Determining RNA Probe Specific Activity and Yield

The specific activity of a probe in a transcription reaction is independent of the yield of RNA. However, both the theoretical and experimental specific activity of a RNA probe can be determined.

Specific activity may be broadly defined as the level of radioactivity (in counts per minute or "cpm") exhibited by the products of a reaction in relation to the total mass of those products. In an in vitro transcription reaction such as MAXIscript™, specific activity refers to the counts per minute (and therefore the sensitivity to autoradiography) of the transcription product. This radioactivity is contributed by radiolabeled nucleotides incorporated into the transcript during the course of the reaction. Specific activity may be determined either by calculation based on reaction parameters and numbers supplied by the vendor of the radiolabeled nucleotide (theoretical specific activity), or by direct measurement via scintillation counting of the product after removal of unincorporated radioisotope (experimental specific activity).

The specific activity of a probe in a transcription reaction is determined solely by the ratio of [³²P]-NTP to unlabeled NTP added and is, therefore, independent of the yield of RNA. Specific activity may be calculated by assuming that any fixed amount of the radioactivity added to a reaction is incorporated into RNA, and then solving for the mass amount of product that would be made in the reaction, and how many cpm of radioactivity that product would contain. It is also helpful to assume that nucleotides are incorporated in equimolar ratios, so that the final fraction of the limiting nucleotide (e.g. "U") in the transcript is 0.25. If it is known that the composition of the RNA product differs significantly from a 1 to 4 ratio, a correction factor may be applied to reflect the actual amount of product synthesized.

The actual yield of product must be determined experimentally. This is usually done by measuring the amount of radioisotope incorporated into synthesized transcript. Based on the proportion of labeled to unlabeled limiting nucleotide, yield is then calculated.

This article will walk you through the calculations to determine RNA probe specific activity and yield.

Calculating Theoretical Yield and Specific Activity

To calculate the theoretical specific activity and yield of a probe, the amount of limiting nucleotide in the transcription reaction must be known. This is determined from the specific activity and amount of the isotope used in the transcription reaction plus, the amount of any unlabeled form of this nucleotide added. For example, if 5 µl of [alpha-32P]UTP with a specific activity and concentration of 800 Ci/mmol, 10mCi/ml, and 2 µl of 50 µM unlabeled UTP are added to a 20µl reaction,

1. The amount of radioactivity available to the reaction is determined:
Since the UTP has a concentration of 10 mCi/ml,
0.005 ml x 10 mCi = 0.05 mCi

Given that 1 µCi = 2.2x10⁶ cpm,
0.05 mCi or 50 µCi = 1.1x10⁸ cpm

2.The moles [alpha-32P]UTP in the reaction:
Since 800 Ci (8x10⁸ µCi) = 1 mmol, 50 µCi = 62.5 pmol

3.The moles of unlabeled UTP in the reaction:

4.The total UTP (the limiting nucleotide) in the reaction:
62.5 pmol + 100 pmol = 162.5 pmol UTP

5.The maximum yield of RNA from this reaction is determined:
Since the amount of total UTP in the reaction is limiting, each of the other three nucleotides can be incorporated only up to this same level. Therefore, one can assume 162.5 pmol of each ribonucleotide was incorporated.The sum of the molecular weights of the four ribonucleotides is about 1320 daltons. (The average molecular weight of a nucleotide in RNA is 330 daltons.)

(If the composition of the RNA product is known to differ significantly from that of the idealized product containing equimolar amounts of all 4 ribonucleotides, a correction factor can be applied to more accurately reflect the amount of product synthesized.)

6.Therefore, the theoretical specific activity for this probe is given by:
(1.1 x 10⁸ total cpm input into the reaction, from Step 1)

Calculating Experimental Yield and Specific Activity

The actual yield of RNA must be determined experimentally by establishing how much [³²P]UTP was incorporated into transcript product (the ratio of labeled to unlabeled UTP incorporated is assumed to reflect the ratio of labeled to unlabeled UTP available in the reaction). The amount of ³²P incorporated into RNA can be determined by trichloroacetic acid (TCA) precipitation and subsequent counting. Alternatively, an aliquot of product that has been separated from unincorporated nucleotides can be counted directly. Separation of the probe transcript from unincorporated nucleotides can be accomplished with a spin column, precipitation with NH₄OAc and EtOH or LiCl, or gel purification. (Note that yield based on product that has been gel purified only takes into account full length probe whereas the other methods will include any prematurely terminated transcripts.) In the following example, TCA precipitation will be used to determine the amount of radiolabel incorporated.

