In vitro Assessment of RNA Polymerase I Activity

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Feb 2016



In eukaryotic cells transcriptional processes are carried out by three different RNA polymerases: RNA polymerase I which specifically transcribes ribosomal RNA (rRNA), RNA polymerase II which transcribes protein-coding genes to yield messenger RNAs (mRNAs) and small RNAs, while RNA polymerase III transcribes the genes for transfer RNAs and for the smallest species of ribosomal RNA (5S rRNA). This protocol describes an in vitro assay to evaluate the rRNA transcriptional activity of RNA polymerase I. The method measures the quantity of radiolabelled uridine 5’ triphosphate incorporated in ex novo synthesized rRNA molecules by RNA polymerase I, in optimal conditions for the enzyme activity and in the presence of a toxin, α-amanitin, which inhibits RNA polymerase II and III without affecting RNA polymerase I (Novello and Stirpe, 1970).

Keywords: RNA polymerase I (RNA聚合酶I), Ribosomal RNA transcription (核糖体RNA转录), Nuclei isolation (细胞核分离), Radiolabelled uridine incorporation (放射性标记的尿苷结合), α-amanitin (α-鹅膏蕈碱)


In eukaryotic cells the RNA polymerase I transcribes ribosomal genes, which are located in the nucleolus, producing 45S rRNA precursor molecules. These are processed to form the mature 18S, 5.8S and 28S rRNA. They are essential for the assembly of the 60S and the 40S subunits of mature ribosomes. Recent evidence indicates that the ribosome biogenesis rate is related to cell cycle length (Derenzini et al., 2005) and may play a role in tumorigenesis by controlling the expression of the tumour suppressor protein p53. Cells with an up-regulated ribosome biogenesis are rapidly proliferating and are characterized by a down-regulated p53 expression (Donati et al., 2011). Moreover, the ribosome biogenesis rate influences the sensitivity of cancer cells to chemotherapeutic agents which hinder rRNA transcription: higher the rate of ribosome biogenesis, higher the cytotoxic effect induced by the drug (Scala et al., 2016). Therefore, the evaluation of the ribosome biogenesis rate will become a more and more utilized procedure both in tumour pathology and in clinical oncology (Montanaro et al., 2013). Since the rate of ribosome production is tightly conditioned by the rate of 45S precursor molecules transcription, all the methods used for the evaluation of ribosome biogenesis rate measure the synthesized 45S rRNA. The used methods are: quantitative evaluation of 45S rRNA transcripts by real time PCR analysis; quantitative analysis of 45S rRNA, separated by gel electrophoresis of total RNA extracted from cells labelled with 32P-orthophosphate, and visualized by autoradiography; and quantitative evaluation of radiolabelled uridine 5’ triphosphate incorporated in ex novo synthesized rRNA molecules by RNA polymerase I. The first two methods measure the quantity of 45S rRNA present in the cells, that may be influenced by changes of rRNA processing mechanism, whereas the method described here quantifies the transcriptional activity of the RNA polymerase I and it is indicative of the rRNA transcription rate. This method is very complex and time-consuming and requires special accuracy, but it is still the only one method to selectively measure the rRNA transcription rate.

Materials and Reagents

  1. Cell scraper, 40 cm handle, 16 mm blade (Sigma-Aldrich, catalog number: C6106 )
  2. Plastic tubes 50 ml conical base (114 x 28 mm) (SARSTEDT, catalog number: 62.547.254 )
  3. Plastic tubes 15 ml conical base (120 x 17 mm) (SARSTEDT, catalog number: 62.554.002 )
  4. Plastic tubes 1.5 ml safe lock (Eppendorf, catalog number: 022363204 )
  5. Glass microfiber filters, diameter 25 mm, Whatman grade GF/C (GE Healthcare, catalog number: 1822-025 )
  6. Scintillation plastic vials (Sigma-Aldrich, catalog number: V6755 )
  7. Microscope slides (26 x 76 mm) (Sigma-Aldrich, catalog number: Z692247 )
  8. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  9. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  10. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  11. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
  12. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 )
  13. Tris base (Sigma-Aldrich, catalog number: T1503 )
  14. Ammonium sulfate [(NH4)2SO4] (Sigma-Aldrich, catalog number: A4418 )
  15. Sucrose (Merck Millipore, catalog number: 107687 )
  16. 1 M magnesium chloride (MgCl2) solution (Sigma-Aldrich, catalog number: M1028 )
  17. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  18. 1 M manganese(II) chloride (MnCl2) solution (Sigma-Aldrich, catalog number: M1787 )
  19. Guanosine 5’-triphosphate sodium salt (GTP) (Sigma-Aldrich, catalog number: 10106399001 )
  20. Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A7699 )
  21. Cytidine 5’-triphosphate disodium salt (CTP) (Sigma-Aldrich, catalog number: C1506 )
  22. Uridine 5’-triphosphate tri salt (UTP) (Sigma-Aldrich, catalog number: U6875 )
  23. Uridine 5’ triphosphate [5,6-3H] tetrasodium salt (3H-UTP) (PerkinElmer, catalog number: NET380250UC )
  24. α-amanitin (Sigma-Aldrich, catalog number: A2263 )
  25. Liquid scintillation cocktail Hionic Fluor (PerkinElmer, catalog number: 6013319 )
  26. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T4885 )
  27. Perchloric acid (PCA) 65% w/w (10.35 N) (CARLO ERBA Reagents, catalog number: 306091 )
  28. Potassium hydroxide (KOH) (EMD Millipore, catalog number: 105033 )
  29. 2-mercaptoethanol (EMD Millipore, catalog number: 805740 )
  30. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S7920 )
  31. PBS (pH 7.3) (see Recipe 1)
  32. 1 M Tris-HCl solution (pH 7.4 or pH 8.0) (see Recipe 2)
  33. 3 M ammonium sulfate (see Recipe 3)
  34. Homogenization-solution (see Recipe 4)
  35. Washing solution (see Recipe 5)
  36. Suspension solution (see Recipe 6)
  37. High ionic strength 5x reaction mixture (see Recipe 7)
  38. Nucleoside triphosphate mixture (see Recipe 8)
    1. Non-radioactive solution
    2. Radioactive solution
  39. α-amanitin reconstitution (see Recipe 9)
  40. 10% (w/w) TCA (see Recipe 10)
  41. 0.6 N PCA (see Recipe 11)
  42. 0.3 N KOH (see Recipe 12)
  43. 6 N PCA (see Recipe 13)
  44. 7% (w/w) PCA (see Recipe 14)
  45. 0.5 M NaF (see Recipe 15)
  46. Low ionic strength 5x reaction mixture (see Recipe 16)


