automatic pipette aid

    2. Set up the electrophoresis apparatus and pre-run the gel
      1. Dismount the gel from the casting chamber and assemble it to the gel apparatus according to the manufacturer’s instructions.
      2. Fill the lower buffer chamber with 1x TBE running buffer, so that the glass plates will be submerged 2-3 cm with the buffer. Fill the upper buffer chamber up to the top of the gel with 1x TBE running buffer.
      3. Carefully remove the comb and rinse the wells with running buffer by using a pipette and gel loading tips.
      4. Attach the lid of the gel system and plug in the cables to a high voltage power supply. Before loading the samples you have to prerun the gel for at least 30 min to heat up the gel and to remove the remaining urea from the gel. The optimal temperature should be between 45-55 °C. Avoid temperatures higher than 60 °C as bands could smear or the glass plates could crack. Choose a constant watt for the prerun (30 W). The assembled electrophoresis apparatus is indicated as in Figure 3.


        Figure 3. A picture of the assembled electrophoresis apparatus

    3. Sample preparation
      1. Add 15 µl 2x gel loading mix to 15 µl sample.
      2. Denature the sample by heating at 95 °C for 3 min.
    4. Load and run the gel
      1. Remove the lid and rinse the wells thoroughly as described earlier as urea will leach into the wells.
      2. Load 3 µl of samples carefully from the bottom of the wells. Avoid introducing air bubbles.
      3. Assemble the lid and run the gel at 30 W to maintain a gel temperature of 55 °C similar to the prerun. Observe the migration of the marker dyes until the dye front reached the lower end of the gel. A run can last 2-4 h.
    5. Process the gel
      1. Remove the gel cassette from the chamber by loosening the clamps. Pull away the spacers and carefully disassemble the glass plates. If necessary, cut away the upper well containing the gel part.
      2. Cut a Whatman 3MM paper to the same size of the gel, attach it to the gel, and carefully remove the gel from the glass plate.
      3. Carefully attach a saran wrap on top of the gel, seal it with scotch tape. A picture of the Whatman 3MM paper cut and saran wrapped is indicated as in Figure 4.
      4. Move the sealed gel to the X-ray cassette, put in a single piece of X-ray film, expose at -80 °C for 16 h.


        Figure 4. A picture showing the Whatman 3MM paper cut the same size as the outer plate or gel, saran wrapped sealed with scotch tape placed in the X-ray cassette

      5. After the exposure time, take out the cassette from the refrigerator, wait about 1 h until it warms up to the room temperature.
      6. Develop the X-ray film in a dark room. Firstly, dilute one-part concentrate of the developer to 4 parts of water. Mix and store it in a bottle. Prepare the fixer in the same way. Then, pour the developer into a container, take the exposed X-ray film out of the cassette, immerse the film into the developer. After several minutes, when the dark bands appear, rinse the film with distilled water and then fix the film in the fixer.

Data analysis

The data can be scanned and quantified using the software ‘ImageJ’ (https://imagej.nih.gov/ij/). ImageJ is a Java based software by Wayne Rasband and others from National Institute of Health (USA).

  1. Scanning the film and setting the measurement criteria
    1. Create high resolution scans from the film in .tiff file format. Keep this as your source image, and use it for the quantification analysis.
    2. Start the ImageJ analysis software and load the previously saved .tiff file from the destination folder using the ‘File’ menu. Change the picture format to .jpeg and the picture mode to ‘grayscale’.
    3. Under the ‘Analyze’ menu, choose ‘Set Measurements’. From the checkboxes have the ‘Area’ checked (Figure 5).


      Figure 5. ImageJ software interface showing the Menu and tool features. The bottom picture explaining the different types of ‘Set Measurements’ options, checked ‘Area’ for our specific quantification of the 3’ end bands.

  2. Selection for measurements
    1. ImageJ selection of measurements defines a rectangle area of interest across the lanes of the final PE products of the 3’ RACE. Choose the ‘rectangle’ tool from ImageJ and draw a frame around the bands of the samples.
      Notes:
      1. You can trail around and resize the frame.
      2. Adjust it so that it covers the area to contain the whole of the largest and the smallest band of the sample row.
    2. For each of the analysis to take measurements, for instance in Figure 6, the first lane will be the control and all the other bands across the lanes will be the samples to quantify and compare. Centre the band inside the frame and utilize the ‘Ctrl’ + ‘M’ to record a measurement or choose ‘Measure’ from the ‘Analyze’ menu.
    3. This will spread out the measurement window and display the area peaks of the samples in order. Make a closed peak of the samples by using the ‘Straight’ tool to draw freehand lines to find the area of the individual sample peaks. Applying the ‘Wand’ tool obtains the area of the individual sample peaks as shown in Figure 6.


