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Oct 2019
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Transcriptional Run-on: Measuring Nascent Transcription at Specific Genomic Sites in Yeast
连缀转录:测定酵母特定基因组位点的新生转录   

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Abstract

DNA transcription by RNA polymerases has always interested the scientific community as it is one of the most important processes involved in genome expression. This has led scientists to come up with different protocols allowing analysis of this process in specific locations across the genome by quantitating the amount of RNA polymerases transcribing that genomic site in a cell population. This can be achieved by either detecting the total number of polymerases in contact with that region (i.e., by chromatin immunoprecipitation (ChIP) with anti-RNA polymerase antibodies) or by measuring the number of polymerases that are effectively engaged in transcription in that position. This latter strategy is followed using transcription run-on (TRO), also known as nuclear run-on (NRO), which was first developed in mammalian cells over 40 years ago and has since been adapted to many other different organisms and high-throughput methods. Here, we detail the procedure for performing TRO in Saccharomyces cerevisiae for single genomic regions to study active transcription on a single gene scale. To do so, we wash the cells in the detergent sarkosyl, which prevents new initiations at the promoter level, and then perform an in situ reaction, leading to the radiolabeling of transcripts by RNA polymerases that were already engaged in transcription at the moment of harvesting. By subsequently quantitating the signal of these transcripts, we can determine the level of active transcription in a single gene. This presents a major advantage over other forms of transcription quantitation such as RNA polymerase ChIP, since in the latter, both active and inactive polymerases are measured. By combining both ChIP and TRO, the amount of inactive or paused polymerases on a particular gene can be estimated.


Graphic abstract:



Transcriptional run-on scheme


Keywords: Run-on (连缀), Nascent transcription (新生转录), Nascent RNA (新生RNA), RNA polymerase II (RNA聚合酶II), Saccharomyces cerevisiae (酿酒酵母), Yeast (酵母)

Background

Transcription is an essential step in gene expression. It is a highly regulated process during which an RNA molecule is produced from a template DNA sequence by the action of RNA polymerases. Many scientists are interested in the complex process of transcription, which still remains to be fully understood and many others need to quantitate gene transcription in a specific region of the genome for practical purposes. Thus, several methods have been developed over the years to study eukaryotic transcription (reviewed in Pérez-Ortín et al., 2012).


Each developed method reveals different aspects of the transcription process. In this method article, we detail the protocol for transcription run-on (TRO), also known as nuclear run-on (NRO), which is particularly useful for detecting RNA polymerases that are engaged in active transcription at the moment of the experiment (Smale, 2009). The addition of sarkosyl detergent permeabilizes cells and prevents new initiations of transcription at the promoter level. Therefore, once new substrates (NTPs) are provided for the transcription reactions, only previously fully engaged RNA polymerases will act, incorporating a radiolabeled analog of UTP into the newly produced nascent RNA. This radiolabeling then allows for the detection of elongating RNA polymerases by hybridizing the nascent RNA onto nylon membranes that contain immobilized DNA fragments corresponding to the genomic sites of interest.


TRO was introduced over 40 years ago and has since experienced many upgrades for its use in different organisms and high-throughput sequencing (Greenberg and Ziff, 2004). The first attempt to take TRO genome-wide in yeast was the Genomic Run-On (GRO) method developed in 2004 (Garcia-Martinez et al., 2004), in which the labeled RNAs are hybridized to arrays. The protocol was then adapted to become high-resolution (Bio-GRO) and high-throughput (GRO-seq) (Core et al., 2008; Jordán-Pla et al., 2016 and 2019).


Here, we describe in detail the TRO method developed for Saccharomyces cerevisiae used in one of our recent publications (Corzo et al., 2019), which detects active RNA polymerases in single genes.

Materials and Reagents

  1. Whatmann filter paper, 180 µm thickness (Macherey-Nagel, catalog number: 742213)

  2. 1.5 ml tubes (Eppendorf, catalog number: 22 36 411-1)

  3. 50 ml and 15 ml Falcon tubes (Eppendorf, catalog numbers: 0030122178 and 0030122151)

  4. FastPrep 2 ml Lysing Matrix tubes (MP Biomedicals, catalog number: 115076200-CF)

  5. ProbeQuantTM G-50 Micro Column (GE28-9034-08)

  6. Saran wrap

  7. Acetate film

  8. Glass beads (Sigma, catalog number: G9268) washed in acid following the manufacturer’s protocol

  9. HCl 37% (Sigma, catalog number: 320331)

  10. Pfu DNA polymerase (Promega, catalog number: M7741)

  11. QIAquick PCR Purification Kit (Qiagen, catalog number: 28104)

  12. QIAquick Gel Extraction Kit (Qiagen, catalog number: 28115)

  13. Yeast DNA Extraction Kit (Thermo Fisher, catalog number: 78870)

  14. E. coli gDNA extraction (see He, 2011)

  15. Hybond-N membrane (Amersham, catalog number: RPN303N)

  16. NTP Set 100 mM (Invitrogen, catalog number: R0481)

  17. Sarkosyl N-lauroylsarcosina (Sigma, catalog number: L5125)

  18. DTT (Roche, catalog number: 11583786001)

  19. α-32P UTP (Perkin-Elmer, catalog number: NEG507H001MC, 10 µCi/µl)

  20. Cold MilliQ water

  21. Acid phenol pH 4.3 (Sigma, catalog number: P4682)

  22. Sodium acetate (Sigma, catalog number: 127-09-3)

  23. 100% ethanol (JT Baker, catalog number: 8025)

  24. Chloroform (Sigma, catalog number: C2432)

  25. NaOH pellets (Sigma, catalog number: 221465)

  26. Random hexamers 50 µM (Invitrogen, catalog number: N8080127)

  27. dNTP set (100 mM) (Invitrogen, catalog number: 10297018)

  28. Klenow fragment (NEB, catalog number: M0212S)

  29. α-32P dCTP (Perkin-Elmer, catalog number: NEG513H250UC, 10 µCi/µl)

  30. NaCl (Labotaq, catalog number: SO0227005P)

  31. Sodium citrate (Prolabo, catalog number: 27833.363)

  32. MgCl2 (Prolabo, catalog number: 25108295)

  33. EDTA (Sigma, catalog number: E5134-1KG)

  34. SDS (Amresco, catalog number: 0227)

  35. Sodium dihydrogen phosphate monohydrate (Sigma, catalog number: 10049-21-5)

  36. Di-sodium hydrogen phosphate (Sigma, catalog number: 7558-79-4)

  37. Potassium phosphate (Merck, catalog number: 1048731000)

  38. Tris base (Sigma, catalog number: T1503-10KG)

  39. Boric acid (Panreac, catalog number:131015.1211)

  40. Yeast extract (Pronadisa, catalog number: 1702)

  41. Glucose (Prolabo, catalog number: 24379.363)

  42. Agarose low EEO (Sigma, catalog number: A0576-100G)

  43. Red safe (Labotaq, catalog number: 8014)

  44. DNA 1 Kb ladder (Invitrogen, catalog number: 10787-026)

  45. DNA gel loading dye 6× (Thermo Scientific, catalog number: R0611)

  46. Denaturalization buffer (see Recipes)

  47. Neutralization buffer (see Recipes)

  48. SCC 20× (see Recipes)

  49. Transcription buffer 2.5× (see Recipes)

  50. TES (see Recipes)

  51. Hybridization solution (see Recipes)

  52. Wash solution I (see Recipes)

  53. Wash solution II (see Recipes)

  54. Neutralization solution (stripping) (see Recipes)

  55. Stripping solution (see Recipes)

  56. 10× TBE buffer (see Recipes)