  1. Protective gloves/protective clothing/eye protection/face protection
  2. Centrifuge (Beckman Coulter, model: TJ-25 )
    Note: This product has been discontinued.
  3. Teflon® pestle PYREX® Potter-Elvehjem tissue grinder (Thomas Scientific, catalog number: 1234F )
  4. Forceps
  5. High speed tissue grinder (homogenizer) (IKA, model: Eurostar )
  6. Inverted microscope (Zeiss, model: Primovert, catalog number: 491206-0001-000 )
  7. 10 ml glass tubes
  8. Shaking water bath (JULABO, model: SW22 )
  9. 2,000 ml filter flask
  10. Spectrophotometer UV-VIS (CHROMSERVIS, model: Nanogenius )
  11. Liquid scintillation counter (Guardian, Wallac, PerkinElmer)
  12. Vacuum glass filter holder for 25 mm disc filters elements: glass funnel, clamp, base for 25 mm glass/filter holder and a perforated stopper (available from many companies, for example: Sigma-Aldrich, catalog number: Z290467 ; EMD Millipore, catalog number: XX1002500 )
  13. Vacuum pump


Note: The experiment utilizes radioactive material and operators require experience in personal and environmental radiation protection and in the management of radioactive waste.

  1. Cell nuclei isolation
    RNA polymerase I activity can be measured in nuclei isolated from cultured cell lines or from fresh or frozen tissues, in this context, the standard procedure of nuclei isolation from cultured cells is described.
    1. Grow the desired cell line in the required culture medium on cell culture flasks until reaching about 50 x 106 cells at least.
      Note: Cells may be subjected to specific treatment, grown under special conditions or various cell lines compared.
    2. Harvest cells by scraper in phosphate buffered saline (PBS), using 10 ml of PBS every 10 x 106 cells and collect the volume of scraped cells in 50 ml plastic tubes.
    3. From this point keep cells cold at all time.
    4. Centrifuge for 10 min at 3,000 x g at 4 °C to pellet cells. Discard the supernatant.
    5. Wash the cell pellet with 20 ml of cold PBS, then centrifuge for 10 min at 3,000 x g at 4 °C and discard the supernatant.
    6. Suspend the cells in 10 ml of homogenization solution and disrupt cytoplasmic membranes and cytoplasm by homogenization with pestle and potter tissue grinder with a motor driven device: 5 strokes might be sufficient.
    7. Let the homogenized cells stand on ice for 10 min.
    8. At this point check whether the cytoplasmic membranes are broken up and the nuclei are intact and cytoplasm free. Mount approximately 10-20 μl of homogenized cells on a slide and observe with an optical microscope (Figure 1). If necessary you can repeat the homogenization step without exceeding the 10 strokes.

      Figure 1. Microscopic images showing PLC whole and homogenized cells. A. PBS washed cells; B. Homogenized cell. By comparing with figure A, in B it is observable the breakage of cytoplasmic membranes and the loss of cell morphology. The nuclei are evident and intact. Arrowheads indicate cytoplasmic debris around nuclei, these residues can be removed by washing. A Zeiss Primovert inverted microscope was used. 200x magnification.

    9. Transfer the homogenized sample to a tube and centrifuge for 10 min at 3,000 x g at 4 °C to pellet nuclei and discard any cytoplasmic residues in the supernatant. Wash the nuclei by adding 5 ml of washing solution and centrifuge for 10 min at 3,000 x g at 4 °C. Remove supernatant completely.
    10. Add 1 ml of suspension solution, mix by pipetting, take 0.1 ml of suspended nuclei and transfer it into another tube to extract and quantify hydrolyzed DNA. Use the remaining volume to assay in vitro RNA polymerase I activity.

  2. In vitro assessment of RNA polymerase I activity
    A high ionic strength medium in presence of α-amanitin is used to evaluate RNA polymerase I. In a high ionic strength medium both RNA polymerase I and II are active, however, RNA polymerase II is inhibited by α-amanitin (Novello and Stirpe, 1970).
    The measurement of each sample is carried out in triplicate accompanied by two blanks.
    1. Prepare 10 ml glass tubes, each tube must contain a final volume of 500 μl.
      Add in the following order:
      100 μl of 5x high ionic strength buffer
      90 μl of nucleoside triphosphate mixture
      1 μl of 1 mg/ml of α-amanitin solution
      MilliQ H2O to 400 μl
      100 μl of nuclei
      Note: Add nuclei to the tubes as the last element, vortex very fast and start reaction time immediately.
    2. Incubate all the samples for 10 min at 37 °C in a water bath with shaking at 100 rpm.
    3. After 10 min, stop the reaction using 5 ml of 10% (w/w) TCA and putting tubes on ice. Keep tubes on ice until the filtration step.
      1. To prepare blank samples, add nuclei to the tubes and immediately block the reaction with 5 ml of 10% (w/w) trichloroacetic acid (TCA), then incubate for 10 min at 37 °C and after that put tubes on ice.
      2. Whereas RNA polymerase I activity accounts for at least 60% of the cell transcriptional activity, RNA polymerase III contributes to a very small percentage of the cell transcriptional activity and this assay is not optimal to discriminate its activity, so it is not taken into account. To completely exclude RNA polymerase III activity an α-amanitin dose ten times more concentrated than the one which inhibits RNA polymerase II is required (at least a final concentration of 10 μg/ml).
      3. Using a high ionic strength medium it is possible to measure both RNA polymerase I and II: RNA polymerase II activity may be obtained from the difference between the measurements in the absence and in the presence of α-amanitin.
      4. It is also possible to discriminate and evaluate the RNA polymerase I activity using a low ionic strength reaction mixture (see Recipe 15), which is optimal for RNA polymerase I enzyme, but not for RNA polymerase II.

