A runoff transcription say is a useful in-vitro analysis of the transcription site. The outputs of this assay are the position of the transition starting site and the promoter regions. It can also be used to measure the effect the modification in promoter position on in-vitro transcription. Since it is in vitro assay it is limited to not find cell-specific genetic expression.
A brief procedure for performing the run-off transcription is generalized below:
1) First, a gene of interest (including promoter) is cloned into a plasmid.
2) Digestion of the plasmid is carried out. It is done in the downstream direction the transcription site, which we have inserted. From this cut sequences, the desired mRNA can be separated in the steps to follow.
3) Before running the assay the DNA is purified.
4) RNA polymerase, radiolabeled UTP, and other nucleotides are added and incubated with the linearized DNA. This starts the transcription process. The transcription runs the full length of the DNA producing the mRNA fragment of the desired length.
5) Gel electrophoresis is used to separate this mRNA fragment. The separation is aided alongside by size standards and autoradiography. The standard size gives the size of the mRNA fragment and the intensity gives the amount of mRNA produced.
To minimize RNase contamination, all reagents should be made with water which contains less than 20 ppm of total organics. Alternately, Diethyl pyrocarbonate (DEPC) treated water may be used: add 1 mL of DEPC per L of water, shake well, and autoclave after 1 h. Wear gloves during all operations to avoid contamination with finger RNases. Pipetting devices should be wiped down with ethanol and never used with solutions containing RNase. All reagents can be stored at –20°C.
- Phosphate-buffered saline (PBS): 1.54 mM KH2PO4, 155.17 mM NaCl, 2.71 mM Na2HPO4.
- Buffer A: 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonate (HEPES) (pH 7.9 at 4°C), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitrol (DTT).
- Low-salt buffer: 20 mM HEPES (pH 7.9 at 4°C), 1.5 mM MgCl2, 20% glycerol, 20 mM KCl, 0.2 mM ethylene diamine tetraacetic acid (EDTA), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM DTT.
- High-salt buffer: 20 mM HEPES (pH 7.9 at 4°C), 1.5 mM MgCl2, 20% glycerol, 1.2 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT.
- Buffer D: 20 mM HEPES (pH 7.9 at 4°C), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT.
- QIAGEN buffer A: 100 mM NaH2PO4, 10 mM Tris-HCl, pH 8.0, 6 M guanidine- HCl (adjusted to pH 8.0 with NaOH).
- 0.5X buffer D: 10 mM HEPES (pH 7.9 at 4°C), 10% glycerol, 50 mM KCl, 0.1 mM EDTA, 0.1 mM PMSF, 0.25 mM DTT.
- 20 mM HEPES, pH 7.2.
- 1 M Ethanolamine, pH 8.0.
- Forward exchange buffer (10X): 500 mM Tris-HCl (pH 7.5), 100 mM MgCl2, 50 mM DTT, 1 mM spermidine.
- Transcription buffer (10X): 40 mM HEPES (pH 7.9 at 4°C), 40 mM creatine phosphate, 100 mM MgCl2, 200 mM KCl, 5 mM DTT, 0.2 mM EDTA.
- Stop mix: 20 mM EDTA, pH 8.0, 200 mM NaCl, 1% sodium dodecyl sulfate, 0.2 mg/mL glycogen.
- PE buffer (2X): 100 mMTris-HCl (pH 8.3 at 42°C), 100 mMKCl, 20 mMMgCl2, 20 mM DTT, 2 mM dNTPs, 1 mM spermidine.
- TBE (10X): 1 M Tris base, 900 mM boric acid, 10 mM EDTA.
- Formamide loading mix: 98% formamide, 10 mM EDTA (pH 8.0), 0.01% xylene cyanol, 0.01% bromophenol blue.
- Acrylamide gel mix: 40% acrylamide/bis-acrylamide (29:1).
- Corning polypropylene centrifuge tubes, 250-mL.
- Corning polypropylene disposable centrifuge tubes, 50-mL.
- Corning polypropylene disposable centrifuge tubes, 15-mL.
- Kontes B pestle.
- Spectro-Por dialysis tubing (18-mm flat width, molecular-weight [MW] cutoff)
- Slide-A-Lyzer, 3000 MW cut-off.
- Centiprep YM-3, 3000 MW cut-off.
- Centricon YM-3, 3000 MW cut-off.
- Affi-Gel 10.
- 32P-ATP (approx 1000 Ci/mmol).
- T4 DNA ligase.
- ATP, GTP, CTP, UTP: 5 mM each.
- 0.3 M Sodium acetate.
- Phenol:chloroform:isoamyl alcohol (50:50:2), saturated with RNase-free water.
- 80% Ethanol.
- 40 mM Sodium pyrophosphate.
- AMV reverse transcriptase.
