For the most sensitive and reliable Nuclease Protection Assays (NPAs), we strongly recommend using probes that have been gel purified.While precipitations and “spin columns” remove freenucleotides and DNase treatments can remove template DNA, these techniques do not allow qualitative analysis of the probe. Only by gel assessment can we reliably verify probe integrity. Gel purification is the only method for removing DNA template when making single-stranded DNA probes (e.g. by primer extension).

For both RNA and DNA probe preparations, gel purification, in onestep, removes free nucleotides, DNA template, buffer components, and enzymes, as well as “prematurely terminated” products. Prematurely terminated products can form during extension or transcription if the polymerase encounters significant secondary structure or a homopolymeric stretch of one nucleotide, or if there is an inadequate amount of limiting nucleotide (usually the labeled nucleotide in the reaction).

Prematurely terminated products, if not removed, will increase background bands and smearing on the NPA gel after hybridization and digestion. With this in mind, we suggest that all probes used in NPAs be gel purified.

Procedure Description

Gel purification is straight forward and easy. Really! After transcription or primer extension, the reaction is run on a denaturing polyacrylamide gel (a “mini” vertical gel apparatus can be used) to separate the DNA template, full-length RNA or DNA probe, any prematurely terminated products and freenucleotides, by size. The gel is exposed to film, stained or UV shadowed (depending on the nature and quantity of the probe made, e.g. radioactive, nonisotopic or unlabeled probe). Full-length probe is identified and the band is cut from the gel. The probe is eluted by passive diffusion from the gel fragment and is ready for use. Note that while many researchers use an overnight incubation to elute probe, the procedure can produce enough probe for many applications, ready for hybridization in just 1–4 hours.

Reagents and Equipment Needed

  • PAGE apparatus (0.75 - 1.5 mm spacers and comb)
  • Reagents for denaturing polyacrylamide gel (see recipe below)
  • Gel Loading Buffer (95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue, 0.5 .mM EDTA, 0.025% SDS)
  • Elution buffer (0.5 M NH4OAc, 1 mM EDTA, 0.1% SDS), TE, or nuclease-free H2O
  • Incubator or water bath capable of 37°C or 65°C

For Radioisotopic Probes:
Autoradiographic film

For Nonisotopic/Unlabeled Probes:
For UV shadowing Fluor-coated TLC plate or intesifying screen and short-wavelength (254 nm)
hand-held UV light source
For Staining Acridine Orange (Available from Amresco, Prod. Code 0360) or Ethidium Bromide
and UV transilluminator

Protocol for Gel Purification

We routinely use mini gels (e.g. Ambion’s Vertical Gel Apparatus or BioRad’s Mini-Protean) for gel purification as larger gels are not required. While larger gels provide greater resolution, this is usually not necessary since the predominant probe band should easily resolve from premature terminations on using a small gel. Compared to larger gels, small gels offer the advantages of being quick and easy to prepare (30 min) run (30 min), and of being inexpensive.

TIP: The same mini-gel apparatus can often be used to analyze NPA products after hybridization and digestion.


1. Preparation of 5% Acrylamide/8M Urea denaturing polyacrylamide gel (makes 15 ml, enough for a 13 cm x 15 cm x 0.75 mm thick gel)
a.
Set up glass plates and spacers. Have comb ready.

b. Mix the following:
7.2 g                   high-quality urea
1.5 ml                10X TBE
1.875 ml            40% acrylamide
                           (acrylamide: bis acrylamide = 19:1)
c. Add dH2O to a final volume of 15 ml
d. Stir at room temperature until urea dissolves (this stock solution can be stored for up to 1
week at 4°C)
e. Then add:
120 μl                10% ammonium persulfate in dH2O (fresh)
16 μl                   TEMED
f. Mix briefly and pour.
g. Allow to set (about 30 min.).

2. Loading and Running of Gel
a.
Add an equal volume of Gel Loading Buffer (see Reagents list) to the probe or, if the probe has been precipitated, resuspend directly in Gel Loading Buffer.
b. Heat at 95°C for 3-5 minutes to denature any secondary structure, then place on ice to prevent renaturation. Secondary structure will cause some or all of the RNA to migrate aberrantly through the gel giving a smear, multiple bands, or bands of the wrong size.
c. After flushing any urea from the wells, load the probe in the gel and run the gel until the more rapidly moving blue dye front (bromophenol blue) reaches the bottom of the gel (200 mA for about 30 minutes for minigels).