  3. Newly synthesized rRNA collection and measurement
    TCA precipitates nucleic acid molecules, including newly synthesized 3H-labelled rRNA, which can be collected on a glass-fiber filter and measured in a liquid scintillation counter.
    1. To prepare for a vacuum filtration, gather together a 2,000 ml filter flask and a vacuum glass filter holder for 25 mm disc filters, connect the side arm of filter flask to a vacuum pump using vacuum tubing. Assemble the apparatus placing a wet glass microfiber filter disc on the base (Figure 2). The filter discs are wetted with 5% (w/w) TCA.
    2. Pour a sample in the funnel, turn the vacuum pump on until the entire sample has passed through the filter and funnel base. Rinse the filter with 5% (w/w) TCA, 10 ml each wash for 5 washings.
      Note: The collected liquid in the filter flask contains most of the radioactivity.
    3. Turn the vacuum pump off, remove the funnel and using forceps place the filter in a scintillation plastic vial containing 10 ml of scintillation liquid.
    4. Repeat steps C2 and C3 for each sample tube.
    5. Shake the vials vigorously and put them in a liquid scintillation counter to measure the radioactivity of the samples.

      Figure 2. Assembly of the filtration system. A and B. Apparatus components: filter flask (1), glass microfiber filter discs (2), funnel (3), glass support base (4) inserted in a perforated stopper (5), forceps (6) and clamp (7). C. The stopper with glass support base is inserted in the flask opening. TCA wet glass microfiber filter is placed on the base. D. The funnel is put on the filter and stopped with the clamp. The side arm of the filter flask is connected to a vacuum pump by a vacuum tubing.

  4. Nuclear DNA extraction and quantification
    The following procedure is based on what reported by Munro and Fleck (1966).
    1. Use 0.1 ml of suspended nuclei to extract and quantify hydrolyzed DNA.
    2. Collect 0.1 ml of nuclei and transfer it into a 10 ml glass tube.
    3. Add 0.5 ml of 0.6 N perchloric acid (PCA), vortex the sample a few seconds and centrifuge for 10 min at 3,000 x g at 4 °C. Remove and discard the supernatant containing single nucleotides and oligonucleotides, not included in DNA and RNA molecules that are soluble in weakly acid solution. Maintain pellet containing nuclear RNA molecules and DNA.
    4. Add 0.5 ml of 0.3 N potassium hydroxide (KOH) to pellet, vortex the sample and incubate for 30 min at 37 °C in a water bath with shaking to hydrolyze RNA.
      Note: In alkaline environment RNA is susceptible to base-catalyzed hydrolysis while DNA is chemically stable.
    5. Add 50 μl of 6 N PCA to solubilize ribonucleotides and precipitate macromolecules that are still present. Centrifuge for 10 min at 3,000 x g at 4 °C, discard supernatant containing hydrolyzed RNA and retain pellet containing DNA.
    6. Add to pellet 0.5 ml of 7% (w/w) PCA, vortex the sample to suspend and incubate for 15 min at 70 °C in a water bath with shaking to hydrolyze DNA into deoxynucleotides.
      Note: Acid solution and heating liberate deoxynucleotides, which are recoverable in supernatant after centrifugation.
    7. Centrifuge for 10 min at 3,000 x g at 4 °C. Transfer the supernatant containing hydrolyzed DNA to a 1.5 ml tube.
    8. Use spectrophotometric measurements at the 260 nm wavelengths to quantitate the amount of hydrolyzed DNA.
    9. DNA concentration is calculated multiplying absorbance at 260 nm, normalized to a 10 mm path length, by a factor of 20:
      DNA (μg/ml) = (OD260nm/NF) x 20
      OD260nm (Optical Density): absorbance at the indicated wavelength,
      NF (Normalization Factor): factor to normalize to a 10 mm path length, for example, if path length is 1 mm the normalization factor is 10,
      Factor 20: 1 OD at 260 nm corresponds to approximately 20 μg/ml for oligonucleotides.

Data analysis

RNA polymerase I activity is evaluated by the measurement of radioactivity, expressed in disintegrations per minute (dpm), incorporated into newly synthesized rRNA. Each cell sample is evaluated in triplicate.

  1. Calculate the dpm mean of the blanks.
  2. Subtract the blank mean from each replicated sample.
  3. Divide the results by the μg of DNA previously quantified and calculate the mean and the standard deviation. The mean is expressed in dpm/μg DNA.

Assuming that the DNA quantity is directly proportional to the number of nuclei, the result represents the quantity of 3H labelled rRNA newly synthesized by RNA polymerase I per nucleus.

Representative data

An example of RNA polymerase I activity measurement is described below. The representative data were obtained measuring the enzymatic activity in two human cancer cell lines, HCT11 and LoVo. These cell lines are characterized by different level of rRNA synthesis as reported in Scala et al. (2016) and have different cell cycle length.

  1.  Figure 3 shows the output of radioactivity measurements by the liquid scintillation counter instrument.

    Figure 3. Picture of the radioactivity values obtained by reading the samples in the liquid scintillation counter (Guardian, Wallac, PerkinElmer). Radioactivity values are expressed in dpm. 1-5: HCT116 samples; 6-10 Lovo samples. Blue circle encloses two blanks, whereas red circle corresponds to the radioactivity measures of 3H uridine 5’ triphosphate amount incorporated in ex novo synthesized rRNA molecules by RNA polymerase I. The instrument provides both disintegrations per minute (dpm) and counts per minute (cpm); counts per minute are the counts received by an instrument from the source.