- Siliconizing reagent: Rain-X.
- 3 MM filter paper sheets.
- 32P-UTP (800–1000 Ci/mmol).
Preparation of the Nuclear Transcription Extract
Nuclear extracts are made from cells grown in monolayer or in suspension culture. There is no fixed volume of cells or nuclear extract in this procedure. For reproducibility and convenience, the majority of nuclear extracts are from liter quantities of HeLa cell suspension culture. We use suspension cultures of HeLa cells grown in minimum essential medium (ICN) supplemented with 10% calf serum.
- Six liters of HeLa cells in suspension are grown by feeding cells every day while maintaining a cell density of about 6 × 105 per mL. Cells should be harvested for the preparation of nuclear extracts when they are growing well, i.e., doubling nearly every 24 h.
- Harvest cells in six 250-mL Corning polypropylene centrifuge tubes by repeated centrifugation at 4°C for 10 min at 180g. Carefully pour off the supernatant and add fresh suspension culture on top of the existing cell pellet and repeat centrifugation until the entire 6 L of suspension culture are harvested.
- All subsequent steps are done at 4°C using precooled buffers. Gently resuspend cell pellets into 40 mL of PBS by pipetting, combine the suspended pellets in a single 50-mL Corning polypropylene centrifuge tube, and centrifuge again. Gently resuspend the packed cell pellet (usually 5–8 mL) in five times the pellet volume of hypotonic buffer A and incubate on ice for 10 min. Centrifuge the swollen cells for 10 min at 180g and carefully withdraw the supernatant with a pipet so as not to disturb the soft, swollen cell pellet. Using buffer A, resuspend the cells in twice the original volume of packed cells. A microscopic examination of a small aliquot should reveal that cells are greatly swollen but largely intact.
- Dounce-homogenize the cells using 10 strokes of a Kontes B pestle. A microscopic examination should show that more than 90% of the cells have been disrupted and that the vast majority of nuclei are intact
- Centrifuge the nuclear preparation in a 50-mL polypropylene disposable centrifuge tube at 1000g for 10 min. Remove the supernatant and recentrifuge at 1000g for 5 min. Remove and discard the small amount of remaining supernatant above the nuclear pellet. Note the volume of the nuclear pellet.
- To prepare the nuclear extract, resuspend the nuclear pellet in a volume of low-salt buffer representing exactly one-half the nuclear pellet volume. Add dropwise the same volume of high-salt buffer as low-salt buffer while gently vortexing the tube. Tightly cap the tube and continue to extract the pellet by rotation for 30 min.
- Centrifuge the extract at 9000g for 30 min. Transfer the supernatant into dialysis tubing and dialyze for 3–5 h against 1 L of buffer D.
- Remove the extract from the dialysis tubing and clarify it by centrifugation for 10 min at 10,000g. Aliquot the supernatant (100–500 μL) into precooled microfuge tubes (4°C) and freeze by immersion in liquid nitrogen. Store in a –70°C freezer or in a vapor-phase nitrogen freezer for long-term storage.
Preparation of Biologically Active E1A Functional Domains
- Biologically functional His6-tagged E1A 243R and E1A 1-80 polypeptides are prepared. Harvest a culture of isopropylthio-β-D-galactoside-induced bacterial cells (500 mL) expressing an appropriate pQE E1A construct by centrifugation at 2000g for 10 min at 4°C, and freeze the cell pellet at –20°C.
- Thaw the pellet at room temperature and lyse in 40 mL of buffer A with gentle mixing for 1 h. Clarify the supernatant by centrifugation at 9000g for 30 min at 4°C.
- Bind the His6-tagged polypeptide to 2 mL of Ni-NTA resin by rotating the clarified lysate with the resin overnight at 4°C. Batch wash the resin five times with 20 mL of buffer A and five times with buffer A adjusted to pH 6.3.
- Load the resin into two 5-mL columns (Image Molding) and wash each column with 20 mL (10 column volumes) of the same buffer and then with 50 mL of buffer A adjusted to pH 5.9.
- Elute the His6-tagged polypeptide from the resin with 20 mL of buffer A adjusted to pH 4.5. Collect 2-mL fractions. Generally, sufficient polypeptide is produced so that protein-containing fractions can be identified by adding 2 μL of each fraction to 100 μL of Protein Assay Reagent B. Detectable color develops after 10 min at room temperature.
- To prepare biologically active E1A 1-80 polypeptides, it is necessary to remove guanidine-HCl slowly from the preparation to facilitate proper folding. Pool fractions containing eluted polypeptide and adjust to 6 mL with elution buffer (buffer A at pH 4.5 containing 6 M guanidine-HCl). Dilute the sample 1:1 with 0.5X buffer D.