      Figure 6. An example of quantifying 3’ RACE-PE results using ImageJ. A. A schematic of the galactose operon. The transcription initiation site from the P1 promoter is marked as + 1. The positions of primers used to amplify the 3’ ends of mT1 are indicated by small arrows. The gel image showing the 3’ ends of galT mRNA in WT and Δspf strains by 3’ RACE-PE. The numbers on the sides of each gel picture indicate relative positions to the P1 transcription start site (+1) (Lee et al., 2008). B. The peaks and area table of the mT1 bands. Bands framed by the ‘Rectangle’ tool in yellow is used to obtain the peaks and to find the area of these 3’ end bands.

  3. Spreadsheets and calculations
    1. When the area of the peaks of the bands along with the loading control band is obtained, the data are transferred to an Excel sheet.
    2. When the area of sample bands with respect to the control band is recorded, a ratio of samples band value over the control band of that lane is calculated (Figure 6. Lane 2, 3, 4 over Lane 1 (Control)).
    3. The final relative quantitative values are the proportion of the sample bands to the control band.
    4. A bar (or other) graph is constructed to compare the samples with respect to the control.

Notes

  1. The amount of starting sample should not exceed the capacity of Trizol-reagent. Overloading will reduce the quality of RNA.
  2. Use RNase-free reagents and filter tips during the whole process of the experiment. Wear gloves and keep the tubes closed throughout the procedure.
  3. PCR cycles can be modified depending on the amount of product.
  4. The time of exposure on the X-ray films from the gel at -80 °C can be modified according to the intensity of band.
  5. A specific area of the laboratory should be designated for P-32 handling. When handling radioactive materials, always wear protective gloves and clothing, use a Plexiglas shield to prevent radiation. Try to keep your exposure time as short as possible. The radioactive materials should be kept in lead-lined containers, and experiment wastes should be discarded in designated containers. Use the Geiger counter to check the radiation level in front of the shield and your gloves frequently for contamination.

Recipes

  1. LB + 0.5% galactose (1 L)
    10 g tryptone
    5 g yeast extract
    10 g NaCl
    Add distilled water up to 1 L
    Autoclave
    Add 25 ml filtered 20% galactose
    Store at room temperature
  2. 8% sequencing solution (1 L)
    76 g acrylamide
    4 g Bis-acrylamide
    200 ml 5x TBE
    500 g urea
    Add distilled water up to 1 L
    Dissolve by heating and stirring
  3. 8% Urea-PAGE gel (50 ml)
    49.67 ml 8% sequencing solution
    300 µl 10% APS solution
    30 µl TEMED
  4. 2x gel loading mix
    9 ml formamide
    200 µl 0.5 M EDTA
    100 µl 1% xylene cyanol
    100 µl 1% bromophenol blue
    Add RNase and DNase-free water up to 10 ml
  5. Protoplasting buffer
    750 µl 1 M Tris-HCl (pH8.0)
    7.7 g sucrose
    800 µl 0.5 M EDTA (pH8.0)
    Add RNase and DNase-free water up to 50 ml

Acknowledgments

This work was supported by the National Science Foundation for Young Scientists of China (grant 31600061) and the China Postdoctoral Science Foundation (grants 2015M582234, 2017T100562). This research was also supported by a grant from Chungnam National University (2015-1420-01). This protocol was adapted from our publication in J Mol Biol (Lee et al., 2008), J Bacteriol (Wang et al., 2014) and PNAS (Wang et al., 2015). The authors declare no conflict of interest.