  2. In Table 1 the data analysis is shown step by step. Once the radioactivity measures were obtained, the dpm means of the blanks were calculated and subtracted from each respective replicate. The results were divided by the μg of DNA present in 0.1 ml of nuclei. Then, the mean and the standard deviation of the blank subtracted dpm values normalized to 1 μg of DNA were calculated. All data were processed using Microsoft Excel program.

    Table 1. Data analysis step by step. The table reports the data analysed in a worksheet of Excel program (Microsoft Excel). All data processed are in bold. The means of radioactivity values normalized to 1 μg of DNA are in red. The DNA content in 0.1 ml of nuclei suspension was calculated as indicated in Nuclear DNA extraction and quantification paragraph.

  3. In Figure 4 the final results are reported in a histogram, which makes clearer the different quantity of 3H labelled rRNA newly synthesized by RNA polymerase I in the two cancer cell lines.

    Figure 4. Histogram of representative data from Table 1. The histogram shows the final results: 3H-uridine 5’ triphosphate incorporated in rRNA newly synthesized by RNA polymerase I normalized to 1 μg of DNA (dpm/μg DNA). Data are shown as means ± standard deviation.


  1. PBS (pH 7.3)
    8.00 g NaCl
    0.20 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Dissolve in 800 ml of ddH2O
    Adjust pH to 7.3 with HCl and add ddH2O to a final volume of 1 L
    Final concentration: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,1.8 mM KH2PO4
  2. 1 M Tris-HCl solution (pH 7.4 or pH 8.0)
    12.11 g of Tris base
    Dissolve in 80 ml of MilliQ H2O
    Adjust pH to 7.4 (or pH 8.0) with the appropriate volume of concentrated HCl
    Add MilliQ H2O to a final volume of 100 ml
  3. 3 M ammonium sulfate
    39.64 g of ammonium sulfate
    Dissolve in 80 ml of MilliQ H2O
    Add MilliQ H2O to a final volume of 100 ml
  4. Homogenization-solution
    8.56 g of sucrose
    Dissolve in 50 ml of ddH2O
    Add 1 ml of 1 M Tris-HCl pH 7.4 (see Recipe 2), 1 ml of 1 M MgCl2 and 0.5 g of Triton X-100
    Add ddH2O to a final volume of 100 ml
    Final concentration: 0.25 M sucrose, 10 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.5% Triton X-100
  5. Washing solution
    Note: This solution is the same as homogenization solution without detergent.
    8.56 g of sucrose
    Dissolve in 50 ml of ddH2O
    Add 1 ml of 1 M Tris-HCl pH 7.4, 1 ml of 1 M MgCl2
    Add ddH2O to a final volume of 100 ml
  6. Suspension solution
    4.28 g of sucrose
    Dissolve in 40 ml of ddH2O
    Add 50 μl of 1 M MgCl2 and reach 50 ml of final volume with ddH2O
    Final concentration: 0.25 M sucrose, 1 mM MgCl2
  7. High ionic strength 5x reaction mixture
    2.5 ml 1 M Tris-HCl pH 8.0 (see Recipe 2)
    0.1 ml 1 M MnCl2 (see Reagent 18)
    2.0 ml 3 M ammonium sulfate (see Recipe 3)
    0.4 ml MilliQ H2O
    Final concentration in enzyme assay: 0.1 M Tris-HCl (pH 8.0), 4 mM MnCl2, 0.24 M ammonium sulfate
  8. Nucleoside triphosphate mixture
    Mix non-radioactive nucleoside triphosphate solution with radioactive solution in the proportions shown below.
    1. Non-radioactive solution:
      14.4 mg of GTP
      12 mg of CTP
      13.5 mg of ATP
      0.48 mg of UTP
      Dissolve in 2.5 ml of MilliQ H2O
    2. Radioactive solution:
      50 μCi of 3H-UTP diluted in
      0.5 ml of MilliQ H2O
    Final concentration in enzyme assay: 0.5 mM GTP, 0.5 mM CTP, 0.5 mM ATP, 0.02 mM UTP and 0.5 μCi 3H-UTP
  9. α-amanitin reconstitution
    1 mg/ml aqueous solution of α-amanitin
    Store it at -20 °C
    Note: You may store for a long time. It does not degrade with repeated freeze-thaw cycles.
  10. 10% (w/w) TCA
    Dilute 10 g of concentrated TCA (≥ 99% w/w) in 100 g of ddH2O
  11. 0.6 N PCA
    Dilute 2.9 ml of 65% (w/w) perchloric acid in 50 ml of ddH2O
  12. 0.3 N KOH
    Dissolve 0.842 g of KOH in 50 ml of ddH2O
  13. 6 N PCA
    Dilute 29 ml of 65% (w/w) PCA in 50 ml of ddH2O
  14. 7% (w/w) PCA
    Dilute 5.39 g of 65% (w/w) PCA in 50 g of ddH2O
  15. 0.5 M NaF
    Dissolve 0.3 g of NaF in 10 ml of MilliQ H2O
  16. Low ionic strength 5x reaction mixture
    2.5 ml 1 M Tris-HCl (pH 8.0) (see Recipe 2)
    0.1 ml 1 M MgCl2 (see Reagent 16)
    18 μl 2-mercaptoethanol (14 M)
    0.3 ml 0.5 M NaF (see Recipe 15)
    2.082 ml MilliQ H2O
    Final concentration in enzyme assay: 0.1 M Tris-HCl (pH 8.0), 4 mM MgCl2, 50 μM 2-mercaptoethanol, 6 mM NaF
    Note: Using low ionic strength medium the α-amanitin is not necessary and it is recommended a reduced volume of nucleoside triphosphate mixture (30 μl) because the blanks may be too high.


