Methods for Enzymatic Nonisotopic Labeling of RNA by in Vitro Transcription

• A wide variety of modified nucleotides can be used to make nonisotopic RNA probes
• Probes are compatible with nuclease protection assays, Northerns, Southerns and dot blots, regardless of the type of label used
• Yields approximately 4 µg of nonisotopically labeled probe per small-scale transcription reaction - enough for up to 2000 RPA reactions or 400 ml of Northern (or Southern) hybridization buffer
• Only make probes once. Nonisotopic probes do not undergo radiolysis and are stable for at least a year
• Biotinylated RNA probes are ideal for detection by Ambion's BrightStar® BioDetect™ Kit
• Synthesize fluorescent probes for direct detection by FISH

There is an increasing interest in the use of nonisotopically labeled probes for RNA analysis due to the high cost of radioactive waste disposal and possible health concerns. Also, nonisotopic probes do not undergo radiolysis, so they are stable for at least a year when stored correctly. Nonisotopic RNA probes can be generated by chemical crosslinking (e.g., Ambion's BrightStar® Psoralen-Biotin Nonisotopic Labeling Kit) or by incorporating modified nucleotides in an in vitro transcription reaction (using Ambion's MAXIscript® Kit). Once labeled, nonisotopically modified probes can be used in ribonuclease protection assays, Northerns, Southerns and dot blots. When transcribing with nonisotopically labeled nucleotides, synthesis is robust since there is no limiting nucleotide, and approximately 4 µg of transcript can be synthesized from each reaction. Since Northern, Southern and dot blot analysis require only about 10 ng of probe per ml of hybridization solution (for a 300 nt probe; 0.1 nM final concentration), and RPAs require only 200-800 pg of probe per reaction, a single nonisotopic MAXIscript reaction generates enough probe for hundreds of assays. Note that blot hybridizations performed with Ambion's ULTRAhyb® will require 10-100 fold less nonisotopic probe than hybridizations carried out in standard hybridization solutions.

Will Nonisotopic Systems Be Sensitive Enough?

Ambion has put considerable effort into reducing the background and increasing the signal-to-noise ratio in nonisotopic detection methodology. This has led to a detection system that rivals the sensitivity of ³²P-labeled probes. When Ambion's BrightStar BioDetect Kit is used with a biotinylated RNA probe made by either chemical crosslinking or enzymatic incorporation of the label, there is a very simple rule of thumb to determine whether this nonisotopic assay will be sensitive enough: In any system (Northern, Southern, RPA, etc.) in which a signal can be detected in less than two days with ³²P, you can generally switch to this biotinylated system without compromising the quality of your data. This represents the current effective upper limit of sensitivity for nonisotopic systems, corresponding to the detection of a few hundred femtograms. If longer exposures with ³²P are required, switching to RNA probes from DNA probes, from Northerns to RPAs, or from total RNA to poly(A) RNA may very well decrease exposure time sufficiently to allow the use of nonisotopic detection.

Issues Concerning the Use of Modified Nucleotides

There are several issues that should be considered when synthesizing a nonisotopically labeled probe. The presence of the modified nucleotide may affect the efficiency of the labeling reaction, as well as the effectiveness of the probe in subsequent applications. The type, size and location of the modification on the nucleotide will determine how well it is incorporated into the transcript by the RNA polymerase. The modified nucleotides within the probe might also interfere with hybridization of the probe to the target sequence through steric hindrance. Nuclease digestion, such as in RPAs, may also be affected by modified nucleotides.

Ratio of Modified Nucleotide Incorporated

Researchers using in vitro transcription to incorporate modified nucleotides are usually interested in obtaining the highest possible yield from their reactions. Therefore, the final nucleotide concentration is kept at 0.5 mM in the transcription reaction (no limiting nucleotide) which is equal to 1 µl of each 10 mM NTP stock in a 20 µl MAXIscript reaction. Ideally, the modified nucleotide is used at a level that allows the highest percent incorporation without inhibiting either the transcription reaction or subsequent hybridization. We have found that in most cases a ratio of modified nucleotide to unlabeled nucleotide of 1:1 to 1:3 works well (i.e., 0.2 mM Biotin-14-CTP and 0.3 mM CTP). Ambion has tested a variety of modified nucleotides in our MAXIscript Kit. Recommended ratios and sources for these modified nucleotides are listed below. For nucleotides not listed below, it may be helpful to perform a pilot experiment to determine the optimum amount of a given modified nucleotide to be used in transcription and subsequent hybridization reactions. An example of a pilot experiment is given below.

Standard Protocol for In Vitro Transcription Reaction

The following recipe assumes labeling with a modified UTP nucleotide and can be adjusted for modified CTP nucleotides. The ratio of modified:unlabeled NTP can also be adjusted.

Protocol:

1. Thaw all non-enzyme reagent solutions at room temperature and place on ice. The phage RNA polymerase enzyme should be placed directly on ice.
   