References

  1. Altman, S. and Robertson, H. D. (1973). RNA precursor molecules and ribonucleases in E. coli. Mol Cell Biochem 1(1): 83-93.
  2. Holden, H. M., Rayment, I. and Thoden, J. B. (2003). Structure and function of enzymes of the Leloir pathway for galactose metabolism. J Biol Chem 278(45): 43885-43888.
  3. Lee, H. J., Jeon, H. J., Ji, S. C., Yun, S. H. and Lim, H. M. (2008). Establishment of an mRNA gradient depends on the promoter: an investigation of polarity in gene expression. J Mol Biol 378(2): 318-327.
  4. Nudler, E. and Gottesman, M. E. (2002). Transcription termination and anti-termination in E. coli. Genes Cells 7(8): 755-768.
  5. Sambrook, J. and Russell, D. W. (2006). Rapid amplification of 3' cDNA ends (3'-RACE). CSH Protoc 2006(1).
  6. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
  7. Wang, X., Ji, S. C., Jeon, H. J., Lee, Y., Lim, H. M. (2015). Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene. Proc Natl Acad Sci U S A 112: 7581-7586.
  8. Wang, X., Ji, S. C., Yun, S. H., Jeon, H. J., Kim, S. W., and Lim, H. M. (2014). Expression of each cistron in the gal operon can be regulated by transcription termination and generation of a galk-specific mRNA, mK2. J Bacteriol 196: 2598-2606.
  9. Zhao, J., Hyman, L. and Moore, C. (1999). Formation of mRNA 3' ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol Mol Biol Rev 63(2): 405-445.

简介

在该测定中,使用缩写为3'RACE-PE的3'RACE(cDNA3'末端快速扩增),随后是PE(引物延伸),以鉴定mRNA3'末端。以下方案描述E中半乳糖操纵子的mRNA 3'末端的扩增。并通过PE对相应的PCR产物进行可视化。在PE中,使用[γ-(32)P] ATP和T4多核苷酸激酶对确定的引物进行5'末端标记,其退火至3'RACE的PCR产物内的特定DNA分子。由于逆转录酶(RTase)仅在5'→3'方向聚合,常规PE只能用于定位mRNA转录物的5'末端。因此,使用Taq聚合酶代替RTase,进行PCR。因此,我们能够使用此测定法定位mRNA的3'末端。通过在变性8%尿素-PAGE(聚丙烯酰胺凝胶电泳)凝胶中分离DNA产物,可以直接显示和定量3'末端的相对量。 3'末端的确切位置可以通过比较这些最终的DNA产物与相应的DNA测序阶梯进行测序。


【背景】mRNA 3'末端的合成是E中的重要步骤。产生稳定的信使RNA(mRNA)的大肠杆菌。在真核细胞中,mRNA 3'末端形成是通过从内部磷酸二酯键切割,然后加入聚(A)尾;而在原核细胞中,通过终止转录或通过加工初级转录产生mRNA的3'末端(Altman和Robertson,1973; Nudler和Gottesman,2002; Zhao等人,1999年)。因此,分析mRNA 3'端的确切位置和相对数量以了解mRNA产生的机制是重要的。

3'RACE测定是获得确定的RNA转录物的3'末端序列信息的特定程序(Sambrook和Russell,2006)。通常,实验步骤始于将RNA的3'末端连接至合成的RNA寡聚物,随后使用RTase和针对RNA寡聚物的互补引物(3RP)合成cDNA。随后,使用基因特异性引物和引物3RP通过PCR扩增特定的cDNA。通常,RACE产物是直接测序的,然而,根据我们的修改程序,PCR产物经历了另一个引物延伸(PE)的结束步骤,其使用Taq聚合酶代替RTase。整合到PCR产物中的标记引物在变性PAGE凝胶中延伸,这使得我们可视化和量化每种产物。 3'RACE-PE的方案如图1所示。多顺反子gal操纵子编码用于糖D-半乳糖角裂的两亲性酶(Holden等, 2003)。使用这种方法,我们已经鉴定和定量了野生型和突变株中的gal操纵子mRNA的3'末端(Lee等人,2008; Wang等人, et al。,2014和2015)。

“”src
图1. E中的程序3'RACE-PE的示例。大肠杆菌

关键字:3' RACE, 引物延伸, RNA 3' 末端, 大肠杆菌, 半乳糖操纵子

材料和试剂

  1. 移液器吸头(DNase / RNase-free,Sorenson Bioscience)