3. Preparation of Gel Before Visualization
Radioisotopic probes (32P-, 33P-, or 35S labeled)–Separate the glass plates, leaving the gel adhered to the larger bottom glass plate. Wrap a piece of plastic wrap over the gel. If the glass and gel will not fit into the film cartridge, then the bottom glass plate should be carefully removed and the gel wrapped entirely in plastic wrap (for easier handling). The gel is now ready to expose to film. Nonisotopic probes (unlabeled, biotin-, or digoxygenin- labeled) – The gel needs to be removed from both of the glass plates since glass blocks UV light and will prevent visualization by either UV shadowing or staining. The gel is wrapped in plastic wrap to aid in handling and marking. Remove the top glass plate, and lay a sheet of plastic wrap over the gel, then flip the gel and glass plate over and carefully peel the gel away from the bottom glass plate. Wrap the gel entirely in the plastic wrap.

TIP: Use just a single layer of plastic wrap and try to prevent any bubbles from forming between the gel and plastic wrap. These bubbles can scatter the UV light and make visualization difficult.

4. Visualization of radioisotopic probes
a.
Place the gel (sandwiched between the glass and plastic wrap) against the X-ray film. The film can simply be aligned with one corner of the glass plate, the corners and sides of the glass plate marked directly on the film with a permanent marker, or alternatively, radioactive ink or fluorescent stickers can be used for orientation. One corner of the film (e.g. bottom right corner) is usually snipped or folded up so that the glass and gel can be precisely aligned with the film after developing.
b. Expose the gel to autoradiographic film, about 30 seconds for a high specific activity 32Plabeled probe and 10 minutes for a low specific activity 32P-labeled probe or a high specific activity 35S-labeled probe. The goal is to get an exposure which produces a light gray band on the film so that a thin gel fragment can be excised from the gel. Realign the glass plate and gel with the developed film (using the guide marks made earlier) and carefully excise the most intense (usually highest molecular wight) band using a nuclease-free scalpel or razor blade. The smaller the size of this gel fragment, the better the elution efficiency (i.e.more probe will be recovered more quickly). The gel can be re-exposed to insure that the gel and film were properly aligned and that the probe was excised.

TIP: If possible, run markers or a known size standard so that the appropriate band is selected. If no markers have been run, the bromophenol (dark blue) and xylene cyanol (light blue) dyes can serve as size references. In a denaturing 5% polyacrylamide gel, bromophenol blue runs at 35 nt and xylene cyanol runs at 130 nt.

5. Visualization of Nonisotopic Probes
Nonisotopic and unlabeled probes containing Gels cannot be visualized by direct exposure to film. Note, however, that a much greater mass of probe (several micrograms as compared to nanogram amounts of radiolabeled probe) is usually synthesized and can be visualized by either UV-shadowing with a short-wavelength hand-held UV light source and fluor-coated TLC plate or staining with acridine orange or EtBr and UV transillumination.
Note: Any surface that the gel comes in contact with should be treated to remove RNase contamination (e.g. RNase Zap™)


UV shadowing
Place the gel on top of the dull white side of the fluor-coated TLC plate and remove the plastic wrap on top of the gel. Hold a hand-held shortwavelength (254 nm) UV light source over the gel. (Long wavelength UV light will not work). The TLC plate beneath the gel should glow a yellow-green color. Any nucleic acid bands in the gel will cast a shadow on the TLC plate which will appear as a purple band. The limit of sensitivity is about 0.4 μg in a single band. Alternatively, if a TLC plate is not handy, an intensifying screen may be used.

TIP: UV shadowing works for either DNA or RNA, labeled or unlabeled, so this technique has many other applications; e.g. for visualizing restriction enzyme digests.

Staining
It is important that if a stain is used to visualize the probe in the gel, that it be completely removed before hybridization as it will compromise hybridization efficiency. We recommend staining with acridine orange as opposed to EtBr since acridine orange be removed from the probe by EtOH precipitation. EtBr can be used, but requires multiple butanol extractions to subsequently remove it. Remove the gel from the plastic wrap and place in a 2.0 μg/ml acridine orange in nuclease-free water for 15 minutes. Destain the gel in distilled water for 10 minutes. Rewrap the gel in plastic wrap for easier handling, and place the gel on a UV transilluminator to visualize the probe.

TIP: If possible, run markers or a known size standard so that the appropriate band is selected. If no markers have been run, the bromophenol (dark blue) and xylene cyanol (light blue) dyes can serve as size references. In a denaturing 5% polyacrylamide gel, bromophenol blue runs at 35 nt and xylene cyanol runs at 130 nt.

6. Probe Elution
Transfer the gel fragment to a nuclease-free tube and add enough Elution Buffer (see Reagents list) to submerge it (usually about 350 μl). Any buffer or dH2O can be used; however, we recommend 0.5 M NH4OAc, 1 mM EDTA, 0.1% SDS. The presence of SDS helps inactivate low level nuclease contamination and the presence of EDTA chelates Mg++, thereby reducing RNase activity. The 0.5 M NH4OAc allows an optional EtOH precipitation to be performed more easily and quickly since he salt is already present.