- Dialyze the diluted sample (now at 3 M guanidine-HCl) in a 3000 MW cutoff Slide-A-Lyzer dialysis cassette against 0.5X buffer D containing 2 M guanidine- HCl. After 6–8 h of dialysis, remove one-half of the dialysis buffer and replace it with fresh 0.5X buffer D without guanidine-HCl, thereby reducing the guanidine- HCl concentration by half.
- Continue dialysis with buffer replacement in the manner described above until the guanidine-HCl concentrations are reduced to 50–100 mM. Complete the dialysis against several changes of 0.5X buffer D for 8 h.
- Concentrate the E1A polypeptides by size exclusion centrifugation using Centriprep YM-3 followed by Centricon YM-3 to a final concentration of about 1 mg per mL of the polypeptide.
Preparation of Nuclear Transcription Extracts Affinity Depleted With E1A Functional Domain Polypeptides
- Affi-Gel 10 immobilized E1A polypeptides are prepared as follows. Exchange by dialysis into 20 mM HEPES, pH 7.2, the E1A 1-80 polypeptides purified as described above.
- Prepare 1.5 mL of packed Affi-Gel 10 beads immediately prior to use as follows. Take 3 mL of Affi-Gel suspension (comes with a 1:1 slurry in isopropanol) and centrifuged in a 15-mL centrifuge tube at 2000 rpm (1000g) for 5 min. Wash beads three times with 12 mL of cold water by centrifugation at 2500 rpm (1700g) for 2 min. Incubate the packed beads with 1.5 mg of the polypeptide in 1.5 mL of buffer for 4 h at 4°C with rotation. Retain 10 μL of polypeptide prior to addition to beads and after 4 h of incubation to determine the efficiency of polypeptide binding as follows.
- Centrifuge the aliquots in a microfuge tube at 1700g for 5 min and add 5 μL of 1 N HCl to the supernatant. Add 40 μL of water and read the absorbance at 280 nm. Efficient binding is reflected by a reduction in absorbance at 280 nm of 80–90%. If an efficient binding is not attained, continue rotation of the beads with the polypeptide.
- When satisfactory binding of the polypeptide to beads is obtained, centrifuge the beads at 2000 rpm for 5 min and resuspend the beads in 800 μL of PBS. Add 200 μL of 1 M ethanolamine, pH 8.0, and rotate for 1 h at 4°C to block reactive groups remaining on the beads. Centrifuge to remove the ethanolamine and wash three times with buffer D. Store the polypeptide-immobilized beads at 4°C.
- Prepare affinity column (5-mL) by loading 250 μL of the packed polypeptide-immobilized beads. Recirculate 1 mL of HeLa cell nuclear extract through each column for 2 h at 4°C at a flow rate of 0.1 mL per min. Aliquots of the nuclear extract before and after affinity chromatography (as well as the proteins bound to the beads) can be analyzed by immunoblot analysis with specific antibodies to candidate E1A cellular partners by use of a sensitive Western blotting kits utilizing chemiluminescent substrates. The depleted nuclear extracts can now be used by in vitro transcription to probe the function of the E1A polypeptide interacting domains.
Analysis of In Vitro Transcription by Primer Extension and by Run-Off Assay
The run-off assay uses a DNA template cut with a restriction enzyme downstream of the transcription start site. It simply works on creation of a site in the DNA template in which the RNA polymerase falls off. This terminates the transcription at a certain point. The assay uses a radio-labeled rNTP precursor. The RNA of a specific length is synthesized and is resolved as a discrete band by denaturing polyacrylamide gel electrophoresis (PAGE). Autoradiography or phosphor image analysis is done following the PAGE. The product of the assays can be quantitated by scanning densitometry or by more sensitive phosphor image analysis.
Preparation of DNA Templates for In Vitro Transcription
Plasmid templates containing the promoter of interest must be of high purity for in vitro transcription. Plasmids purified by double CsCl density gradient centrifugation are of high quality and can be used directly. Plasmid templates prepared by standard alkaline lysis procedures or by use of commercial plasmid preparation kits may require further purification. It is often important to further purify those templates by phenol-chloroform extraction followed by ethanol precipitation. For primer extension, superhelical plasmid templates are used. For run-off analysis, templates are linearized with a restriction enzyme in order to terminate transcription at a known site.
Preparation of Radiolabeled Deoxyoligonucleotides for Primer Extension
Deoxyoligonucleotide primers are designed to be about 30 nucleotides (nt) in length and to be complementary to a region from 100 to 200 nt downstream of the transcription initiation site. Primers with self-complementary sequences are avoided. To label a primer, incubate 10 pmol of deoxyoligonucleotide at 37°C for 30 min in a 10-μL reaction containing 1 μL of 10X forward exchange buffer, 6 μL of γ32P-ATP (1000 Ci/mmole), and 1 μL of T4 polynucleotide kinase (10 U). Heat the reaction mixture at 100°C for 2 min and add 190 μL of water. Store at –20°C. The radiolabeled primer may be used as long as a suitable transcription signal is obtained, usually 4–6 wks.