  2. 1.5 ml离心管(SARSTEDT,目录号:72.690.001)
  3. 细胞培养瓶(康宁,目录号:3056)
  4. 20 x 150 mm试管(Karter Scientific Labware Manufacturing,产品目录号:212W5)
  5. Sephadex G-50柱(GE Healthcare,目录号:27-5330-01)
  6. Whatman 3MM纸(GE Healthcare,目录号:3017-915)
  7. 柯达CL-XPosure薄膜(Thermo Fisher Scientific,Thermo Scientific TM TM,目录号:34090)
  8. 手套(Ultra TEX手套,Taeshin生物科学,产品编号:UT-11)
  9. 电子。 (CGSC,大肠杆菌基因库中心)(例子)
  10. 溶菌酶(Roche Diagnostics,目录号:10837059001)
  11. TRIZOL(分子研究中心,目录号:TR 118)
  12. 氯仿(Merck,Sigma-Aldrich,目录号:C2432)
  13. 异丙醇(Merck,Sigma-Aldrich,目录号:I9516)
  14. 乙醇(Merck,Sigma-Aldrich,目录号:E7023)
  15. RNA储存溶液(Thermo Fisher Scientific,Invitrogen TM,目录号:AM7001)
  16. 碱性磷酸酶(Takara Bio,目录号:2250A)
  17. DNase I(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2222)
  18. RNasin核糖核酸酶抑制剂(Promega,目录号:N2111)
  19. PCI(苯酚:氯仿:异戊醇)溶液25:24:1(Merck,Sigma-Aldrich,目录号:77617)
  20. 乙酸钠(Merck,Sigma-Aldrich,目录号:S2889)
  21. RNase和DNase-free水(Bioneer,目录号:C-9011)
  22. T4 RNA连接酶(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2141)
  23. 合成的3'RACE RNA寡核苷酸:3'-反式脱氧胸苷(3'-idT)RNA(Dharmacon)
  24. 3RP引物(5'AGCATGCGGCCGCTAAGAAC3')
  25. dNTP混合物(各10mM)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0191)
  26. HotStarTaq Plus DNA聚合酶(QIAGEN,目录号:203603)
  27. Gal特异性引物(表1)

    表1.每个半乳糖操纵子基因特异性引物列出的序列


  28. T4多核苷酸激酶(New England Biolabs,目录号:M0201L)
  29. ATP,[γ-32 P] -6,000Ci / mmol(PerkinElmer,目录号:NEG002Z250UC)
  30. 5'末端标记的PE引物(5'GAGAGTAGGGAACTGCCA 3')
  31. 5x显影剂(Vivid X-RAY显影剂,Duksan(DS)实验室,目录号:0514_00004)
  32. 5x定影剂(Vivid X-RAY RAPID FIXER,Duksan(DS)Lab,目录号:0514_00003)
  33. 胰蛋白胨(BD,Difco TM,目录号:211705)
  34. 酵母提取物(BD,Difco TM,目录号:212750)
  35. 氯化钠(NaCl)(Merck,Sigma-Aldrich,目录号:746398)
  36. 半乳糖(Merck,Sigma-Aldrich,目录号:G5388)
  37. 丙烯酰胺(Merck,Sigma-Aldrich,目录号:V900845)
  38. 双丙烯酰胺(Merck,Sigma-Aldrich,目录号:V900301)
  39. 5倍TBE(Bioneer,目录号:C-9002)
  40. 尿素(Merck,Sigma-Aldrich,目录号:U5128)
  41. 过硫酸铵(Merck,Sigma-Aldrich,目录号:248614)
  42. TEMED(Merck,Sigma-Aldrich,目录号:T9281)
  43. 甲酰胺(Merck,Sigma-Aldrich,目录号:F9037)
  44. 0.5M EDTA,pH8.0(Bioneer,目录号:C-9007)
  45. 二甲苯蓝(Sigma-Aldrich,目录号:X4126)
  46. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  47. 1M Tris-HCl,pH8.0(Bioneer,目录号:C-9006)
  48. 蔗糖(Merck,Sigma-Aldrich,目录号:S7903)
  49. Omniscript RT Kit(QIAGEN,产品目录号:205111)
  50. Sigmacote(Sigma-Aldrich,目录号:SL2)
  51. LB + 0.5%半乳糖(见食谱)
  52. 8%测序解决方案(请参阅食谱)
  53. 8%尿素-PAGE凝胶(见食谱)
  54. 2次凝胶加样混合(见食谱)
  55. 原生质体缓冲液(见食谱)

设备

  1. 自动移液器辅助装置(Thermo Fisher Scientific,Thermo Scientific TM,型号:S1移液器填料)
  2. 移液器(美国Gilson公司)
  3. 37℃摇动培养箱(Hanbaek Scientific Co.,Korea(S))
  4. 37°C和42°C加热块(FINEPCR,韩国(S))
  5. 分光光度计(美国BECKMAN COULTER)
  6. Vortex-Genie 2(Scientific Industries,型号:Vortex-Genie 2)
  7. 4°C微型离心机(Beckman Coulter,USA)
  8. NanoDrop 1000(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 1000)
  9. PCR仪(Bio-Rad Laboratories,USA)
  10. 浅的固定器托盘(Bio-Rad Laboratories,USA)
  11. 测序凝胶电泳仪(LABREPCO,USA)
  12. X射线盒(Duksan(DS)Lab,Korea(S))
  13. 电源(Bio-Rad Laboratories,USA)