This protocol was adapted from the research article of Novello and Stirpe (1970). Work was supported by the Roberto and Cornelia Pallotti’s Legacy for Cancer Research. The author thanks Prof. Massimo Derenzini for the opportunity to describe this protocol and for help and suggestions. The author is also thankful to Dr. Christine M. Betts for language revision of the manuscript. The author declares no conflicts of interest.


  1. Derenzini, M., Montanaro, L., Chilla, A., Tosti, E., Vici, M., Barbieri, S., Govoni, M., Mazzini, G. and Trere, D. (2005). Key role of the achievement of an appropriate ribosomal RNA complement for G1-S phase transition in H4-II-E-C3 rat hepatoma cells. J Cell Physiol 202(2): 483-491.
  2. Donati, G., Bertoni, S., Brighenti, E., Vici, M., Treré, D., Volarevic, S., Montanaro, L. and Derenzini, M. (2011). The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells. Oncogene 30: 3274-3288.
  3. Montanaro, L., Trere, D. and Derenzini, M. (2013). The emerging role of RNA polymerase I transcription machinery in human malignancy: a clinical perspective. Onco Targets Ther 6: 909-916.
  4. Munro, H. N. and Fleck, A. (1966). Recent developments in the measurement of nucleic acids in biological materials. A supplementary review. Analyst 91(79): 78-88.
  5. Novello, F. and Stirpe, F. (1970). Simultaneous assay of RNA polymerase I and II in nuclei isolated from resting and growing rat liver with the use of alpha-amanitin. FEBS Lett 8(1): 57-60.
  6. Scala, F., Brighenti, E., Govoni, M., Imbrogno, E., Fornari, F., Treré, D., Montanaro, L. and Derenzini, M. (2016). Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate. Oncogene 35(8): 977-989. Let the homogenized cells stand


在真核细胞中,转录过程由三种不同的RNA聚合酶:特异性转录核糖体RNA(rRNA)的RNA聚合酶I,转录蛋白质编码基因以产生信使RNA(mRNA)和小RNA的RNA聚合酶II进行转录,RNA聚合酶III转录转录RNA和最小核糖体RNA(5S rRNA)的基因。该方案描述了用于评价RNA聚合酶I的rRNA转录活性的体外实验方法。该方法测量了合并的rRNA分子中掺入的放射性标记的尿苷5'-三磷酸的量通过RNA聚合酶I,在酶活性的最佳条件和毒素α-amanitin存在下,其抑制RNA聚合酶II和III而不影响RNA聚合酶I(Novello和Stirpe,1970)。

背景 在真核细胞中,RNA聚合酶I转录位于核仁中的核糖体基因,产生45S rRNA前体分子。这些被处理以形成成熟的18S,5.8S和28S rRNA。它们对于成熟核糖体的60S和40S亚基的组装是必需的。最近的证据表明,核糖体生物发生率与细胞周期长度有关(Derenzini等人,2005),并且可能通过控制肿瘤抑制蛋白p53的表达在肿瘤发生中发挥作用。具有上调核糖体生物发生的细胞正在快速增殖,其特征在于下调的p53表达(Donati等人,2011)。此外,核糖体生物发生率影响癌细胞对阻碍rRNA转录的化学治疗剂的敏感性:核糖体生物发生率越高,药物诱导的细胞毒性效应越高(Scala等,2016) 。因此,核糖体生物发生率的评估将在肿瘤病理学和临床肿瘤学中成为越来越多的应用程序(Montanaro等人,2013)。由于核糖体产生速率受45S前体分子转录速率的限制,所有用于评估核糖体生物发生率的方法都是测定合成的45S rRNA。使用的方法是:通过实时PCR分析定量评估45S rRNA转录物; 45S rRNA的定量分析,通过凝胶电泳分离,由从32号正磷酸标记的细胞提取的总RNA分离,并通过放射自显影显像;和通过RNA聚合酶I合并的rRNA分子中的放射性标记的尿苷5'-三磷酸的定量评估。前两种方法测量存在于细胞中的45S rRNA的量,其可能受到rRNA处理机制,而这里描述的方法量化了RNA聚合酶I的转录活性,并且其指示rRNA转录速率。该方法非常复杂且耗时,需要特殊的准确度,但仍然是选择性测定rRNA转录率的唯一方法。