  57. Tris-HCl, pH 7 (see Recipes)

  58. Phosphate buffer, pH 7 (see Recipes)

Equipment

  1. Slot Blot blotting manifold (Hoefer Scientific, catalog number: PR648)

  2. Thermocycler (Bio-Rad, catalog number: T100)

  3. NanoDrop (Thermo Scientific, catalog number: 840274100)

  4. Thermoblock heater (Labnet)

  5. Vacuum pump

  6. Spectrophotometer (Eppendorf, catalog number: EP6135000923)

  7. Falcon centrifuge fixed rotor (Eppendorf, model: 5810R), max speed 3,220 × g

  8. Microcentrifuge fixed rotor (Eppendorf, model: 5424), max speed 17,000 × g

  9. UV crosslinker (Stratagene, model: UV Stratalinker 1800)

  10. FastPrep-24TM 5G instrument (MP Biomedicals, catalog number: 116005500)

  11. Hybridization oven (UVP, model: HB-1000 Hybridizer)

  12. PhosphoImager screen and cassette (FUJIFILM BAS)

  13. STORM-840 imaging system (GE Healthcare)

  14. Vortex mixer (Jencons, model: VX100), use at max speed

  15. Geiger counter (Thermo, model: Mini 900)

  16. Microwave oven

  17. Orbital shaker (Appleton Woods Stuart), slow shaking at about 50 rpm

  18. Heat-resistant gloves

  19. Electrophoresis chamber and power supply (Bio-Rad, catalog number: 1640300)

  20. Gel casting tray and comb (Bio-Rad)

Software

  1. GelQuant.NET software (http://biochemlabsolutions.com/GelQuantNET.html)

Procedure

Notes:

  1. The procedure uses 32P; appropriate equipment, facilities, and training for radioactive work is needed.

  2. Filter tips should be used when manipulating radioactivity or RNA.

    The whole protocol takes 10 days to complete without stopping. The membranes and DNA fragments of interest, yeast and E. coli gDNA, and yeast cell pellets can be made and stored in advance. Mayor stopping points would be after membrane preparation (A); after labeled RNA hybridization, exposition, and scanning (B-D); and after labeled gDNA hybridization, exposition, and scanning (F).


  1. Preparing the membranes

    1. Treat the Slot Blot (SB) blotting manifold with 0.1 M HCl for 1 h to eliminate any contaminants. Wash with distilled H2O abundantly and leave to dry in a fume hood on top of a clean filter paper to avoid contamination.

    2. Prepare the DNA fragments corresponding to the genes of interest (i.e., the genes you would like to check are being actively transcribed and would like to quantitate) to be loaded onto the membrane:

      1. Design PCR oligos to obtain fragments of DNA of around 300 bp to be analyzed.

      2. Perform PCR reactions using Pfu DNA polymerase to ensure fidelity of the DNA product (following the manufacturer’s protocol) and to obtain 100 ng DNA per membrane (usually 3-4 50 µl PCR reactions for 12 membranes). Template yeast gDNA can be isolated using a kit.

      3. Combine the PCR reactions of the same DNA fragment, purify using QIAquick PCR purification columns according to the manufacturer’s protocol, and measure the DNA concentration using a NanoDrop. Check the sizes on a 1% agarose gel by loading approximately 500 ng DNA measured by the NanoDrop to ensure there are no other non-specific bands at a size different from that expected (~300 bp). If there are other bands, you will need to perform an extra step to purify the 300 bp band from the gel by loading all your DNA on a 1% agarose gel, cutting the 300 bp band, and purifying using QIAquick Gel extraction columns. DNA can be stored for up to 1 year at -20°C.

      4. Apart from your PCR DNA of interest, we recommend that you include a negative control consisting of DNA from another organism (e.g., E. coli genomic DNA) and a positive control consisting of genomic DNA from the organism you are using (e.g., yeast genomic DNA).

      5. Mix 100 ng DNA and 60 μl SSC 20× (final concentration of 6×), and add MilliQ H2O up to a final volume of 200 μl per sample and membrane.

      6. Boil DNA at 95°C on a thermoblock for 10 min and immediately place on ice.

    3. Mount the SB blotting manifold (see Figure 1):

      1. Connect the lower fitting to the vacuum pump to draw samples through the SB.

      2. Place the middle block of the SB on top of the bottom block with the red rubber facing downward.

      3. Dampen an 11 × 3 cm Whatman in 6× SSC and place it on top of the SB.

      4. Carefully place an 11 × 3 cm Hybond N membrane on top, with the top left corner cut in order to know the orientation of the membrane.

      5. Place the top block of the SB on top of this, making sure that the number 1 and letter A of the SB are also on the top left, and screw in the red screws bit by bit, making sure all screws are tightened at the same time. Be careful not to tighten too hard so as not to make indentations in the membrane.

      6. Turn on the vacuum pump (use at maximum setting).



      Figure 1. The Hoefer PR648 Slot Blot (SB) set up for standard use (taken from the Hoefer PR648 manual)


    4. Blotting:

      1. Add 500 μl 6× SSC to each well and make sure the liquid is being correctly sucked through the membrane and Whatmann paper.

      2. Wait until the wells are dry.

      3. Add each boiled DNA sample, previously placed on ice, into the assigned well, being careful not to touch the sides of the well, and wait until it dries.

      4. Once all the DNA has been sucked through the SB, turn the vacuum pump off and unscrew the SB.

      5. Dampen a 12 × 4 cm Whatmann in denaturalization buffer and carefully place the membrane on top, with the DNA facing upward. Incubate at room temperature for 10 min.

      6. Repeat the previous process (4e) using neutralization buffer, but incubate for 5 min.

      7. Place the membrane on a dry Whatmann and label it with a pencil.

      8. Fix the DNA to the membrane by UV-crosslinking at 70000 μJ/cm2. Membranes can now be stored for up to a year after fully drying them out between Whatmann filter paper.


    Note: From Step B9 onward, you need to work in a radioactive facility.

  2. Run-on reaction

    1. Grow your yeast strains in 25-ml YPD cultures or similar until they reach an OD600nm of 0.5 from a dilution of a small 5-ml culture grown overnight. Make sure you have the same volume of cells across samples.

    2. Centrifuge at 3,220 × g for 3 min at RT and discard the supernatant. At this point, you can flash freeze your cells in liquid nitrogen and store the pellets in a -80°C freezer for up to 1 month.

    3. Prepare the transcription mix: 60 μl transcription buffer, 8 μl NTPs (A,C,G) at 10 mM each, and 3 μl 0.1 M DTT per sample. Do not add UTP. Place on ice until use.