TIP: Elution can be accelerated by dicing or crushing the gel fragment to increase its surface area. We routinely incubate the gel fragment in elution buffer overnight at 37°C. However, the elution time is dependent on the size of the gel fragment, transcript size and the temperature of the incubation. We find that about 50% of a 400 nt transcript will elute in about 4 hrs at 37°C. By increasing the incubation temperature to 65°C, most of the probe will elute in 1 hr. Larger transcripts will take longer to elute. It is not necessary to elute all the probe before proceeding to the hybridization, only what will be needed (i.e. 2-8 x 104 cpm of a high specific activity probe per RPA reaction, or ~1 x 107 cpm for Northern Blots). You can remove the elution buffer (containing some of the probe) at any time during the elution and proceed with the post elution cleanup (optional) and yield determination. Fresh elution buffer can be placed back in the tube with the gel fragment and the rest of the probe eluted over a longer period of time. This allows setup of hybridization reactions on the same day as probe preparation. Note that elution of a high specific activity probes in 350 μl of elution buffer should yield approximately 1-4 x 104 cpm/ml.
a. Spin the tube at 14K rpm for about 3-5 minutes to pellet the gel fragment.
b. Carefully transfer the supernatant to a clean microfuge tube using a micropipetter, leaving behind the gel fragments.

7. Post Elution Cleanup (optional)
Probe is usually concentrated enough to be used directly in the elution buffer. However, the following steps may be performed to further concentrate or clean up the probe.

TIP: Since nonisotopic probes will last 6 mos. - 1 yr. or more, we highly recommend this optional cleanup step.
a. An optional phenol:chloroform step can be per formed at this time.
NOTE: If using digoxigenin as the label, do not treat with phenol:chloroform as the RNA/DNA probe will partition into the organic phase.
b. Perform a standard EtOH precipitation using 2-3 volumes of ice-cold Ethanol and a final concentration of 0.5 M NH4OAc. If working with radioisotopic probes, 5-10 μg of an optional carrier such as yeast RNA, glycogen or linear acrylamide can be added to aid with precipitation and pellet visualization. If working with nonisotopic probes, a carrier should not be needed since microgram amounts are usually eluted. More importantly, nucleic acid carriers should not be used with nonisotopic probes as their presence will affect absorbance readings and probe quantitation; glycogen and linear acrylamide however, do not absorb in the range of 260 - 280 nm, therefore they can be used if needed.

8. Probe Quantitation
Radioiotopic Probes
Quantitate the gel-purified probe by scintillation counting to determine the concentration (cpm/ml). A typical transcription reaction should yield approximately 1-4 x 104 cpm/μl in 350 μl of gel elution buffer. Probe should be stored at -20°C or -80°C.
Nonisotopic Probes
Quantitate the probe by checking the absorbance readings at 260 and 280 nm. The A260/280 ratio should be 1.9-2.0. If it is much lower (< 1.6), then an additional LiCl precipitation should be performed (Request Ambion’s Technical Bulletin #160). Low A260/280 ratios indicate protein and/or acrylamide contamination
which can lead to inaccurate concentration calculations. Based on the 260 reading and the conversion: 1 A260 unit = 40 μg/ml RNA, calculate the μg/μl concentration of the probe. A typical 20 μl transcription reaction (containing 0.5 mM of each NTP) which has been gel purified should yield 4-8 μg of RNA. The probe should be stored at -20°C or -80°C. We start seeing degradation after 5-10 freeze-thaw cycles, so the probe should be stored as aliquots.


TIP: For faster set-up of hybridization assays, dilute the probe to 1 ng/μl for use in NPAs or 10 ng/μl for use in Northern or Blot hybridizations and store this working concentration of probe as 10+ aliquots. Each aliquot will experience fewer freeze-thaws and will last longer. When the probe is needed it will already be diluted and ready for use.


9. Amount of Probe to use
Once quantitated, the probe is ready for use in hybridization assays, and can usually be used directly in the elution buffer.


Nuclease Protection Assays
The amount of probe added depends on the specific activity of the probe, mass amount of sample RNA and expression level of the target message. For moderately abundant messages (e.g. ß-actin or GAPDH), 2-8 x 104 cpm of a 300 base, high specific activity radiolabeled probe or 1 ng of a 300 base, high specific activity nonisotopic probe should be added per 10 μg of total RNA. As the specific activity is decreased (by addition of “cold”/unlabeled form of the limiting nucleotide during preparation of probe) or as the probe size decreases, the mass amount of probe added to the hybridization reaction should be decreased linearly. However, the molar amount of the probe added to a hybridization reaction is dependent on the amount of target RNA and should remain the same even as specific activity is adjusted.


Membrane/Blot Hybridizations
If performing membrane hybridizations, 1 x 106 cpm of a radioisotopic probe or 10 ng of nonisotopic probe should be added to each ml of hybridization solution.