Gel Purification of Probes for Nuclease Protection Assays

Introduction

For the most sensitive and reliable Nuclease Protection Assays (NPAs), we strongly recommend that the probes used be full-length and purified. While precipitations and "spin columns" remove free nucleotides, and DNase treatments can remove template DNA, these techniques do not allow qualitative analysis of the probe. Only by gel assessment can probe integrity be reliably verified. Gel purification is the only method for easily removing DNA template when making single-stranded DNA probes (e.g. by primer extension). For both RNA and DNA probe preparations, gel purification, in one step, 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

After transcription or primer extension, the reaction is run on a denaturing polyacrylamide gel (a "mini" protein gel apparatus can be used) to separate by size the DNA template, full-length RNA or DNA probe, any prematurely terminated products, and free nucleotides. The gel is exposed to film and 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 ready for hybridization in 1-4 hours.

Reagents and Equipment Needed

	PAGE apparatus (0.75 - 1.5 mm spacers and comb)
	PAGE reagents for denaturing 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 and short-wavelength (254 nm) handheld 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 small mini gels (e.g. 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 only one band (the full length probe) is expected. Compared to larger gels, small gels offer the advantage that they are quick and easy to prepare (~30 min), run quickly (~30 min) and are inexpensive.

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

1. Preparation of 5% Acrylamide/8 M Urea denaturing polyacrylamide gel (makes 15 ml, enough for a 13 cm x 15 cm x 0.75 mm thick gel)

1. Mix the following:

   7.2 g	high-quality urea
   1.5 ml	10X TBE
   1.875 ml	40% acrylamide (acrylamide: bis acrylamide = 19:1)

   
   
2. Add dH₂O to a final volume of 15 ml
   
   
3. Stir at room temperature until urea dissolves
   
   
4. Then add:

   120 µl	10% ammonium persulfate in dH2O (fresh)
   16 µl	TEMED

   
   
5. Mix briefly and pour.
   
   
6. Allow to set (about 30 min.).
   

2. Loading and running of gel

1. Add an equal volume of Gel Loading Buffer (see Reagents list above) to the probe or, if the probe has been precipitated, resuspend directly in Gel Loading Buffer.
   
   
2. 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.
   
   
3. 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 (20 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 ready to expose to film.

Nonisotopic probes (unlabeled, biotin-, or digoxigenin-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, 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 only 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

1. Place the gel (sandwiched between the glass and plastic wrap) against the film so that the film is closest to the gel. 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.
   
   
2. Expose the gel to autoradiographic film, about 30 seconds for a high specific activity ³²P-labeled probe and 10 minutes for a low specific activity ³²P-labeled probe or high specific activity ³⁵S-labeled probe. The goal is to get an exposure of a light gray band 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 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). This band should be the most intense band present. 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
With nonisotopic and unlabeled probes, the gel cannot be directly exposed to film as with radioisotopic probes. Note, however, that a much greater mass of probe (several micrograms, vs. nanogram amounts of radiolabeled probe) is usually synthesized. Therefore, the gel can either be UV-shadowed with a short-wavelength hand-held UV light source and fluor-coated TLC plate, or stained with acridine orange or EtBr and held over a UV transilluminator to visualize the probe's location within the gel.

Note: Any surface with which the gel comes in contact should be treated to remove RNase contamination (e.g. use Ambion's RNaseZap for this purpose).

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 short-wavelength (254 nm) UV light source over the gel. (Long-wavelength UV light will not work). The TLC plate beneath the gel should glow bright purple wherever nucleic acids are present. The limit of sensitivity is about 0.3 µg in a single band.

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, 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 can 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 solution for 15 minutes. Destain the gel in distilled water for 10 minutes. Re-wrap the gel in plastic wrap for easier handling, and place the gel on a UV transilluminator to visualize the probe.

Carefully cut out (using a nuclease-free scalpel or razor blade) the smallest gel fragment possible containing the probe. (This will correspond to the bright purple band on the TLC plate, or the band in the gel; the smaller the size of this gel fragment, the better the elution efficiency (i.e. more probe will be recovered more quickly). This band should be the most intense band present. If you are concerned that not all the probe was cut out, visualize the gel again with UV light to verify that the probe band is gone.

6. Probe elution
Transfer the gel fragment to a nuclease-free tube and add enough Elution Buffer (see reagents list above) to cover it (usually ~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 Mg²⁺, thereby reducing RNase activity. The 0.5 M NH₄OAc allows an optional EtOH precipitation to be performed more easily and quickly since the salt is already present.

 TIP: Elution can be accelerated by dicing up 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, the transcript size and the temperature of the incubation. We find that about 50% of a 400 nt transcript should elute in about 4 hrs at 37°C. By increasing the incubation temperature to 65åC, most of the probe will elute in 1 hr. Larger transcripts will take longer to elute. It is not necessary to elute all of 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 quantitation. 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/µl.

Spin the tube at 14K rpm for about 3-5 minutes to pellet the gel fragment.

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 from 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.

An optional phenol:chloroform step can be performed at this time.
NOTE: If using digoxygenin as the label, DO NOT phenol:chloroform extract, as the RNA/DNA probe will separate into the organic phase.

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 (Ambion offers these; see below) 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 spectrophotometer readings and probe quantitation; however, glycogen and linear acrylamide do not absorb in the range of 260 - 280 nm, so these coprecipitants can be used if needed.

8. Probe quantitation
Radioisotopic Probes
Quantitate the eluted 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 for greater stability.

Nonisotopic probes
Quantitate the probe by checking the absorbance at 260 and 280 nm with a spectrophotometer. The A₂₆₀/A₂₈₀ ratio should be 1.9-2.0. Low A₂₆₀/A₂₈₀ ratios indicate protein/acrylamide contamination, which can lead to inaccurate concentration calculations. Based on the A₂₆₀ reading and the conversion: 1 A₂₆₀ unit = 40 µg RNA, calculate the µg/µl concentration of the probe. A typical 20 µl transcription reaction (containing 0.5 mM of each NTP) and subsequent gel purification should yield about 4-8 µg of RNA. The probe should be stored at -20åC or -80åC for greater stability. 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 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 from 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 particular 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 probe size decreases, the amount of probe added to the hybridization reaction should be decreased in a linear manner.

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.

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