    4. Resuspend the cell pellets in 5 ml 0.5% sarkosyl.

    5. Centrifuge at 1,800 × g for 3 min at RT and discard the supernatant carefully.

    6. Resuspend the pellet in 1 ml 0.5% sarkosyl and move to a 1.5-ml Eppendorf tube.

    7. Centrifuge at 700 × g for 4 min at RT and discard the supernatant using a pipette.

    8. Centrifuge the samples again at 700 × g and RT for 1 min to remove any remaining supernatant.

    9. Resuspend in 60 μl MilliQ H2O and incubate at 30°C until the samples are warm.

    10. Add 8 μl α-32P UTP (Perkin-Elmer NEG507H001MC, 10 µCi/µl) per sample to the transcription mix and place at 30°C to warm up.

    11. Once the samples and transcription mix are warm (around 5 min at 30°C), add 79 μl transcription mix to each sample and incubate for exactly 5 min at 30°C. To achieve this, it is best to leave 30 s between samples.

    12. After the 5 min incubation, quickly add 800 μl cold MilliQ H2O and place on ice to stop the reaction.

    13. Centrifuge at 3,500 × g for 1 min at 4°C if possible (if not, at RT) and discard the supernatant. At this point, you can stop and freeze the samples at -20°C before continuing. Samples should be used as soon as possible since the 32P will decay over time. Use within a few days.


  3. RNA isolation

    Note: A commercially available RNA isolation kit could be used instead.

    1. Resuspend the pellet in 400 μl TES and transfer to a FastPrep tube containing 400 μl glass beads and 400 μl acid phenol.

    2. Break the cells using the FastPrep (3 cycles of 30 s at 5 m/s) at RT.

    3. Centrifuge for 5 min at 16,200 × g (if possible at 4°C; if not, at RT).

    4. Transfer the supernatant to an Eppendorf tube and add 400 μl chloroform.

    5. Centrifuge for 5 min at 16,200 × g (if possible at 4°C; if not, at RT).

    6. Transfer the supernatant to a fresh Eppendorf tube and add 1 ml 100% ethanol and 40 μl sodium acetate 3M pH 5.5.

    7. Precipitate RNA overnight at -20°C.

    8. The next day, centrifuge at 16,200 × g for 15 min at 4°C if possible (if not, at RT). In the meantime, start pre-hybridizing the prepared membranes (step D1) so that the labeled RNA can be used immediately.

    9. Discard the supernatant.

    10. Wash with 1 ml 70% ethanol and centrifuge at 16,200 × g for 5 min at 4°C if possible (if not, at RT).

    11. Discard the supernatant, then centrifuge again and eliminate any leftover ethanol.

    12. Dry the pellet at room temperature for 5-10 min.

    13. Add 100 μl MilliQ H2O and incubate at 65°C (for approximately 5 min), vortexing the sample every 30 s until all the RNA has been resuspended.

    14. Add 25 μl NaOH 0.2 N and incubate for 5 min on ice. This step fragments the RNA and helps it to hybridize with the DNA on the membranes.

    15. Neutralize by adding 25 μl HCl 0.2 N.


  4. Hybridization

    1. Pre-hybridize each membrane by incubating at 65°C inside a 15-ml Falcon tube containing 5 ml hybridization solution for at least 1 h (the longer, the better). Make sure you place the membrane with the fixed DNA toward the inside of the tube and try to minimize overlapping of the membrane. A 50-ml Falcon tube can be used for larger membranes so that they do not overlap. For this and the following steps, a hybridization oven was used; the Falcon tubes where inserted into a glass tube and rotated. If you have only a few membranes, you can use the glass tube directly. Make sure you screw the lids on well to prevent leakage.

    2. Once the RNA is ready, discard the hybridization solution and add 3 ml fresh hybridization solution and the labeled RNA.

    3. Incubate at 65°C for 48 h while rotating.

    4. Reuse or save the labeled RNA by storing at 4°C. Reuse within 1 week since you will lose the signal over time. When reusing, heat the solution in the hybridization oven to ensure that it liquifies.

    5. Rinse the membrane in 5 ml wash solution I. Discard the solution in an appropriate radioactive recipient.

    6. Add 5 ml fresh wash solution I and incubate for 20 min at 65°C while rotating.

    7. Discard the solution, add 5 ml wash solution II, and incubate for 10 min at 65°C.

    8. Repeat the previous step.

    9. Carefully take each membrane out of the Falcon tube and remove the excess liquid using tissue paper before placing it with the fixed DNA side facing down on a piece of Saran wrap. Place a previously cut 12 × 4 cm acetate film on top and wrap the Saran wrap around the membrane, making sure that no creases are formed.

    10. Place the wrapped membrane in a cassette with a Fuji screen and expose for around 72 h at RT (the Geiger should indicate 2-5 cps per membrane). Make sure that the DNA-fixed side of the membrane is facing up and is in contact with the screen. We recommend placing the membranes with the cut edge at the top left-hand side.

    11. After scanning the Fuji screen, the membranes must be stripped and hybridized a second time with genomic DNA to normalize the signal.


  5. Stripping

    1. Incubate each membrane in 5 ml 50 mM NaOH in a 15-ml Falcon tube for 30 min at 45°C inside a hybridization oven while rotating.

    2. Discard the NaOH and neutralize the membranes by adding 5 ml neutralization solution for an extra 15 min under the same conditions.

    3. Transfer the membranes to a single glass tray that can hold 100 ml stripping solution previously boiled in a microwave. Caution when manipulating the boiling solution; use heat resistant gloves.

    4. Incubate the membranes in boiling stripping solution at room temperature for 10 min on an orbital shaker at slow speed to ensure no loss of liquid.

    5. Wash the membranes once with distilled H2O and leave to dry at RT on a clean filter paper; save them or proceed to the next step. Measure the membranes with a Geiger counter to make sure the radioactive signal has disappeared.

    6. When they are ready to be re-hybridized, transfer the stripped membranes into a fresh 15-ml Falcon tube and pre-hybridize (Step D1).


  6. Radioactive labeling of genomic DNA

    1. Extract yeast genomic DNA using your preferred method (a kit can be used) and dilute 1 μg in a final volume of 37 μl MilliQ H2O.

    2. Boil for 5 min at 95°C on a thermoblock and immediately transfer to ice. Add 5 μl random hexamers, 5 μl each 10 mM dNTP (A, T, G), 1 μl Klenow fragment, and 5 μl α-32P dCTP (Perkin-Elmer NEG513H250UC, 10 µCi/µl).

    3. Incubate at 37°C for 45 min on a thermoblock.

    4. Purify the labeled genomic DNA using a ProbeQuantTM G-50 Micro Column following manufacturer’s protocol.

    5. If to be used immediately, boil the labeled DNA at 95°C for 5 min; and if not, freeze at -20°C. Labeled DNA should be used as soon as possible since 32P decays over time. Use within a few days.

    6. The labeled DNA obtained will be sufficient for 12 membranes. Dilute the obtained DNA with MilliQ H2O until 20 μl per membrane is obtained. For example, if you have 12 membranes, you need to add 190 μl MilliQ H2O to the resulting 50 μl labeled DNA to obtain 20 μl DNA per membrane.

    7. Add 3 ml fresh hybridization solution and 20 μl diluted labeled DNA to a 15-ml Falcon containing the membrane, and hybridize for 24 h at 65°C.