  14. 高压灭菌器(Dong Won Scientific Corp,韩国(S))

软件

  1. ImageJ的

程序

  1. RNA提取
    1. 拍出 E。用于在LB平板上提取RNA并在37℃孵育过夜。
    2. 从有条纹的LB琼脂平板上选择单个菌落。接种 E。大肠杆菌菌株在3ml添加有0.5%半乳糖的新鲜LB培养基中培养,然后生长16小时。第二天,通过测量OD 600 600来评估细菌生长,通常是在4左右。
    3. 在50ml预热(37℃)LB培养基中稀释培养物,补充0.5%半乳糖以获得约0.05的OD 600读数。
    4. 在振荡培养箱中37°C培养细菌培养物。评估OD 600达到0.6,通常野生型大肠杆菌细胞需要1小时50分钟。此时,1ml培养物中的细胞数约为2×10 8个。
    5. 在离心管中收集1ml细胞,离心沉淀细胞(12,000×g,1分钟,20℃)。
    6. 弃去上清液,用50μl原生质体缓冲液重悬细胞沉淀,加入5μl溶菌酶(50mg / ml),充分混匀,在室温下孵育5分钟。
    7. 加入500μlTrizol,涡旋振荡15秒。
      在室温下孵育裂解的样品5分钟
    8. 加入100μl氯仿溶液,剧烈振荡15秒。在室温下将所得混合物温育5分钟,并在4℃下以12,000×gg离心15分钟。
    9. 离心过程后,混合物分成3层:透明的上层水层(含RNA),中间层和红色下层有机层小心地将250μl上层水相转移到新鲜管中。
    10. 通过混合250μl(等体积)的异丙醇,从水相中沉淀RNA。
    11. 将样品在室温下储存10分钟,并在4℃下以12,000×gg离心15分钟。
    12. 滗析上清液,用1 ml 75%乙醇振荡洗涤RNA沉淀。

    13. 在12,000xg g离心RNA悬浮液5分钟4℃。
    14. 通过重复离心过程去除剩余的乙醇,并短时间空气干燥RNA沉淀5分钟(空气干燥可以在室温下进行)。
    15. 将RNA溶解在30μlRNA储存溶液中。
    16. 用NanoDrop测量RNA的浓度。通常,可以从1ml的E中分离出800-1,000ng /μl的RNA。大肠杆菌文化。

  2. 脱氧核糖核酸酶I和碱性磷酸酶处理

    1. 在1x AP反应缓冲液中重悬25μgRNA至终体积50μl。
    2. 加入3U脱氧核糖核酸酶I和45U碱性磷酸酶,20U RNasin核糖核酸酶抑制剂,充分混合并在37℃孵育30分钟。
    3. 加入50μlPCI,涡旋混合10秒。

    4. 12000×g 离心5分钟
    5. 收集30μl上清液,转移至Sephadex G-50柱。

    6. 在2,000 em x g离心2 min,收集通过。
    7. 用NanoDrop测量RNA浓度。通常,可以收集300-400ng /μl的RNA。

  3. 结扎
    1. 3'-idT RNA寡聚物的去保护
      1. 短暂离心试管。
        加入400μl去保护缓冲液至管中,涡旋10秒并离心10秒。
      2. 在60°C孵育30分钟。
      3. 按如下方法进行苯酚提取和乙醇沉淀:向管中加入400μlPCI溶液,涡旋5秒钟。

      4. 在室温下12,000×g g离心5分钟。

      5. 小心地将300μl上层水相移至新管中。

      6. 加入2.5体积的冰冷的100%乙醇,0.1体积的3M乙酸钠,涡旋混匀。
      7. 在-80°C下沉淀20分钟。

      8. 12,000×g g离心20分钟,4℃。
      9. 滗析上清液并小心吸取剩余的上清液,通过加入500μl冰冷的70%乙醇洗涤沉淀两次。

      10. 在室温下风干颗粒并重悬于100μlRNase和DNase-free水中。
    2. 建立一个20μL的反应如下:
      2μl10x RNA连接酶缓冲液
      0.25μlRNasin(40U /μl)
      1μlT4 RNA连接酶(5 U /μl)
      2.5μgRNA
      0.5μl3'RACE RNA oligo(100 nM)
      无RNase水