Consider again the example
A 20 µl transcription reaction contains 5 µl of ³²P-UTP (800 Ci/mM, 10 mCi/ml) and 2 µl of 50 µM unlabeled UTP. At the end of the synthesis reaction, 1 µl of DNase I is added; after incubation, 21 µl of gel loading buffer is added. A 2 µl aliquot of the final reaction is removed and diluted into 198 µl of TE containing 100 µg of carrier RNA; 100 µl of this dilution is counted directly in a scintillation counter (total radioisotope input into the reaction) and found to contain 2.6 x 10⁶ cpm. The amount of ³²P incorporated into RNA is determined by TCA precipitation and filtration of the remaining 100 µl of the diluted reaction as indicated in the sidebar on this page. The filter is counted and found to have 1.3 x 106 cpm. (The counting efficiency of the ³²P isotope in liquid scintillation cocktail is assumed to be 100%.) The specific activity of the RNA probe is calculated as follows:

1. The proportion of UTP incorporated into RNA (i.e. TCA-precipitable material):

2. The moles [alpha-32P]UTP in the reaction:
Again, this is calculated by converting the volume of [alpha-³²P]UTP added (5 µl) to the number of mCi of ³²P added, and then converting the amount of [alpha-³²P]UTP in mCi to a molar amount using the known specific activity and concentration of the [alpha-³²P]UTP (800 Ci/mmol, 10 mCi/ml).

3. The moles of unlabeled UTP in the reaction:

4.The total UTP in the reaction:
62.5 pmol [alpha-³²P]UTP + 100 pmol unlabeled UTP = 162.5 pmol total UTP

5. Total UTP incorporated into RNA:
162.5 pmol UTP x 50% incorporation = 81.5 pmol UTP incorporated

(This should not be confused with pmol of transcript, which is equal to pmol/#nucleotides)

6. Amount of RNA synthesized, in picograms:
Assume that the RNA synthesized contained equal molar amounts of all four ribonucleotides (ATP, CTP, GTP, and UTP). Therefore, one can assume 81.5 pmoles of each ribonucleotide was incorporated. The sum of the molecular weights of the four ribonucleotides is about 1320 daltons. (The average molecular weight of a nucleotide in RNA is 330 daltons.)

7. Cpm incorporated into the RNA product:
The final reaction volume from which the 2 µl sample was removed to determine label incorporation was 42 µl (20 µl transcription reaction + 1 µl DNase + 21 µl loading buffer). The amount of sample TCA precipitated and found to contain 1.3 x 10⁶ cpm was 1 µl (half of the 2 µl sample diluted 1:100 in 198 µl TE and carrier).

42 µl x 1.3 x 10⁶ cpm/µl = 55 x 10⁶ cpm of TCA-precipitable material (i.e. RNA)

8.Specific activity of the product:

Note that this number correlates well with the theoretical specific activity (5.12 x 10⁸ cpm/µg) calculated in Step 6.

Assessing Transcription Efficiency by TCA Precipitation

The efficiency of the transcription reaction can be determined by trichloroacetic acid (TCA) precipitation of the reaction products. Following is an example of how a reaction is assessed by TCA precipitation. The specific volumes and amounts can be varied depending on individual requirements and preferences.

At the completion of the reaction, remove a small aliquot, for example 2 µl, and dilute into 198 µl of TE containing 100 µg of RNase-free carrier nucleic acid (for example, sheared fish sperm DNA or yeast RNA). Add 100 µl of this mixture to an aqueous fluor and count in a scintillation counter to determine the total amount of radioactive nucleotide in the sample.

Add the remaining 100 µl to about 2 ml of cold 10% TCA in a 12 x 75 mm borosilicate tube, vortex briefly, and place on ice for about 5 minutes. The precipitated nucleic acid is then collected by vacuum filtration through a Whatman GF/C glass fiber filter, or its equivalent, which has been wetted with TCA solution. Wash the tube successively with 2 ml cold 10% TCA and twice with 2 ml per wash of 95% ethanol, passing the washes through the filter. (The exact amount of TCA solution used in not critical, but reproducibility is improved if all samples are washed with the same amount). Immerse the filter in aqueous fluor and count to determine the amount of radioactive nucleotide incorporated into RNA. (Note, if using toluene-based scintillation cocktail, the filter must be dried before counting.) The ratio of cpm on the filter to total cpm is the fraction of labeled nucleotide incorporated into RNA. This value should generally be at least 50%.