    8. Follow the same steps as above (steps in Procedure D).

    9. Expose the membrane overnight or longer depending on the signal obtained (48 h if the Geiger indicates 5-10 cps).


  7. Scanning the membranes and quantitation

    1. Scan the Fuji screens using a STORM-840 imaging system or similar.

    2. Be careful to remember the order of the membranes and to not let the Fuji screen be exposed to light for long, since light erases the screen.

    3. If the signal observed is not high enough, you can re-expose the screen for longer.

    4. Quantitate the radioactive signal using the free GelQuant.NET software (http://biochemlabsolutions.com/GelQuantNET.html) or similar.

    5. Normalize the RNA radioactivity intensity to the DNA radioactivity intensity.



      Figure 2. Results from a typical TRO experiment. A. Membrane after labeled RNA hybridization. Both top and bottom membranes have the same DNA fragments crosslinked on them: several fragments of the pGAL1-YLR454w gene construct, positive (yeast gDNA) and negative (E. coli gDNA) controls, and several fragments of the GAL1 gene. The top membrane was hybridized with labeled RNA from WT yeast cells and the bottom from mutant (Mut) yeast cells. The mutant in this example corresponds to pdf1Δ. B. The same membranes shown in A were stripped and re-hybridized with labeled yeast gDNA. C. Graph showing the quantitation of the results shown in A and B.

Data analysis

  1. The phosphoimages taken by STORM or a similar imaging system should be in .gel format.

  2. First, we need to quantitate the labeled RNA membranes (as seen in Figure 2A), load the image into the GelQuant.NET program (see Figure 3), and adjust the contrast by pressing the .GEL scale button. Afterwards, rotate and center the image using the “Rotate 90” and zoom buttons.

  3. Next, proceed to quantitate each line by drawing a small red square around the line in question. Then, press the control button on the bottom panel to ensure that you use the same sized square for all the lines, and press the quantify button. A number should appear on the right-hand side, which corresponds to the quantitation of the square minus the background. We usually use the automatic local background option (bottom right), but you can determine your own background by clicking on the manual background option.

  4. Repeat this on the next line by dragging the fixed red square over it and pressing quantify; repeat until you have quantitated every relevant line (the positive and negative controls do not need to be quantitated, they just need to be visible or absent, respectively).

  5. On the top right, you will see a series of numbers corresponding to the volume quantitation of your lines. You can copy these numbers into a clipboard by selecting the box under the numbers, highlighting the numbers that you want to copy, and pressing the clipboard button at the bottom. Then, copy these numbers into an Excel sheet.

  6. Repeat the steps 2-5 for the DNA-labeled membranes (as seen in Figure 2B). These numbers should be more similar to each other.

  7. Once you have all the numbers for each fragment and each membrane in Excel, you can now finish analyzing them. First, normalize the RNA membranes by the number of uridines (or thymines) in each DNA fragment loaded onto the membranes, as this can affect the amount of labeled signal obtained. Then, normalize the RNA numbers to the DNA membranes. Finally, normalize them to the number of cells harvested in each sample using the O.D. numbers.

  8. We repeat each experiment 3 times and perform a Student’s t-test on each fragment in the WT with respect to the mutant to identify significant changes in active RNA polymerases on each DNA fragment studied.



    Figure 3. Screenshot of the GelQuant program used to quantitate the hybridized labeled RNA signal. At the top-right, the .GEL scale, zoom, brightness, and contrast buttons can be found. At the top, the open, save as, rotate, etc buttons can be found. At the bottom, buttons to draw the red quantitation square (z) can be found, and next to it, the control button that is selected to allow the same red square to be dragged over the different bands for quantitation. The quantitate button is next to it, which when selected, will give numbers on the right-hand side of the page. At the bottom-right, the auto local background option can be found, and above this is the clipboard option.

Recipes

Note: Autoclave all stock solutions to sterilize.

  1. Denaturalization buffer

    1.5 M NaCl

    0.5 M NaOH

  2. Neutralization buffer

    1 M NaCl

    0.5 M Tris-HCl, pH 7

  3. SSC 20×

    150 mM NaCl

    20 mM Na citrate

    Add HCl until pH 7

  4. Transcription buffer 2.5×

    550 mM Tris-HCl, pH 7

    500 mM KCl

    80 mM MgCl2

  5. TES

    10 mM Tris-HC,l pH 7.5

    10 mM EDTA, pH 8

    0.5% SDS

  6. Hybridization solution

    0.5 M phosphate buffer, pH 7

    7% SDS, 0.5 M EDTA pH 8

  7. Wash solution I

    1× SSC

    0.1% SDS

  8. Wash solution II

    0.5× SSC

    0.1% SDS

  9. Neutralization solution (stripping)

    550 mM Tris-HCl, pH 7

    0.1× SSC

    0.1% SDS

  10. Stripping solution
    55 mM potassium phosphate, pH 7

    0.1% SDS

  11. 10× TBE buffer

    13 M Tris base

    450 mM boric acid

    25 mM EDTA, pH 8

  12. Tris-HCl 1M, pH 7

    1 M Tris base

    add HCl until pH 7

  13. Phosphate buffer 1 M, pH 7

    99.4 g Na2HPO4

    41.4 g NaH2PO4·H2O

    Check pH is at 7

Acknowledgments

This work was funded by the Spanish Ministry of Economy and Competitiveness, and European Union funds (FEDER) [BFU2016-77728-C3-1-P].

The original research article in which this protocol was used is Begley et al. (2019).

Competing interests

None declared.

References

  1. Begley, V., Corzo, D., Jordan-Pla, A., Cuevas-Bermudez, A., Miguel-Jimenez, L., Perez-Aguado, D., Machuca-Ostos, M., Navarro, F., Chavez, M. J., Perez-Ortin, J. E. and Chavez, S. (2019). The mRNA degradation factor Xrn1 regulates transcription elongation in parallel to Ccr4. Nucleic Acids Res 47(18): 9524-9541.
  2. Core, L. J., Waterfall, J. J. and Lis, J. T. (2008). Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters.Science 322(5909): 1845-1848.
  3. Garcia-Martinez, J., Aranda, A. and Perez-Ortin, J. E. (2004). Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms. Mol Cell 15(2): 303-313.
  4. Greenberg, M. E. and Ziff, E. B. (1984). Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature 311(5985): 433-438.
  5. He, F. (2011). E. coli Genomic DNA Extraction. Bio-protocol 1: e97.
  6. Jordan-Pla, A., Miguel, A., Serna, E., Pelechano, V. and Perez-Ortin, J. E. (2016). Biotin-Genomic Run-On (Bio-GRO): A High-Resolution Method for the Analysis of Nascent Transcription in Yeast. Methods Mol Biol 1361: 125-139.
  7. Jordan-Pla, A., Perez-Martinez, M. E. and Perez-Ortin, J. E. (2019). Measuring RNA polymerase activity genome-wide with high-resolution run-on-based methods. Methods 159-160: 177-182.
  8. Perez-Ortin, J. E., de Miguel-Jimenez, L. and Chavez, S. (2012). Genome-wide studies of mRNA synthesis and degradation in eukaryotes. Biochim Biophys Acta 1819(6): 604-615
  9. Smale, S. T. (2009). Nuclear run-on assay.Cold Spring Harb Protoc 2009(11): pdb prot5329.