    3. 在37°C孵育3小时 3'RACE RNA寡聚物的序列是5'P. UUCACUGUUCUUAGCGGCCGCAUGCUC.idT
    4. 将连接混合物应用于G-50柱以进行缓冲液交换,以2,000×g离心2分钟并收集通过新微量离心管的通过。

  4. 逆转录
    1. 装配3RP引物,将引物溶于无RNase和DNase的水中,制成100μM储备液(=浓度)。
      25μM的工作溶液是通过稀释RNase和DNase-free水而制成的。
      3RP引物序列(5'AGCATGCGGCCGCTAAGAAC3')
    2. 建立一个20μL的反应如下:
      2μl10x RTase缓冲液
      0.25μlRNasin(40U /μl)
      1μlRTase(4 U /μl)
      连接产物13.75μl
      1μl3RP引物(25μM)
      2μldNTP混合物(5 mM)
    3. 在37°C孵育2小时。

  5. PCR
    1. 组装引物,如表1所示。
    2. 建立一个50μL的反应如下:
      5μl10x PCR缓冲液
      0.5μlHotStarTaq加DNA聚合酶(5U /μl)
      1μldNTP混合物(10 mM)
      1μl3RP引物(25μM)
      2.5μlgal特异性引物(10μM)
      2μlcDNA
      38μlRNase-free水
    3. 将PCR管置于预热至105°C的PCR仪上并运行热循环过程。表2中显示了PCR的热循环条件。

      表2. PCR的热循环条件


  6. 引物末端标记
    1. 设置以下反应至最终体积为20μl:
      2μl10x激酶缓冲液
      1μl激酶(10U /μl)
      2μl引物(10μM)
      2μl[γ-32 P] ATP

      13μlRNase-free水

    2. 在37°C孵育30分钟

    3. 在65°C灭活酶30分钟
    4. 向反应混合物中加入30μlRNase和DNase-free水,通过离心机以2000×g / em的速度通过G-50柱2分钟。
    5. 将标记的引物保存在4°C。

  7. 引物扩展
    1. 设置以下反应至最终体积为20μl:
      2μl10x PCR缓冲液
      0.2μlHotStarTaq加DNA聚合酶(5 U /μl)

      0.3μldNTP混合物(10 mM)
      0.3μl5'末端标记的PE引物 2μlDNA模板

      15.2μlRNase-free水
    2. 将PCR管置于预热至105°C的PCR仪上并运行热循环过程。表3列出了PCR的热循环条件。

      表3. PE的热循环条件


  8. 凝胶电泳
    1. 凝胶铸造
      1. 根据制造商的说明组装凝胶并将凝胶固定在凝胶浇铸室中。使用0.4毫米厚的垫片,玻璃板(内板x外板(宽x高):31 x 38.5厘米),4 x 0.4毫米(14厘米)凝胶梳)。
      2. 准备50毫升的8%尿素-PAGE溶液。
      3. 立即使用两块玻璃板之间的自动移液器将凝胶倒入。避免引入气泡。插入梳子,让凝胶聚合60分钟。组装后的凝胶如图2所示。


        图2.使用自动移液器辅助装置的凝胶注射室中的凝胶装配图

    2. 设置电泳装置并预凝胶

      1. 根据制造商的说明将凝胶从流延室中取出并将其组装到凝胶设备中。
      2. 用1x TBE运行缓冲液填充下部缓冲液室,以便玻璃板用缓冲液浸没2-3cm。
        使用1x TBE运行缓冲液将上部缓冲液室填充到凝胶顶部。

      3. 。小心取出梳子,用移液器和凝胶加样针头冲洗缓冲液
      4. 连接凝胶系统的盖子并将电缆插入高压电源。加载样品前,您必须预先凝胶至少30分钟以加热凝胶并从凝胶中除去剩余的尿素。最佳温度应在45-55°C之间。避免温度高于60°C,因为条带可能会弄脏或玻璃板可能会开裂。为预运行选择恒定功率(30 W)。组装的电泳装置如图3所示。