   
2. Assemble the following components in the indicated order at room temperature, as the spermidine present in the transcription buffer can cause precipitation of template DNA at lower temperatures.
   
    Final volume of 20 µl:
    -- µl Nuclease-free water to 20 µl final volume
    2 µl 10X Transcription Buffer
    1 µl 10 mM ATP
    1 µl 10 mM CTP
    1 µl 10 mM GTP
    -- µl 10 mM UTP (e.g., 0.6 µl)
    -- µl 10 mM labeled UTP (e.g., 0.4 µl)
    -- 0.5–1 µg linearized template DNA
   2 µl SP6, T3, or T7 RNA Polymerase (5 U/µl) + RNase Inhibitor (5 U/µl)
   _____
    20 µl
   
   
3.  Incubate the reaction mixture at 37°C for 2 hours.

Each 20 µl reaction will yield approximately 4 µg of transcript (10 µl reactions will yield approximately 2 µg).

Removal of Template DNA

If the in vitro synthesized transcript is not going to be gel purified, it is important to remove the DNA template prior to using the transcript as a probe. It should be noted that gel purification is not essential if the RNA is going to be used a probe for hybridization to target sequences bound to a solid support (e.g., membrane, filter, slides). Even if truncated probe molecules are generated in the transcription reaction, they will still hybridize and yield a positive signal. However, it is desirable to gel purify the probe if it is going to be used in a nuclease protection assay.

Protocol:

1. Add 1 µl DNase I to the transcription reaction.
   
   
2. Bring to 37°C and incubate for 15 minutes.

Note: In some transcription reactions, the template DNA may prove to be refractory to complete digestion by the DNase I. It is presumed that a small amount of transcript hybridizes to the template from which it was transcribed, protecting it from both DNase I and RNase digestion. In Northern blotting, such DNA/RNA hybrids will not present a problem. In nuclease protection assays, they may be more apparent. It may help to reduce the amount of template used in the transcription reaction when this occurs.

Removal of Unincorporated Nucleotides

There are three common methods for removing unincorporated nucleotides:

1. Precipitation with LiCl or ammonium acetate/ethanol,
   
   
2. Spin column filtration, and
   
   
3. Gel purification.

Gel Analysis and Gel Purification

Gel purification is straightforward and easy. After transcription the reaction is run on a denaturing polyacrylamide gel (a "mini" protein gel apparatus can be used) to separate the DNA template, full-length RNA probe, any prematurely terminateproducts and free-nucleotides by size. The gel is either stained or UV shadowed. Full-length probe is then 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 usually produce enough probe for hybridization in just 1–4 hours.

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)

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 to the probe or, if the probe has been precipitated, resuspend direct 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 onto the gel. Run the gel until the more rapidly moving blue dye front (bromophenol blue) reaches the bottom of the gel (200 volts for about 30 min for minigels).

3. Visualization of the gel
Nonisotopic and unlabeled probes cannot be directly visualized by exposing the gel to film as with radioisotopic probes. However, since a large mass of transcript is generated (several micrograms), these probes may be visualized by UV shadowing or by staining (with ethidium bromide or acridine orange).

UV Shadowing
After electrophoresis, remove one of the glass plates and cover the gel with plastic wrap. Place the gel, gel side down, on a flat surface and slowly remove the gel from the second glass plate. The gel should then be covered with another sheet of plastic wrap so that both sides of the gel are now covered. In a darkened room, place the plastic-wrapped gel on top of a fluor-coated TLC plate (or a standard intensifying screen) and visualize the bands by shining a hand-held UV light source (set on short wavelength or 254 nm) on the surface of the gel. The RNA transcripts absorb the UV light and appear as purple "shadow" bands. Excise the band representing the full-length transcript (cutting out the smallest gel slice possible) using an RNase-free razor blade or scalpel.

Staining
As an alternative to UV shadowing, the RNA transcripts may be visualized by staining the gel with ethidium bromide or acridine orange. It is important to note, however, that the stain should be removed before hybridization, as it may compromise hybridization efficiency.

4. Probe elution
Transfer the gel fragment, either as a whole piece or cut into several smaller pieces, to a nuclease-free microfuge tube containing enough Elution Buffer to completely submerge it (about 350 µl). Since the RNA moves out of the gel slice by passive diffusion, any RNase-free solution should work. However, we recommend 0.5 M NH₄OAc/1mM EDTA/0.2% SDS, because the EDTA and SDS will inactivate low levels of nuclease and the NH₄OAc allows easy precipitation with the addition of 3 volumes of 100% ethanol. Incubate the tube at 37°C. Approximately 50% of a probe <400 nt will have eluted after about 2 hours (dependent on length of probe). However, we routinely incubate overnight for convenience and to maximize recovery of the probe.