简介

[摘要] RNA 聚合酶的 DNA 转录一直受到科学界的关注,因为它是基因组表达中最重要的过程之一。这使得科学家们想出了不同的协议,允许analys是这个过程中通过孔定量整个基因组的特定位置达荷兰国际集团的RNA聚合酶转录该基因组位点在细胞群的量。这可以通过检测与该区域接触的聚合酶总数(即通过染色质免疫沉淀 ( ChIP ) 与抗 RNA 聚合酶抗体)或通过测量在该位置有效参与转录的聚合酶数量来实现. 这后一种策略埃及随后使用转录运行的(TRO),也被称为(NRO)的核上运行,这是首次在哺乳动物细胞中超过40年前开发并一直以来被改编到许多其他不同的生物体和高通量方法。在这里,我们详细介绍了在酿酒酵母中对单个基因组区域进行 TRO以研究单个基因规模上的活性转录的过程。Ť ø这样做,我们洗细胞在洗涤剂十二烷基肌氨酸钠,其防止新灌顶启动子水平的,和然后执行原位反应,铅ING由那些已经在此刻从事转录的RNA聚合酶转录物的放射性标记的收获。通过随后孔定量达荷兰国际集团这些转录的信号,我们可以判断的转录活性的单个基因的水平。这呈现出优于其他形式的转录的主要优点定量如RNA聚合酶的ChIP ,因为在LAT吨ER ,活动和非活动的聚合酶被测量。通过结合ChIP和 TRO,可以估计特定基因上失活或暂停的聚合酶的数量。

图文摘要:

转录连续方案

【背景】转录是基因表达的重要步骤。这是一个高度受管制的过程,在此过程中,RNA 分子通过RNA聚合酶的作用从模板 DNA 序列中产生。许多科学家感兴趣的是转录的复杂过程,这还有待充分的理解和许多其他需要QUANTI泰特基因转录基因组中的实际用途的特定区域。因此,多年来已经开发了几种方法来研究真核转录(Pérez-Ortín等人综述,2012 年)。

每种开发的方法都揭示了转录过程的不同方面。在这篇方法文章中,我们详细介绍了转录连续 (TRO) 的协议,也称为核连续 (NRO),它特别适用于检测在实验过程中参与主动转录的 RNA 聚合酶(斯梅尔,2009)。的一个的ddition肌氨酰洗涤剂透化细胞,并防止转录新灌顶启动子水平。因此,一旦为转录反应提供了新的底物 (NTP) ,只有先前完全参与的 RNA 聚合酶才会起作用,将放射性标记的 UTP类似物整合到新产生的新生 RNA 中。这种放射性标记然后允许该检测由杂交的新生RNA上含有对应于感兴趣的基因组位点固定的DNA片段的尼龙膜伸长RNA聚合酶。

TRO在40年前推出,并具有自其在不同的生物体和高通量测序使用经验的多次升级(格林伯格和齐夫,2004年)。第一次尝试采取TRO基因组-宽在酵母是在2004年开发的基因组运行接通(GRO)方法(加西亚-马丁内斯等人,2004。 ),其中所述标记的RNA杂交到阵列。然后将该方案调整为高分辨率(Bio-GRO)和高通量(GRO- seq )(Core et al ., 20 08; Jordán-Pla et al ., 2016 and 2019)

在这里,我们详细描述了为Saccharomyces cerevisiae开发的 TRO 方法,该方法用于我们最近的出版物之一(Corzo等人,2019 年),该方法检测单个基因中的活性 RNA 聚合酶。

关键字:连缀, 新生转录, 新生RNA, RNA聚合酶II, 酿酒酵母, 酵母



材料和试剂


1.的Whatmann ˚F ILTER p纸张,180层μ米厚度(马歇雷- Ñ AGEL,目录号:742213)     

2. 1.5 ml 管(Eppendorf,目录号:22 36 411-1)     

3. 50 ml 和 15 ml Falcon 管(Eppendorf,目录号:0030122178 和 0030122151)     

4. FastPrep 2 ml Lysing Matrix管(MP Biomedicals ,目录号:115076200-CF)     

5. ProbeQuant TM G-50 微型柱 (GE28-9034-08)     

6.萨兰W¯¯说唱     

7.醋酸薄膜     

8.按照制造商的方案在酸中洗涤玻璃珠(Sigma,目录号:G9268)     

9. HCl 37%(Sigma,目录号:320331)     

10. Pfu DNA聚合酶(Promega ,目录号:M7741) 

11. QIA快速PCR纯化试剂盒(Qiagen公司,目录号:28104) 

12. QIAquick Gel Extraction Kit(Qiagen ,目录号:28115) 

13.酵母DNA Ë xtraction ķ它(热费舍尔,目录号:78870) 

14. E.大肠杆菌gDNA的抽行动(见他,2011 ) 

15. Hybond -N 膜(Amersham ,目录号:RPN303N) 

16. NTP Set 100 mM (Invitrogen,目录号:R0481) 

17. Sarkosyl N- lauroylsarcosina (Sigma ,目录号:L5125) 

18. DTT(罗氏,目录号:11583786001) 

19. α - 32 P UTP(Perkin-Elmer ,目录号:NEG507H001MC,10 µCi/µl) 

20.冷MilliQ水 

21.酸性苯酚pH 4.3(Sigma,目录号:P4682) 

22.醋酸钠(Sigma,目录号:127-09-3) 

23. 100%ë THANOL(JT贝克,目录号:8025) 

24.氯仿(Sigma ,目录号:C2432) 

25. NaOH颗粒(Sigma,目录号:221465) 

26.随机h检测器 50 µM (Invitrogen,目录号:N8080127) 

27. dNTP s et(100 mM )(Invitrogen,目录号:10297018) 

28.克列诺˚F ragment(NEB,目录号:M0212S) 

29. α - 32 P dCTP (Perkin-Elmer ,目录号:NEG513H250UC,10 µCi/µl) 

30. NaCl (Labotaq ,目录号:SO0227005P) 

31.柠檬酸钠(Prolabo ,目录号:27833.363) 

32. MgCl 2 (Prolabo ,目录号:25108295) 

33. EDTA(Sigma,目录号:E5134-1KG) 

34. SDS(Amresco ,目录号:0227) 

35.磷酸二氢钠一水合物(Sigma,目录号:10049-21-5) 

36.磷酸氢二钠(Sigma,目录号:7558-79-4) 

37.磷酸钾(默克,目录号:1048731000) 

38.的Tris b酶(Sigma,目录号:T1503-10KG) 

39.硼酸(Panreac ,目录号:131015.1211) 

40.酵母ë XTRACT(Pronadisa ,目录号:1702) 

41.葡萄糖(Prolabo ,目录号:24379.363) 

42.琼脂糖升流EEO(Sigma,目录号:A0576-100G) 

43.红色保险箱(Labotaq ,目录号:8014) 

44. DNA 1 Kb 阶梯(Invitrogen,目录号:10787-026) 

45. DNA凝胶加载染料6 × (Thermo Scientific,目录号:R0611) 

46.变性缓冲液(见食谱) 

47.中和缓冲液(见配方) 

48. SCC 20    ×(见食谱)


49.转录缓冲液 2.5 × (见配方) 

50. TES (见食谱) 

51.杂交溶液(参见配方小号) 

52.洗涤液 I (见配方) 

