Ambion, Inc. - Determining RNA Probe Specific Activity

Technical Resources > Appendix > Tools & Calculators

RNA Probe Specific Activity Calculator

This interactive calculator will walk you through the calculations to determine the specific activity of a radiolabeled RNA probe produced by in vitro transcription.

Step 1. Calculate the Stock Concentration of Radiolabeled Nucleotide

What is the specific activity of your radiolabeled nucleotide stock (Ci/mmol)?
What is the concentration of your radiolabeled nucleotide stock (mCi/ml)?

µM concentration of your radiolabeled nucleotide stock is:
µM

Radionuclide concentration (mCi/ml)		Specific activity of radiolabeled nucleotide (Ci/mmol)		Ci		1000 ml		1000 µmol		µ M radiolabeled nucleotide
	x		x		x		x		=
				1000 mCi		L		mmol

Step 2. Determine the Moles of Labeled and Unlabeled Nucleotide in Your Transcription Reaction

What is the volume of radioactive nucleotide in your transcription reaction (µl)?
What is the volume of unlabeled nucleotide in your transcription reaction (µl)? Consider only the same nucleotide as your labeled nucleotide.
What is the concentration of unlabeled nucleotide added to the transcription reaction (µM)?


The moles of labeled nucleotide in the reaction is:	The moles of unlabeled nucleotide in the reaction is:	The total moles of labeled + unlabeled nucleotide in the reaction is:
pmol	pmol	pmol

Vol radiolabeled nucleotide (µl)		Conc radiolabeled nucleotide (µM)		1 L		mol		10¹² pmol
	x		x		x		x		=	pmol nucleotide
				10⁶ µl		10⁶ µmol		mol

Step 3. Calculate the Maximum Theorectical Yield

The maximum amount of RNA synthesized in the reaction is: µg

Since the total moles of labeled + unlabeled nucleotide is limiting, one can assume that each of the other three nucleotides can be incorporated only up to this amount . The sum of the molecular weights of the four ribonucleotides is about 1320 g/mol.

		1320 g		mol		10⁶µg
pmol nucleotide	x		x		x		=	µg RNA
		mol		10¹²pmol		g

Step 4. Calculate the Theoretical Specific Activity

The theoretical specific activity is: cpm/µg

1 µCi = 2.222 x 10⁶ cpm

Radionuclide stock concentration (mCi/ml)	x	Vol radiolabeled nucleotide added (µl)	x	ml 1000 µl	x	1000 µCi mCi	x	2.222 x 10⁶ cpm 1 µCi		Theoretical specific activity (cpm/µg)
									=
µg RNA

Step 5. Calculate the Efficiency of Radiolabeled Nucleotide Incorporation

To perform this step, you must have counted two aliquots of reaction product: one counted after addition of the radiolabeled nucleotide before any other manipulation, and the other counted after removal of unincorporated nucleotides.

Unincorporated nucleotides can be removed by TCA precipitation or spin column (see Technical Bulletin 174 for details on TCA precipitation). Because spin columns dilute your sample, the calculations are slightly different depending on the method of unincorporated nucleotide removal. Use only one (A or B) of the below sets of calculations.

A. TCA Precipitation Method
(two aliquots are removed after the reaction: the first is counted directly and the second is TCA precipitated and then counted)

What was the final reaction volume just prior to aliquot removal (µl)?

What was the volume of the aliquot counted
with no other manipulation (µl)?

How many counts per minute were found for the aliquot counted
with no other manipulation (cpm)?

What was the volume of the aliquot from which unincorporated nucleotides were removed prior to counting (µl)?

How many counts per minute were found for the aliquot from which unincorporated nucleotides were removed (cpm)?

The percent of radiolabeled nucleotide incorporated is %

cpm/µl after removal of unincorporated nucleotides		100%		percent incorporation
	x		=
cpm/µl before removal of unincorporated nucleotides

B. Spin Column Method
(an aliquot is removed directly after the labeling reaction; the remaining reaction product is put through a spin column)

What was the final reaction volume before spin column treatment (µl)?

What was the volume of the aliquot removed (before spin column treatment) (µl)?

How many counts per minute were found for the aliquot taken prior to spin column treatment (cpm)?

What was the total volume of the reaction after spin column treatment (µl)?

What was the volume of the aliquot removed after spin column treatment (µl)?

How many counts per minute were found for the aliquot taken after spin column treatment (cpm)?

The percent of radiolabeled nucleotide incorporated is %

cpm/µl after removal of unincorporated nucleotides		Dilution factor		100%		percent incorporation
	x		x		=
cpm/µl before removal of unincorporated nucleotides

Step 6. Calculate the Amount of RNA Synthesized

The amount of RNA that was synthesized is µg

maximum theoretical yield (µg RNA)

percent incorporation

100

µg RNA synthesized

Step 7. Calculate the cpm Incorporated into the RNA Product

Counts per minute incorporated into the RNA product is
cpm

Rxn Vol (µl)

cpm/µl after removal of unincorporated nucleotides*

cpm incorporated

Step 8. Calculate the Specific Activity of the Probe

The specific activity of the probe is cpm/µg

cpm incorporated

µg RNA synthesized

specific activity (cpm/µg)