        图3.组装电泳仪的图片

    3. 样品制备
      1. 向15μl样品中加入15μl2x凝胶加样混合物。

      2. 在95°C加热3分钟使样品变性
    4. 加载并运行凝胶

      1. 如前所述,取下盖子并彻底冲洗水井,因为尿素会渗入井中。
      2. 从孔底部小心加载3μl样品。避免引入气泡。
      3. 组装盖子并在30 W下运行凝胶以保持与预运行相似的凝胶温度55°C。观察标记染料的迁移直至染料前端达到凝胶的下端。运行可能持续2-4小时。
    5. 处理凝胶
      1. 松开夹子将凝胶盒从腔室中取出。拉开垫片并仔细拆卸玻璃板。如有必要,切开含有凝胶部分的上部孔。
      2. LI> 将Whatman 3MM纸裁剪成相同尺寸的凝胶,将其粘贴到凝胶上,然后小心地从玻璃板上取下凝胶。
      3. 小心地将凝胶包裹在凝胶顶部,用透明胶带密封。 Whatman 3MM剪纸和saran包装的图片如图4所示。
      4. 将密封的凝胶移至X射线盒,放入单片X光胶片,在-80°C下暴露16小时。


        图4.显示Whatman 3MM纸张与外层纸板或凝胶切割大小相同的图片,用透明胶带密封的saran包装放置在X光盒中 />
      5. 曝光时间后,从冰箱中取出磁带,等待大约1小时,直至温度升至室温。
      6. 在黑暗的房间里开发X光胶片。首先,将显影剂的一份浓缩物稀释到4份水。混合并储存在瓶子里。以相同的方式准备固定器。然后,将显影剂倒入容器中,将曝光后的X光胶片从暗盒中取出,将胶片浸入显影剂中。几分钟后,出现暗带时,用蒸馏水冲洗薄膜,然后将薄膜固定在定影液中。

数据分析

该数据可以被扫描,并使用软件定量“的ImageJ”( https://imagej.nih.gov/ij/ )。 ImageJ是由Wayne Rasband和来自美国国立卫生研究院的其他人提供的基于Java的软件。

  1. 扫描胶片并设置测量标准
    1. 以.tiff文件格式从电影创建高分辨率扫描。将其保存为源图像,并将其用于定量分析。
    2. 启动ImageJ分析软件并使用“文件”菜单从目标文件夹加载以前保存的.tiff文件。将图片格式更改为.jpeg并将图片模式更改为“灰度”。
    3. 在“分析”菜单下,选择“设置测量”。从复选框中选中'区域'(图5)。


      图5.显示菜单和工具功能的ImageJ软件界面下面的图解释了“设置测量”选项的不同类型,选中“区域”以便对3'端特定量化。 br />

  2. 选择测量
    1. ImageJ选择的测量值定义了3'RACE最终PE产品泳道的矩形区域。从ImageJ中选择“矩形”工具,并在样本的条带周围画一个框架。
      备注:
      1. 您可以追踪并调整框架大小。
      2. 调整它以覆盖区域以包含整个样本行的最大和最小带。
    2. 对于每个分析进行测量(例如图6),第一条车道将作为控制对象,车道上的所有其他条带将作为量化和比较的样本。在框架中央对齐乐队,并使用'Ctrl'+'M'记录测量结果或从'分析'菜单中选择'测量'。
    3. 这将展开测量窗口并按顺序显示样本的面积峰值。通过使用“直线”工具绘制手绘线以找到各个样品峰的面积,制作样品的封闭峰。应用'魔杖'工具可获得如图6所示的单个样品峰的面积。


      图6.使用ImageJ量化3'RACE-PE结果的示例。 :一种。半乳糖操纵子的示意图。来自P1启动子的转录起始位点标记为+ 1.用小箭头指示用于扩增mT1的3'末端的引物的位置。通过3'RACE-PE显示WT和Δspff菌株中emTamT mRNA的3'末端的凝胶图像。每张凝胶图片侧面的数字表示与P1转录起始位点(+1)的相对位置(Lee等人,2008)。 B. mT1频带的峰值和面积表。 “矩形”工具以黄色框起的波段用于获取波峰并找到这些3'末端波段的区域。

  3. 电子表格和计算
    1. 当获得带的峰值区域以及加载控制带时,将数据传输到Excel工作表。
    2. 当记录相对于对照带的样品带的面积时,计算样品带值与该泳道的对照带之比(图6.泳道1(对照)中的泳道2,3,4)。 />
    3. 最终的相对定量值是样品带与对照带的比例。
    4. 构建一个条形图(或其他)图形来比较样本与控件的关系。