Calculation of Yield

After precipitation or gel purification of the RNA probe, the yield of the probe can be determined by measuring the A₂₆₀ units. Measure the A₂₆₀ of a diluted aliquot of the RNA probe solution (e.g., 5 µl RNA solution to 500 µl water). Multiply the A₂₆₀ reading by the dilution factor (e.g., 100) and by 40 (1 A₂₆₀ unit = 40 µg/ml RNA). A typical transcription reaction yields approximately 4 µg of RNA.

Alternatively, an ethidium bromide (EtBr) spot assay may be used to obtain an approximate calculation of yield. Dilutions of a known amount of RNA standard can be spotted onto agarose containing EtBr, and labeled RNA of unknown concentration can be spotted next to these dilutions. The intensity of the spots from the unknown concentrations of RNA can be compared to those of the known concentrations of the diluted RNA standard, and the amounts of the unknown concentrations can be found by interpolation.

To avoid freeze-thawing, the majority of the labeled probe should be stored in 5 µl aliquots at -80°C. The currently used aliquot may be stored at -20°C. The probe should be stable for at least a year stored at -80°C in the absence of nuclease contamination.

Amount of Probe to Use in Hybridization Applications

For nonisotopic nuclease protection assays, we recommend using between 200–800 pg of a 300 nucleotide-long RNA probe per 20 µg sample of total RNA. This achieves a sufficient molar excess of probe over target for even moderately abundant mRNAs such as ß-actin (i.e., you need even less for rare messages). The final concentration of the nonisotopically-labeled probe in a Northern, Southern, dot blot or colony hybridization buffer should be 0.1 nM. For a 300 nt-long RNA probe, this is equivalent to 10 ng/ml. Nonisotopic probes used with ULTRAhyb Hybridization Solution should be 10–100 fold more dilute as described previously.

Pilot Experiment

At Ambion, we have already determined optimum ratios of incorporation for the modified nucleotides listed above. However, if a different modified nucleotide will be used, we recommend performing the following pilot experiment, which is analogous to the methods used at Ambion. This experiment will help determine the optimum ratio of modified nucleotide:unlabeled nucleotide that will provide the highest sensitivity probe without interfering with hybridization. Set up several transcription reactions (see Standard Protocol above) each with a different ratio of modified nucleotide:unlabeled nucleotide (e.g., 20%:80%, 25%:75%, 33%:67%, 50%:50%) with a final concentration of each of the four types of nucleotides of 0.5 mM. This means, for example, that if you want to have 25% fluorescein-12-UTP in a transcription reaction, 0.5 mM x 25% or 0.125 mM fluorescein-12-UTP should be added as well as 0.375 mM unmodified UTP. All other components of the transcription reactions should be identical. The template can be an internal control like ß-actin or any sequence abundant in your lab and known to give a good signal with the sample RNA. After performing the transcription reactions and removing the unincorporated nucleotides, the probes can be tested in functional assays each against several replicates of a fixed amount of sample RNA followed by normal detection procedures. The probe preparation that provides the strongest signal or highest sensitivity should indicate the optimum ratio of labeled:unlabeled nucleotide to use.

To avoid running gels and transferring to membranes, dot blot analysis can be used for either membrane or solution hybridization assays. For example, probes to be used in Northerns could be analyzed by hybridizing the probes (0.1 nM or 10 ng of probe per ml of hybridization solution for a 300 nt probe) to membrane strips containing equivalent amounts of RNA (1–10 µg of total RNA for ß-actin). Probes for use in RPAs can be analyzed by hybridizing 200–800 pg of probe to 1–5 µg of sample RNA followed by normal RNase digestion. Each of these reactions can be spotted directly onto a membrane. Normal detection procedures should then be followed.

Alternatively, for quick and easy analysis of the nonisotopically labeled transcription products without having to perform the secondary detection, include a trace amount of ³²P-labeled nucleotide (e.g., 0.1–0.25 µl of 800 Ci/mM, 10 mCi/ml of ³²P-CTP) in each of the above reactions. These probes can be evaluated after performing functional assays or by direct dot blot analysis following transcription and removal of unincorporated nucleotides. Subsequent overnight exposure to X-ray film should reveal the probe preparation that provides the strongest signal or highest sensitivity.

Nucleotide Sources
The MAXIscript Kit has been shown to function well with modified nucleotides from a variety of
sources. Some of these include:

Nucleotide	Company	Cat. #	Ratio Labeled:Unlabeled
Biotin-11-UTP	Ambion	8450	40%:60%
Biotin-16-UTP	Ambion	8452	40%:60%
Digoxigenin-11-UTP	Boehringer Mannheim Corp.	1-209-256	33%:66%
Fluorescein-12-UTP	Boehringer Mannheim Corp.	1-427-857	50%:50%
Guanosine	Sigma Chemical Co.	G6752	2.5 mM:0.5 mM *

*Note: Please call Ambion for more detailed information concerning the use of guanosine for synthesis
of RNA transcripts ready for 5´ end-labeling.

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