53.洗涤液 II (见配方) 

54.中和溶液(剥离)(见配方) 

55.剥离液(见配方) 

56. 10 × TBE 缓冲液(见配方) 

57. Tris-HCl ,pH 7(见配方) 

58.磷酸盐缓冲液,pH 7(见配方) 



设备


Slot Blot印迹歧管(Hoefer Scientific ,目录号:PR648)
热循环仪(Bio-Rad ,目录号:T100)
纳米d ROP (热科学,目录号:840274100)
Thermoblock加热器 ( Labnet )
真空泵
分光光度计(Eppendorf,目录号:EP6135000923 )
Falcon 离心机固定转子(Eppendorf,型号:5810R),最大速度 3,220 × g
微量离心机固定转子(Eppendorf,型号:5424),最大速度 17,000 × g
UV交联剂(Stratagene ,型号:UV Stratalinker 1800)
FastPrep-24 TM 5G我nstrument(MP Biomedicals公司,目录号:116005500)
杂交ø VEN(UVP ,型号:HB-1000混杂)
PhosphoImager屏幕和暗盒(FUJIFILM BAS)
STORM-840 成像系统(GE Healthcare)
涡流混合器(Jencons ,型号:VX100),以最大速度使用
盖革计数器(Thermo ,型号:Mini 900)
微波炉
Orbital s haker (Appleton Woods Stuart),以大约 50 rpm 的速度缓慢摇晃
热火-防护手套
电泳室和电源(Bio - R ad ,目录号:1640300)
凝胶浇铸托盘和梳子(Bio - R ad)


软件


GelQuant.NET 软件 ( http://biochemlabsolutions.com/GelQuantNET.html )


程序


笔记:


程序使用32 P ;适当的设备,设施,以及对放射性工作的培训需要。
处理放射性或 RNA 时应使用过滤器吸头。


整个协议需要 10 天才能完成而不会停止。第m embranes和感兴趣,酵母和DNA片段E.大肠杆菌的gDNA,和酵母细胞沉淀,可以制备和预先存储。市长停止点将在膜制备之后(A);后标记的RNA杂交,博览会,并扫描(B - d); 并经过标记的gDNA杂交、展示和扫描 (F)。


准备膜
用 0.1 M HCl处理Slot Blot (SB) 印迹歧管1小时以消除任何污染物。用蒸馏 H 2 O 大量清洗,并在干净的滤纸顶部的通风橱中晾干,以避免污染。
准备与感兴趣的基因相对应的 DNA 片段(即,您要检查的基因正在积极转录并要定量)以加载到膜上:
                 设计PCR的寡核苷酸,以获得约300的DNA片段碱基是ANALY ž版。
                执行应用Pfu DNA聚合酶,以确保DNA产物的保真度的PCR反应(按照制造商的协议)和以获得每单位膜(通常是3-4个50μl的PCR反应进行12膜)100毫微克DNA。模板酵母 gDNA 可以使用试剂盒进行分离。
                 结合相同的DNA片段的PCR反应中,使用纯化的QIAquick根据制造商的方案PCR纯化柱,并测量了使用DNA浓度Ñ ANO d ROP 。检查的在1%的尺寸琼脂糖凝胶通过加载由测量大约500纳克DNA的Ñ ANO d ROP到连接确保有在没有其他非特异性的条带大小不同的从塔吨EXPE反恐执行(〜300 bp)的。如果有其他条带,您需要执行额外的步骤,通过将所有 DNA 上样到 1% 琼脂糖凝胶上,切割 300 bp 条带,然后使用QIAquick Gel 提取柱进行纯化,以从凝胶中纯化300 bp 条带。
DNA 可在 -20 °C下保存长达1年。
                除了你的PCR DNA兴趣,我们建议是您包括其他生物组成的DNA(为阴性对照例如,大肠杆菌基因组DNA)和阳性对照从生物体由基因组DNA的您正在使用(例如,酵母基因组 DNA)。
                 混合 100 ng DNA和60 μl SSC 20 × (最终浓度为 6 × ),并添加MilliQ H 2 O 至每个样品和膜的最终体积为 200 μl 。
                  煮沸DNA在95℃下在热块10分钟,并立即p花边冰上。
安装 SB 印迹歧管(见图 1):
                 将下部接头连接到真空泵以通过 SB 抽取样品。
                将 SB 的中间块放在底部块的顶部,红色橡胶朝下。
                 在 6 × SSC 中浸湿 11 × 3 cm Whatman并将其放在 SB 的顶部。
                小心地将 11 × 3 厘米Hybond N 膜放在顶部,左上角切开以了解膜的方向。
                 将SB的顶块放在上面,确保SB的数字1和字母A也在左上角,一点一点拧入红色螺丝,确保所有螺丝都同时拧紧时间。小心不要拧得太紧,以免在膜上产生凹痕。
                  打开真空泵(在最大设置下使用)。




图 1. 标准使用的 Hoefer PR648 Slot Blot (SB) 设置(取自 Hoefer PR648 手册)


印迹:
                 加入500 μ升6 × SSC至每个孔,并确保液体被正确地通过膜和吸入的Whatmann纸。
                等到孔变干。
                 每个煮DNA样品添加,以前放在冰上,进入分配好,小心不要触及孔的两侧,并等待其干燥。
                一旦所有的DNA已通过SB吸入,打开真空泵出并拧开SB。
                 在变性缓冲液中浸湿12 × 4 cm Whatmann ,小心地将膜放在上面,DNA 朝上。在室温下孵育 10 分钟。
                  使用中和缓冲液重复前面的过程 (4e) ,但孵育 5 分钟。
                 将膜放在干燥的Whatmann上并用铅笔标记。
                固定DNA通过UV膜-在70000交联μ Ĵ /厘米2 。在Whatmann滤纸之间完全干燥后,膜现在可以储存长达一年。


ñ OTE :从步骤上B9病房,你需要工作放射性设施。


连续反应:
在 25 - ml YPD 培养物或类似物中培养酵母菌株,直到它们从过夜生长的5 - ml小培养物的稀释液中达到0.5的 OD 600nm 。请确保您有细胞相同体积的整个样本。
离心3 ,220 ×克在室温下3分钟,并弃去上清液。在这一点上,你可以刷冻结你的细胞在液氮中,并存储该粒料在-80 ℃的冰箱中长达1一个月。
准备转录组合:60 μ l转录缓冲液、8 μ l NTP(A、C、G),每个 10 mM ,每个样本3 μ l 0.1 M DTT。不要添加 UTP。置于冰上直至使用。
将细胞沉淀重悬在 5 ml 0.5%肌氨酰中。
离心机中以1 ,800 ×克在室温下3分钟,并弃去的仔细上清液。
重悬在1毫升0.5%的沉淀十二烷基肌氨酸钠和移动到1.5 -毫升Eppendorf管中。
离心700 ×克在室温下4分钟并弃去的上清液用移液管。
离心机的样品再次在700 ×克和RT下搅拌1分钟,以除去任何剩余的上清液。
重悬于60 μ升的MilliQ ħ 2 O和孵育在30 ℃下,直到所述样品是温暖。
将每个样品8 μ l α- 32 P UTP(Perkin-Elmer NEG507H001MC,10 µCi/µl)添加到转录混合物中,并置于 30 °C进行预热。
一旦所述样品和转录混合物是温暖的(30约5分钟℃下),加入79 μ升转录混合到每个样品中并孵育,精确至5分钟,在30 ℃下。为此,最好在样本之间留出 30 秒。
在5分钟后的孵育,迅速加入800 μ升冷的MilliQ ħ 2在冰上O和地方以停止反应。
离心3 ,500 ×克1分钟,在4 ℃下,如果可能的(如果不是,在RT)并弃去上清液。此时,您可以停止并在 -20 °C 下冷冻样品,然后再继续。样品应尽快使用,因为在32 P将随时间衰减。几天内使用。