笔记

  1. 起始样本量不应超过Trizol试剂的容量。
    过载会降低RNA的质量
  2. 在整个实验过程中使用不含RNase的试剂和过滤器吸头。
    在整个过程中戴上手套并保持管子关闭
  3. PCR循环可以根据产品的量进行修改。
  4. 可以根据带的强度来修改在-80℃下从凝胶暴露在X射线胶片上的时间。
  5. 应指定实验室的特定区域进行P-32处理。处理放射性物质时,请始终穿戴防护手套和衣服,并使用树脂玻璃罩防止辐射。尽量保持曝光时间尽可能短。放射性物质应保存在铅衬里的容器中,实验废物应放在指定的容器内。
    使用盖革计数器检查防护罩和手套的辐射水平,以防污染。

食谱

  1. LB + 0.5%半乳糖(1 L)
    10克胰蛋白胨
    5克酵母提取物
    10克NaCl

    加蒸馏水至1升 高压灭菌器
    加入25毫升过滤的20%半乳糖
    在室温下储存
  2. 8%测序溶液(1 L)
    76克丙烯酰胺
    4克双丙烯酰胺

    200毫升5x TBE 500克尿素

    加蒸馏水至1升 加热搅拌溶解
  3. 8%尿素-PAGE凝胶(50毫升)

    49.67 ml 8%测序解决方案 300μl10%APS溶液
    30μlTEMED
  4. 2倍凝胶加样混合
    9毫升甲酰胺
    200μl0.5 M EDTA
    100μl1%二甲苯氰醇
    100μl1%溴酚蓝

    加RNase和DNase-free水至10 ml
  5. 原生质体缓冲液
    750μl1 M Tris-HCl(pH8.0)
    7.7克蔗糖
    800μl0.5M EDTA(pH8.0)

    加RNase和DNase-free水至50 ml

致谢

这项工作得到了中国青年科学家基金会(31600061)和中国博士后科学基金(2015M582234,2017T100562)的资助。这项研究也得到了忠南国立大学的资助(2015-1420-01)。该协议改编自我们在J Mol Biol(Lee等人,2008年),J Bacteriol(Wang等人)的出版物(Wang等人。,2014)和 PNAS (Wang et。,2015)。作者宣称没有利益冲突。

参考

  1. Altman,S.和Robertson,H. D.(1973)。在 RNA前体分子和核糖核酸酶中。大肠杆菌。 Mol Cell Biochem 1(1):83-93。
  2. Holden,H.M。,Rayment,I.and Thoden,J.B。(2003)。 Leloir途径的半乳糖代谢酶的结构和功能 J Biol Chem 278(45):43885-43888。
  3. Lee,H.J.,Jeon,H.J.,Ji,S.C.,Yun,S.H。和Lim,H.M。(2008)。 mRNA梯度的建立取决于启动子:基因表达极性的调查。 J Mol Biol 378(2):318-327。
  4. Nudler,E.和Gottesman,M.E。(2002)。 大肠杆菌中的转录终止和反终止 基因细胞 7(8):755-768。
  5. Sambrook,J。和Russell,D.W。(2006)。 3'cDNA末端快速扩增(3'-RACE)。 CSH Protoc 2006(1)。
  6. Schneider,C.A.,Rasband,W.S。和Eliceiri,K.W。(2012)。 NIH Image to ImageJ:25年的图像分析 Nat Methods 9(7):671-675。
  7. Wang,X.,Ji,S.C.,Jeon,H.J.,Lee,Y.,Lim,H.M。(2015)。 斑点42对galK表达的二级抑制:mRNA mK2的降解和增强的转录在galK基因之前终止。 Proc Natl Acad Sci USA 112:7581-7586。
  8. Wang,X.,Ji,S.C.,Yun,S.H.,Jeon,H.J.,Kim,S.W。和Lim,H.M。(2014)。 gal操纵子中每个顺反子的表达可以通过转录终止和产生galk特异性mRNA,mK2。 J Bacteriol 196:2598-2606。
  9. Zhao,J.,Hyman,L。和Moore,C。(1999)。 在真核生物中形成mRNA 3'末端:机制,调控以及与mRNA合成中其他步骤的相互关系。 Microbiol Mol Biol Rev 63(2):405-445。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, X., Jeon, H. J., Abishek N, M. P., He, J. and Lim, H. M. (2018). Visualization of RNA 3’ ends in Escherichia coli Using 3’ RACE Combined with Primer Extension. Bio-protocol 8(5): e2752. DOI: 10.21769/BioProtoc.2752.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。