关键字:RNA聚合酶I, 核糖体RNA转录, 细胞核分离, 放射性标记的尿苷结合, α-鹅膏蕈碱


  1. 细胞刮刀,40厘米手柄,16毫米刀片(Sigma-Aldrich,目录号:C6106)
  2. 塑料管50毫升锥形底座(114 x 28毫米)(SARSTEDT,目录号:62.547.254)
  3. 塑料管15毫升锥形底座(120 x 17毫米)(SARSTEDT,目录号:62.554.002)
  4. 塑料管1.5毫升安全锁(Eppendorf,目录号:022363204)
  5. 玻璃微纤维过滤器,直径25 mm,Whatman级GF/C(GE Healthcare,目录号:1822-025)
  6. 闪烁塑料小瓶(Sigma-Aldrich,目录号:V6755)
  7. 显微镜载玻片(26 x 76 mm)(Sigma-Aldrich,目录号:Z692247)
  8. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  9. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  10. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  11. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P9791)
  12. 盐酸(HCl)(Sigma-Aldrich,目录号:H1758)
  13. Tris碱(Sigma-Aldrich,目录号:T1503)
  14. 硫酸铵[(NH 4)2 SO 4](Sigma-Aldrich,目录号:A4418)
  15. 蔗糖(Merck Millipore,目录号:107687)
  16. 1M氯化镁(MgCl 2)溶液(Sigma-Aldrich,目录号:M1028)
  17. Triton X-100(Sigma-Aldrich,目录号:T8787)
  18. 1M Mn(II)氯化物(MnCl 2)溶液(Sigma-Aldrich,目录号:M1787)
  19. 鸟苷5'-三磷酸钠盐(GTP)(Sigma-Aldrich,目录号:10106399001)
  20. 腺苷5'-三磷酸二钠盐水合物(ATP)(Sigma-Aldrich,目录号:A7699)
  21. 胞苷5'-三磷酸二钠盐(CTP)(Sigma-Aldrich,目录号:C1506)
  22. 尿苷5'-三磷酸三盐(UTP)(Sigma-Aldrich,目录号:U6875)
  23. 尿苷5'三磷酸酯[5,6-三羟基]四钠盐( 3 H-UTP)(PerkinElmer,目录号:NET380250UC)
  24. α-amanitin(Sigma-Aldrich,目录号:A2263)
  25. 液体闪烁混合物Hionic Fluor(PerkinElmer,目录号:6013319)
  26. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:T4885)
  27. 高氯酸(PCA)65%w/w(10.35N)(CARLO ERBA试剂,目录号:306091)
  28. 氢氧化钾(KOH)(EMD Millipore,目录号:105033)
  29. 2-巯基乙醇(EMD Millipore,目录号:805740)
  30. 氟化钠(NaF)(Sigma-Aldrich,目录号:S7920)
  31. PBS(pH 7.3)(参见配方1)
  32. 1M Tris-HCl溶液(pH 7.4或pH 8.0)(参见方法2)
  33. 3 M硫酸铵(见配方3)
  34. 均质溶液(参见配方4)
  35. 洗涤液(参见配方5)
  36. 悬浮液(参见配方6)
  37. 高离子强度5x反应混合物(见方案7)
  38. 核苷三磷酸酯混合物(参见食谱8)
    1. 非放射性溶液
    2. 放射性解决方案
  39. α-淀粉蛋白重组(见方法9)
  40. 10%(w/w)TCA(见配方10)
  41. 0.6 N PCA(见配方11)
  42. 0.3 N KOH(参见配方12)
  43. 6 N PCA(见配方13)
  44. 7%(w/w)PCA(见配方14)
  45. 0.5 M NaF(见配方15)
  46. 低离子强度5x反应混合物(参见配方16)


  1. 防护手套/防护服/眼睛保护/面部护理
  2. 离心机(Beckman Coulter,型号:TJ-25)
  3. 特氟隆®杵PYREX ® Potter-Elvehjem组织研磨机(Thomas Scientific,目录号:1234F)
  4. 镊子
  5. 高速组织研磨机(均质机)(IKA,型号:Eurostar)
  6. 倒置显微镜(Zeiss,型号:Primovert,目录号:491206-0001-000)
  7. 10毫升玻璃管
  8. 水浴(JULABO,型号:SW22)
  9. 2000 ml滤瓶
  10. 分光光度计UV-VIS(CHROMSERVIS,型号:Nanogenius)
  11. 液体闪烁计数器(Guardian,Wallac,PerkinElmer)
  12. 用于25 mm圆盘过滤器的真空玻璃过滤器支架:玻璃漏斗,夹具,25 mm玻璃/过滤器支架底座和穿孔塞子(可从许多公司获得,例如:Sigma-Aldrich,目录号:Z290467; EMD Millipore,目录号码:XX1002500)
  13. 真空泵



  1. 细胞核分离
    1. 在细胞培养瓶中培养需要的培养基中生长所需的细胞系,至少达到约50×10 6个细胞。
    2. 在磷酸盐缓冲盐水(PBS)中通过刮刀收集细胞,每10×10 6细胞使用10ml PBS,并将刮体细胞的体积收集在50ml塑料管中。
    3. 从这一点上来说,一直保持细胞冷。
    4. 在4℃以3,000×g离心10分钟以沉淀细胞。丢弃上清液。
    5. 用20ml冷PBS洗涤细胞沉淀,然后在4℃以3,000×g离心10分钟,弃去上清液。
    6. 将细胞悬浮在10ml匀浆溶液中,并用电机驱动装置用杵和陶器组织研磨机均质化破坏细胞质膜和细胞质:5次行程可能就足够了。
    7. 让均匀的细胞在冰中静置10分钟
    8. 此时检查细胞质膜是否分解,细胞核是否完整和细胞质无关。将大约10-20μl匀浆细胞载于载玻片上并用光学显微镜观察(图1)。如果需要,您可以重复均匀化步骤而不超过10次。

      图1.显示PLC整体和匀浆细胞的显微镜图像。 A。 PBS洗涤细胞;均质细胞。通过与图A比较,在B中观察到细胞质膜的破裂和细胞形态的丧失。细胞核明显而完整。箭头表示核周围的细胞质碎片,这些残留物可以通过洗涤去除。使用蔡司Primovert倒置显微镜。 200倍放大倍率。

    9. 将均质化样品转移到管中,并在4℃以3,000×g离心10分钟以沉淀核并丢弃上清液中的任何细胞质残留物。通过加入5ml洗涤溶液洗涤细胞核,并在4℃以3,000×g离心10分钟。彻底清除上清液
    10. 加入1ml悬浮液,移液混匀,取0.1ml悬浮核,转移至另一管中提取和定量水解DNA。使用剩余体积来测定体外 RNA聚合酶I活性。