RNA分离
Ñ OTE:甲市售RNA提取试剂盒可替代地使用。


重悬在400个粒料μ升TES并转移到一个FastPrep管含有400成μ升玻璃珠和400 μ升酸苯酚。
打破所述细胞使用的FastPrep (30级3个循环小号在室温在5米/秒)。
以 16,200 × g离心 5 分钟(如果可能,在 4 °C 下;如果没有,则在室温下)。
将上清转移到Eppendorf管中,并添加400 μ升氯仿。
以 16,200 × g离心 5 分钟(如果可能,在 4 °C 下;如果没有,则在室温下)。
转移的上清液至新鲜的Eppendorf管中,加入1ml的100%乙醇和40 μ升乙酸钠3M pH5.5的。
在-20 °C 下过夜沉淀 RNA 。
第二天,如果可能,在 4 °C下以 16,200 × g离心15 分钟(如果不是,则在室温下)。在此期间,启动预杂交所制备的膜(步骤D1) ,使得所述标记的RNA可用于立即。
丢弃上清液。
如有可能,用 1 ml 70% 乙醇洗涤,并在 4 °C 下以 16,200 × g离心5 分钟(如果没有,则在室温下)。
丢弃的上清液,然后再次离心和消除任何剩余的乙醇。
干燥的在室温下为丸粒5 - 10分钟。
添加100 μ升的MilliQ ħ 2在65 O和孵育℃(对于约5分钟),涡旋每30级样品小号,直到所有的RNA被重悬。
添加25 μ升的NaOH 0.2N的孵育在冰上5分钟。此步骤片段RNA和帮助小号它到与在膜上的DNA杂交。
中和通过加入25 μ升的HCl 0.2 N.


杂交
通过在65温育预杂交每个膜℃下15内部-毫升猎鹰管含有5为至少1小时毫升杂交溶液(该时间越长,效果越好)。确保将带有固定 DNA 的膜朝试管内部放置,并尽量减少膜的重叠。50 - ml F alcon 管可用于较大的膜,这样它们就不会重叠。对于这一步和接下来的步骤,使用了杂交炉;的猎鹰其中插入玻璃管中,并ROTAT管版。如果你只有几个膜,你可以直接使用玻璃管。确保拧紧盖子以防止泄漏。
一旦 RNA 准备好,丢弃杂交溶液并加入 3 ml 新鲜杂交溶液和标记的RNA。
孵育在65 ℃48 ħ而rotati纳克。
在 4°C 下储存,重复使用或保存标记的RNA。内重用1一周,因为你将失去的信号随着时间的推移。重用时,在加热烘箱中以确保杂交溶液即它液化,。
用 5 ml 洗涤液 I 冲洗膜。将溶液丢弃在适当的放射性容器中。
加入5毫升的新鲜洗涤溶液I和孵育20分钟,在65℃ ,同时rotati纳克。
弃去溶液,加入 5 ml 洗液 II ,65°C 孵育 10 分钟。
重复上一步。
小心取每个膜出的猎鹰管和除去多余的液体用与固定的DNA侧FAC将其放置前棉纸荷兰国际集团在一张萨兰的瓦特说唱。P花边一个预先切割12 ×在上面4厘米酯膜,敷萨兰瓦特周围的膜说唱,确保其被无折痕形成。
P花边的膜包裹在具有富士屏幕的盒并暴露在室温左右72小时(盖革应表明2 -每膜5厘泊)。确保是在DNA -膜固定面朝上,并与屏幕接触。我们建议将薄膜的切边放在左上角。
扫描富士屏幕后,将膜必须剥去和杂交ð与基因组DNA的第二时间来归一化信号。


剥离
孵育在5ml的50mM的NaOH每个膜在15 -毫升猎鹰在45管30分钟℃烘箱杂交内部而rotati纳克。
弃去 NaOH 并在相同条件下再加入 5 ml 中和溶液 15 分钟以中和膜。
将膜转移到单个玻璃托盘上,该托盘可容纳 100 毫升先前在微波炉中煮沸的剥离溶液。操作沸腾溶液时要小心;使用耐热手套。
在室温下在轨道振荡器上以低速将膜在沸腾的剥离溶液中孵育 10 分钟,以确保没有液体损失。
用蒸馏水 H 2 O洗涤膜一次,并在室温下在干净的滤纸上晾干;保存它们或继续下一步。测量膜用盖革计数器,以确保radioactiv Ë信号已经消失。
当他们准备重新杂交,转移剥离膜到新鲜15 -毫升猎鹰管和预杂交(小号TEP D1)。


基因组 DNA 的放射性标记
酵母提取物使用优选的方法(试剂盒可以使用)和稀1基因组DNA μ克在37的终体积μ升的MilliQ ħ 2 O.
煮沸95 5分钟上℃的热块,并立即转移到冰上。加入5个微升随机六聚体,5微升每10毫的dNTP(A,T,G),1微升Klenow酶˚F ragment ,和5微升α- 32 P的dCTP仪(Perkin-Elmer公司NEG513H250UC,10微居里/微升)。
孵育在37 ℃下在45分钟热块。
纯化标记的基因组DNA使用一个ProbeQuant TM下列制造商的方案G-50微柱。
如果要立即使用,将标记的DNA 在 95°C 下煮沸5 分钟;如果不是,则在 -20°C 下冷冻。应尽快使用标记的DNA,因为32 P 会随时间衰减。几天内使用。
获得的标记DNA足以用于 12 个膜。用MilliQ H 2 O稀释获得的 DNA,直到获得每膜20 μl 。例如,如果您有 12 个膜,则需要将 190 μl MilliQ H 2 O 添加到所得的 50 μl标记DNA 中,以获得每张膜20 μl DNA。 
加入3毫升的新鲜杂交溶液和20微升稀释标记的DNA与15 -毫升猎鹰含有该膜,在65和杂交24小时℃下。
执行与上述相同的步骤(程序D中的步骤)。
过夜或更长时间暴露在膜根据所获得的信号(48小时,如果盖革表示5 - 10厘泊)。


扫描膜和孔定量牛逼通货膨胀
使用 STOR M-840 成像系统或类似系统扫描Fuji 屏幕。
小心记住薄膜的顺序,不要让富士屏幕长时间暴露在光线下,因为光线会擦除屏幕。
如果观察到的信号不够高,可以将屏幕重新曝光更长时间。
孔定量泰特使用自由GelQuant.NET软件(放射性信号http://biochemlabsolutions.com/GelQuantNET.html)或类似的。
归一化的RNA radioactiv两者均强度与DNA radioactiv两者均强度。