In Vitro, Transcription Analysis Using the Run-off assay
First, the DNA template is cut at a convenient site with a restriction enzyme.
Reaction mixtures are assembled in RNase-free microfuge tubes containing
2.5 μL of 10X transcription buffer, 2.5 μL of rNTP mixture (5 mM each of
ATP, GTP, CTP, and 0.25 mM UTP), DNA template (typically 500 ng), nuclear extract (typically 10 μL), 32P-UTP (0.5 μL of 10 mCi/mL, 800–1000 Ci/mmol) is added to the reaction mixture and water to give a final volume of 25 μL. The reaction is initiated by the addition of nuclear extract followed by incubation at 30°C for 60 min. For each new DNA template and for each batch of nuclear extract, preliminary titrations are done with different amounts of template (100–1000 ng) and nuclear extract (5.0–12.5 μL) to optimize the transcription signal (strength and authenticity, i.e., correct size). Compensate for volumes of nuclear extract less than 10 μL by the addition of an equivalent volume of buffer D.
Isolation of the RNA transcription product
When the transcription reaction is completed after 60 min, terminate the reaction by addition of 100 μL of stop mix. Then add 300 μL of 0.3 M sodium acetate and 300 μL of phenol/chloroform/isoamyl alcohol (50:50:2). Vortex the sample for 30 s and separate the phases by centrifugation at 10,000g for 2 min. Transfer the upper aqueous phase to a fresh tube containing 1 mL of ethanol, mix, and place on dry ice for 15 min. Centrifuge the sample at 10,000g for 10 min at 4°C, rinse the pellet carefully with 500 μL of 80% ethanol (–20°C), and briefly dry the pellet containing the RNA product in a vacuum desiccator.
Resolution of the labeled primer extension product by denaturing polyacrylamide gel
Resolve the labeled product by electrophoresis on a 0.75-mm-thick/20-cm long 6% urea polyacrylamide gel using a vertical gel chamber. The smaller plate (facing the apparatus) is siliconized with Rain-X to facilitate separation of the plates after electrophoresis. To prepare the gel, stir with a magnetic bar 12 g of urea (ultrapure, RNase-free) with 6.0 mL of 5X TBE, 4.5 mL of 40% acrylamide/bis-acrylamide (29:1), 300 μL of 10% ammonium persulfate, and water to a final volume of 30 mL. When the urea is completely dissolved, add 20 μL of TEMED and pipet or pour the mixture into an assembled gel sandwich held at about a 40° angle. Insert a 15-well comb and then allow the gel to polymerize in a horizontal position for 3 h or overnight. Prior to loading the samples, mount the gel on the electrophoresis chamber, add TBE to the upper and lower chambers, and clean the gel teeth by pipetting TBE up and down. Pre-electrophorese the gel for about 15 min at 400 V constant. Load the samples (10–20 μL) and a labeled size marker (e.g., a 50-bp ladder) into individual lanes on the gel and electrophorese at 400 V constant until the bromophenol blue marker just migrates off the gel. The gel is pulled onto 3MM paper as follows. Briefly, separate the gel sandwich by gentle prying with a fine-bladed spatula. The gel will adhere to the larger, unsiliconized plate. Put the gel-containing plate flat on a bench top and carefully position a sheet of 3MM paper over the gel. Rub the paper gently but firmly and carefully pull off the paper with the adherent gel. The gel can then be dried on a gel dryer prior to autoradiography on X-ray film or simply covered with plastic wrap and autoradiographed with X-ray film and an intensifying screen at –70°C overnight. For quantitation and highly sensitive detection of signals, gels are dried and analyzed by phosphor image analysis.
Phosphor Image Analysis and Quantitation of Transcription Products From Primer Extension and Run-Off Assays
Phosphor image analysis provides for an efficient, accurate, and highly sensitive way to measure radiolabeled transcription products. Phosphor image analysis typically has a linear dynamic range of 5 orders of magnitude as compared to only about 1.5 for X-ray film. The pixels measured by the PhosphorImager can be quantitatively and statistically analyzed using software provided by the manufacturer, e.g., Image Quant for the Molecular Dynamics PhosphorImager system used here. Briefly, cover the dried gel with plastic wrap and place a phosphor screen over the dry gel in a PhosphorImager cassette. Expose the phosphor screen from overnight to 2 d. Place the screen face-down on the PhosphorImager glass plate and select the scanning area using the appropriate software. The image of the gel is visualized and bands of the correct size are selected and quantified using the tools provided by the PhosphorImager software. These images can be printed or saved as digital files for permanent storage and for publication.