  2. RNA聚合酶I活性的体外评估
    在α-淀粉样蛋白存在下使用高离子强度培养基来评估RNA聚合酶I.在高离子强度培养基中,RNA聚合酶I和II都是活性的,然而,RNA聚合酶II被α-amanitin抑制(Novello和Stirpe, 1970) 每个样品的测量一式三份进行,并附有两个空白。
    1. 准备10毫升玻璃管,每个管必须含有最终体积500微升。
      1微升1毫克/毫升的α-淀粉样溶液 MilliQ H 2 O至400μl
    2. 将所有样品在37℃下在水浴中以100rpm摇动孵育10分钟。
    3. 10分钟后,使用5毫升10%(重量/重量)的TCA停止反应并将管放在冰上。将管保持在冰上直到过滤步骤。
      1. 为了制备空白样品,向管中加入核并立即用5ml 10%(w/w)三氯乙酸(TCA)阻断反应,然后在37℃下孵育10分钟,然后将管放在冰。
      2. 尽管RNA聚合酶I活性占细胞转录活性的至少60%,但RNA聚合酶III有助于细胞转录活性的非常小百分比,并且该测定法不能区分其活性,因此不被采用考虑到为了完全排除RNA聚合酶III活性,需要比抑制RNA聚合酶II浓度高10倍的α-淀粉样蛋白(至少最终浓度为10μg/ml)。
      3. 使用高离子强度培养基可以测量RNA聚合酶I和II:RNA聚合酶II活性可以从不存在和存在α-淀粉样蛋白的测量之间的差异获得。
      4. 还可以使用对RNA聚合酶I酶最佳但低于RNA聚合酶II的低离子强度反应混合物(参见方案15)来区分和评估RNA聚合酶I活性。 br />
  3. 新合成的rRNA收集和测量
    TCA沉淀核酸分子,包括新合成的3 H-标记的rRNA,可以在玻璃纤维过滤器上收集并在液体闪烁计数器中测量。
    1. 为了准备真空过滤,将一个2000毫升过滤瓶和一个25毫米圆盘过滤器的真空玻璃过滤器支架聚集在一起,使用真空管将过滤瓶的侧臂连接到真空泵。组装在底部放置湿玻璃微纤维过滤盘的设备(图2)。过滤盘用5%(w/w)TCA润湿。
    2. 将样品倒入漏斗中,打开真空泵,直到整个样品通过过滤器和漏斗底座。用5%(w/w)TCA冲洗过滤器,每次冲洗10 ml洗涤5次 注意:滤瓶中收集的液体中含有大部分放射性物质。
    3. 关闭真空泵,取出漏斗并使用镊子将过滤器放入含有10ml闪烁液的闪烁塑料瓶中。
    4. 对每个样品管重复步骤C2和C3。
    5. 大力摇动小瓶,将其放入液体闪烁计数器中,以测量样品的放射性。

      图2.过滤系统的装配 A和B.装置部件:过滤烧瓶(1),玻璃微纤维过滤盘(2),漏斗(3),玻璃支撑基座(4)穿孔止动器(5),镊子(6)和夹具(7)。 C.带有玻璃支撑底座的塞子插入烧瓶开口。 TCA湿玻璃微纤维过滤器放在基座上。 D.将漏斗放在过滤器上并用夹具停止。过滤瓶的侧臂通过真空管连接到真空泵。

  4. 核DNA提取和定量
    1. 使用0.1ml悬浮核提取并定量水解的DNA
    2. 收集0.1ml细胞核并将其转移到10ml玻璃管中
    3. 加入0.5ml 0.6N高氯酸(PCA),将样品涡旋几秒钟,并在4℃以3,000×g离心10分钟。去除并丢弃含有单核苷酸和寡核苷酸的上清液,不包含在可溶于弱酸溶液的DNA和RNA分子中。保持含核RNA分子和DNA的沉淀。
    4. 加入0.5ml的0.3N氢氧化钾(KOH)沉淀,涡旋样品,并在37℃,水浴中,振荡孵育30分钟,水解RNA。 注意:在碱性环境中,RNA易于进行碱催化水解,而DNA是化学稳定的。
    5. 加入50μl6 N PCA溶解核糖核苷酸并沉淀仍然存在的大分子。在4℃下以3,000×g离心10分钟,弃去含有水解RNA的上清液,并保留含有DNA的沉淀物。
    6. 加入0.5ml 7%(w/w)PCA,旋转样品悬浮并在70℃下在水浴中振荡孵育15分钟,将DNA水解成脱氧核苷酸。
    7. 在4℃以3,000×g离心10分钟。将含有水解DNA的上清液转移到1.5 ml管中
    8. 使用260nm波长的分光光度测量来定量水解DNA的量。
    9. 计算DNA浓度,将260nm处的吸光度乘以20mm路径长度归一化因子20:
      DNA(μg/ml)=(OD 260nm/NF)×20
      OD 260nm(光密度):指示波长处的吸光度,



  1. 计算空白的dpm平均值。
  2. 从每个复制样本中减去空白平均值。
  3. 将结果除以先前量化的DNA,并计算平均值和标准偏差。平均值以dpm /μgDNA表示。




  1. 图3显示了液体闪烁计数仪器的放射性测量值的输出

    图3.通过读取液体闪烁计数器(Guardian,Wallac,PerkinElmer)中的样品获得的放射性值的图片。 放射性值以dpm表示。 1-5:HCT116样品; 6-10 Lovo样品。蓝色圆圈包围两个空白,而红色圆圈对应于通过RNA聚合酶I掺入到合成的rRNA分子中的 3 H尿苷5'-三磷酸含量的放射性测量。仪器提供每分钟崩解(dpm)和每分钟计数(cpm);每分钟的计数是仪器从源头接收的计数。

  2. 在表1中,数据分析逐步显示。一旦获得了放射性测量值,则计算每个空白的dpm均值,并从各自的重复中减去。将结果除以存在于0.1ml核中的DNA的μg。然后,计算标准化为1μgDNA的空白减去dpm值的平均值和标准偏差。所有数据都使用Microsoft Excel程序处理。

    表1.数据分析一步一步。该表报告在Excel程序(Microsoft Excel)的工作表中分析的数据。处理的所有数据均为粗体。标准化为1μgDNA的放射性值的方法为红色。按照核DNA提取和定量段中的指示计算0.1ml核悬浮液中的DNA含量

  3. 在图4中,最终结果以直方图报告,这使得两种癌细胞系中由RNA聚合酶I新合成的3H标记的rRNA的量不同。

    图4.来自表1的代表性数据的直方图。直方图显示最终结果:将由RNA聚合酶I新合成的rRNA中并入的H-尿苷5'三磷酸盐归一化为1μgDNA(dpm /μgDNA)。数据显示为平均值±标准偏差。