图 2. 典型 TRO 实验的结果。A.标记RNA 杂交后的膜。顶部和底部膜具有交联在其上的相同的DNA片段:所述的几个片段PGAL1-YLR454w基因构建体,正(酵母克d NA)和阴性(E.大肠杆菌克DNA )控制,和的几个片段GAL1基因。顶部膜与来自 WT 酵母细胞的标记RNA杂交,底部与突变 (Mut) 酵母细胞杂交。本例中的突变体对应于pdf1Δ 。B. A 中显示的相同膜被剥离并与标记的酵母gDNA重新杂交。C.图显示的孔定量吨A和B中所示的结果的通货膨胀


数据分析


STORM 或类似成像系统拍摄的phosphoimages应为 .gel 格式。
首先,我们需要孔定量泰特所述标记的RNA膜(如看到˚F igure 2A) ,升OAD图像入GelQuant.NET程序(参见图3) ,并调整通过按压.GEL规模按钮对比度。然后,旋转和CENTE ř使用“旋转90”的图像和变焦按钮。
接着,前进到孔定量泰特周围绘制所讨论的行红色小正方形的每一行。然后,按Ç ONTROL按钮底部面板上,以确保该使用相同的尺寸d广场的所有行,然后按q uantify按钮。一些应出现在右手侧,其对应于孔定量吨的平方减去背景的通货膨胀。我们通常使用自动本地背景选项(右下角),但您可以通过单击手动背景选项来确定自己的背景。
通过拖动固定的红色方块并按下 quantify 在下一行重复此操作;重复,直到你有孔定量达ED各相关线(阳性和阴性对照不需要是孔定量达版,他们只需要可见或缺席,分别)。
在右上角,你会看到对应的音量孔定量一系列数字,牛逼你行的通货膨胀。您可以通过数字下选择中的这些号码复制到剪贴板中,突出的数字是要复制,并在底部按下剪贴板按钮。然后,这些数字拷贝我n要一个ê艾克赛尔片。
重复上述步骤2-5的DNA -标记的膜(如看到˚F igure 2B)。这些数字应该更相似。
一旦你为每个片段的所有号码,并在每个膜ë Xcel公司,你现在可以完成ANALY ž荷兰国际集团他们。首先,通过加载到膜上的每个 DNA 片段中的尿苷(或胸腺嘧啶)的数量对 RNA 膜进行标准化,因为这会影响获得的标记信号的数量。然后,将 RNA 编号标准化为 DNA 膜。最后,使用 OD 数将它们标准化为每个样本中收获的细胞数。
我们重复每个实验3次,并执行一个小号tudent的吨-相对于所述突变体,以确定所研究的每个DNA片段活性的RNA聚合酶显著变化对在WT的每一片段的测试。




图3.截图GelQuant程序用于QUANT itate杂交标记的RNA信号。在右上角,可以找到.GEL 比例、缩放、亮度和对比度按钮。在顶部,可以找到打开、另存为、旋转等按钮。在底部,按钮来绘制红色孔定量吨通货膨胀平方(Z),可以发现,与它旁边,所述Ç ONTROL按钮其被选择以允许相同的红色正方形被拖动在不同的频带对孔定量塔季翁。将q uanti泰特按钮旁边,其选择的时候,会给页面右侧的数字。在右下角,可以找到自动本地背景选项,上面是剪贴板选项。


食谱


ñ Ø TE:蒸压所有股票的解决方案,以sterili ž即


变性缓冲液
1.5 M 氯化钠


0.5M氢氧化钠


中和缓冲液
1 M 氯化钠


0.5 M Tris-HCl ,pH 7


SSC 20 ×
150 毫米氯化钠


20 mM 柠檬酸钠


加入HCl 直至 pH 值为 7


转录缓冲液 2.5 ×
550 mM Tris-HCl ,pH 7


500毫米氯化钾


80 mM 氯化镁2


测试服务
10 mM Tris-HC , l pH 7.5


10 mM EDTA ,pH 8


0.5% SDS


杂交方案
0.5 M 磷酸盐缓冲液,pH 7


7% SDS, 0.5 M EDTA pH 8


洗涤液Ⅰ
1 × SSC


0.1% SDS


洗涤液Ⅱ
0.5 × SSC


0.1% SDS


中和溶液(剥离)
550 mM Tris-HCl ,pH 7


0.1 × SSC


0.1% SDS


剥离液
55 mM 磷酸钾,pH 7
0.1% SDS


10 × TBE 缓冲器
13 M Tris 底座


450 mM 硼酸


25 mM EDTA ,pH 8


Tris-HCl 1M ,pH 7
1 M Tris 底座


加入 HCl 直至 pH 值为 7


磷酸盐缓冲液 1 M ,pH 7
99.4 克Na 2 HPO 4


41.4 克 NaH 2 PO 4 · H 2 O


检查pH 值为 7


致谢


这项工作由西班牙经济和竞争力部和欧盟基金 (FEDER) [ BFU2016-77728-C3-1-P ]资助。


原研物品,其中使用该协议是贝格利等。(2019 年)。


利益争夺


没有人声明。


参考


Begley, V., Corzo , D., Jordan- Pla , A., Cuevas-Bermudez, A., Miguel-Jimenez, L., Perez- Aguado , D., Machuca-Ostos , M., Navarro, F., Chavez, MJ、Perez-Ortin , JE 和 Chavez, S. (2019)。mRNA 降解因子 Xrn1 与 Ccr4 平行调节转录延伸。核酸研究47(18):9524-9541。
核心,LJ,瀑布,JJ 和 Lis,JT(2008 年)。新生 RNA 测序揭示了人类启动子的广泛暂停和不同起始。科学322(5909):1845-1848。              
Garcia-Martinez, J.、Aranda, A. 和Perez-Ortin , JE (2004)。Genomic run-on 评估所有酵母基因的转录率并确定基因调控机制。Mol Cell 15(2): 303-313。
Greenberg, ME 和 Ziff, EB (1984)。3T3 细胞的刺激诱导 c-fos 原癌基因的转录。自然311(5985):433-438。
              他,F.(2011)。大肠杆菌基因组 DNA 提取。生物方案1:e97。
Jordan- Pla , A.、Miguel, A.、Serna, E.、Pelechano , V. 和Perez-Ortin , JE(2016 年)。Biotin-Genomic Run-On (Bio-GRO):一种用于分析酵母新生转录的高分辨率方法。 方法Mol Biol 1361:125-139。              
Jordan- Pla , A.、Perez-Martinez, ME 和 Perez- Ortin , JE(2019 年)。使用基于运行的高分辨率方法测量全基因组的 RNA 聚合酶活性。方法159-160:177-182。
Perez- Ortin ,JE,脱米格尔-Jimenez的,L。和查韦斯,S。(2012)。真核生物中 mRNA 合成和降解的全基因组研究。Biochim Biophys Acta 1819(6): 604-615
Smale ,ST (2009)。核连续试验。Cold Spring Harb Protoc 2009(11):pdb prot5329。
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引用:Begley, V., de Miguel-Jiménez, L. and Chávez, S. (2021). Transcriptional Run-on: Measuring Nascent Transcription at Specific Genomic Sites in Yeast. Bio-protocol 11(12): e4064. DOI: 10.21769/BioProtoc.4064.
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