  1. PBS(pH 7.3)
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4
    溶于800毫升ddH 2 O -/- 用HCl将pH调节至7.3,并加入ddH 2 O至最终体积为1升(重量) 终浓度:137mM NaCl,2.7mM KCl,10mM Na 2 HPO 4,1.8mM KH 2 PO 4, sub>
  2. 1M Tris-HCl溶液(pH 7.4或pH 8.0)
    溶解在80毫升MilliQ H< 2> O
    中 用适当体积的浓HCl调节pH至7.4(或pH 8.0) 将MilliQ H 2 O 2添加到最终体积为100ml的
  3. 3 M硫酸铵
    溶解在80毫升MilliQ H< 2> O
    中 将MilliQ H 2 O 2添加到最终体积为100ml的
  4. 均质化解决方案
    溶于50ml的ddH 2 O -/- 加入1ml 1M Tris-HCl pH 7.4(参见方案2),1ml 1M MgCl 2和0.5g Triton X-100
    将ddH 2 O添加到最终体积为100 ml 最终浓度:0.25M蔗糖,10mM Tris-HCl pH 7.4,10mM MgCl 2,0.5%Triton X-100
  5. 洗涤液
    溶于50ml的ddH 2 O -/- 加入1ml 1M Tris-HCl pH 7.4,1ml 1M MgCl 2
    将ddH 2 O添加到最终体积为100 ml
  6. 悬架解决方案
    溶解在40ml的ddH 2 O中 加入50μl的1M MgCl 2,并用ddH 2 O→
    达到50ml终体积 最终浓度:0.25M蔗糖,1mM MgCl 2
  7. 高离子强度5x反应混合物
    2.5 ml 1M Tris-HCl pH 8.0(参见方法2)
    0.1ml 1M MnCl 2(参见试剂18)
    2.0 ml 3 M硫酸铵(见配方3)
    0.4ml MilliQ H 2 O O
    酶测定中的最终浓度:0.1M Tris-HCl(pH 8.0),4mM MnCl 2,0.24M硫酸铵
  8. 核苷三磷酸盐混合物
    1. 非放射性溶液:
      溶解于2.5毫升MilliQ H 2 O -/-
    2. 放射性解决方案:
      50μCi的 3 将H-UTP稀释在
      中 0.5毫升MilliQ H< 2> O
    酶测定中的最终浓度:0.5mM GTP,0.5mM CTP,0.5mM ATP,0.02mM UTP和0.5μCi H-UTP
  9. α-amanitin重组
    1毫克/毫升α-淀粉酶水溶液 储存于-20°C 注意:您可以存储很长时间。它不会随着反复冻融循环而退化。
  10. 10%(w/w)TCA
    在100g ddH 2 O中稀释10g浓缩的TCA(≥99%w/w)
  11. 0.6 N PCA
    稀释2.9ml 65%(w/w)高氯酸在50ml ddH 2 O中的溶液。
  12. 0.3 N KOH
    将0.842g KOH溶解在50ml ddH 2 O中
  13. 6 N PCA
    将29ml 65%(w/w)PCA稀释在50ml ddH 2 O中,
  14. 7%(w/w)PCA
    在50g ddH 2 O中稀释5.39g的65%(w/w)PCA。
  15. 0.5 M NaF
    将0.3g NaF溶解在10ml MilliQ H 2 O
  16. 低离子强度5x反应混合物
    2.5 ml 1M Tris-HCl(pH 8.0)(参见方法2)
    0.1 ml 1M MgCl 2(见试剂16)
    0.3 ml 0.5 M NaF(见配方15)
    2.082ml MilliQ H 2 O
    酶测定中的最终浓度:0.1M Tris-HCl(pH 8.0),4mM MgCl 2,50μM2-巯基乙醇,6mM NaF


这个协议是从Novello和Stirpe的研究文章(1970)改编的。 Roberto和Cornelia Pallotti的遗传癌症研究得到了支持。作者感谢Massimo Derenzini教授有机会描述这个协议,并提供帮助和建议。作者还感谢Christine M. Betts博士对手稿的修订。作者声明没有利益冲突。


  1. Derenzini,M.,Montanaro,L.,Chilla,A.,Tosti,E.,Vici,M.,Barbieri,S.,Govoni,M.,Mazzini,G.and Trere,D。(2005) 实现G1-的适当核糖体RNA补体的关键作用H4-II-E-C3大鼠肝癌细胞中的S期转变。细胞生理学202(2):483-491。
  2. Donati,G.,Bertoni,S.,Brighenti,E.,Vici,M.,Treré,D.,Volarevic,S.,Montanaro,L。和Derenzini,M。(2011)。 rRNA和核糖体蛋白质合成之间的平衡上调和下调哺乳动物细胞中的肿瘤抑制基因p53 。 Oncogene 30:3274-3288。
  3. Montanaro,L.,Trere,D。和Derenzini,M.(2013)。< a class ="ke-insertfile"href =""目标="_ blank"> RNA聚合酶I转录机制在人类恶性肿瘤中的新兴作用:临床观点 Onco Targets Ther 6:909-916。
  4. Munro,HN和Fleck,A.(1966)。生物材料中核酸测量的最新进展。补充审查分析员 91(79):78-88。
  5. Novello,F.和Stirpe,F.(1970)。< a class ="ke-insertfile"href =""target ="_ blank" >使用α-淀粉样蛋白从静息和生长的大鼠肝脏分离的核中同时测定RNA聚合酶I和II。 8(1):57-60。 />
  6. Scala,F.,Brighenti,E.,Govoni,M.,Imbrogno,E.,Fornari,F.,Treré,D.,Montanaro,L。和Derenzini,M。(2016)。由rRNA合成抑制药物和细胞核糖体诱导的p53稳定性水平之间的直接关系生物发生率。癌基因 35(8):977-989。
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引用:Govoni, M. (2017). In vitro Assessment of RNA Polymerase I Activity. Bio-protocol 7(3): e2120. DOI: 10.21769/BioProtoc.2120.