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mRNA calculator

Extinction coefficient of an RNA sequence, and the concentration behind an A260 reading.

RNA concentration
How to turn an A260 reading into an RNA concentration
Absorbance at 260 nm follows the Beer-Lambert law, so the concentration of a pure RNA is its absorbance divided by its extinction coefficient and the path length of the cuvette or pedestal:
ε₂₆₀ = 0.812 × (nA × 15.02 + nC × 7.07 + nG × 12.08 + nU × 9.66)  mM⁻¹cm⁻¹
c (mol/L) = A260 ÷ (ε₂₆₀ × path length)
c (µg/mL) = c (mol/L) × molecular weight
The four coefficients are the revised values for the ribonucleoside 5-prime monophosphates measured by Cavaluzzi and Borer (Nucleic Acids Res 2004, 32:e13), in litres per millimole per centimetre. The factor 0.812 is the hypochromicity correction described below. Molecular weight is the sum of the residue masses, A 329.21, C 305.18, G 345.21 and U 306.17 g/mol, less 61.96 g/mol for a strand with a free 5-prime hydroxyl.
Why the sum of the nucleotides is too high
Bases stacked in a strand absorb less than the same bases free in solution. For unpaired DNA and RNA this hypochromicity is 14 to 24 %, so a coefficient added up from mononucleotides makes the molecule look more absorbing than it is, and the concentration read off it comes out that much too low. The same paper recommends 8.9 A260 units per micromole of residues, or about 37 µg per A260 unit, for complex single-stranded RNA. Scaling the mononucleotide sum by0.812 reproduces that figure while keeping the dependence on base composition. Clear the checkbox to see the uncorrected sum, which is what calculators that ignore hypochromicity report.
Worked example
"Try an example" loads a 720 nt transcript with A 174, C 240, G 203 and U 103. With no reading entered yet the tool reports the sequence itself: a corrected extinction coefficient of 6.30 × 10⁶ M⁻¹cm⁻¹, a molecular weight of 232.1 kDa, and one A260 unit worth36.8 µg/mL, close to the general figure of 37 for complex RNA.
Now type 1.20 into "A260 reading" and 100 into "Sample volume". The concentration is 1.20 ÷ (6.30 × 10⁶) = 1.91 × 10⁻⁷ mol/L, that is 191 nM, and multiplying by the molecular weight of 232,077 g/mol gives 44.2 ng/µL. The 100 µL sample therefore holds 4.42 µg, or 19.1 pmol. Clear the hypochromicity checkbox and the coefficient rises to 7.76 × 10⁶ M⁻¹cm⁻¹ and the concentration drops to 35.9 ng/µL, which is the error that correction avoids.
Reading the A260 sensibly
  • Keep the reading between about 0.1 and 1.0. Below that the blank dominates; above it most instruments leave their linear range, and a pedestal reading of 2 or 3 should be diluted and multiplied back.
  • Blank on the same buffer, not on water. Phenol, guanidinium and EDTA all absorb near 260 nm.
  • A260 does not know what it is measuring. Free nucleotides, residual DNA and degraded RNA all absorb, so a good number is not proof of an intact transcript. Check A260/A280, which should be near 2.0 for RNA, and run a gel or a capillary trace.
  • The result is for a single strand. A duplex, or a structured RNA held in a folded form, absorbs less still.
Related calculators
For DNA mass and molarity, and for oligonucleotides, use thenucleic acid weight and molarity converter. For the Beer-Lambert law on its own, with any extinction coefficient, use theBeer-Lambert calculator. For length, base composition and molecular weight of a sequence, use thesequence statistics tool, and to transcribe a gene into its mRNA,DNA to RNA.
Frequently asked questions
How do you calculate RNA concentration from A260?
Divide the absorbance by the extinction coefficient of the sequence and the path length, which gives the molar concentration, then multiply by the molecular weight to get a mass concentration. The extinction coefficient of the whole molecule is the sum of its nucleotide coefficients reduced by hypochromicity, so it depends on how long the RNA is and on its base composition.
Why does this not use the 40 µg/mL rule?
It gives the same kind of answer but from the sequence rather than from an average. The rule that one A260 unit is 40 µg/mL of RNA comes from measurements on mixed RNA; Cavaluzzi and Borer (2004) revised it to about 37 µg per A260 unit for complex single-stranded RNA. This calculator reports the figure for your own sequence, which for a GC-rich transcript is a little lower and for an A-rich one a little higher.
What is hypochromicity and why does it matter?
Stacked bases absorb less light than the same bases free in solution, so adding up mononucleotide coefficients overestimates the absorbance of an intact strand by 14 to 24 percent. Ignoring it makes the extinction coefficient too large and the reported concentration too low by the same margin. The correction here is a flat factor of 0.812 on the sum, which reproduces the 8.9 A260 units per micromole of residues that Cavaluzzi and Borer recommend for complex single-stranded RNA.
Does it work for modified mRNA with pseudouridine?
The sequence is read as ordinary A, C, G and U. Pseudouridine and N1-methylpseudouridine absorb less at 260 nm than uridine, so a fully substituted transcript reads a little lower than this calculator assumes and its true concentration is slightly higher than reported. For released material, quantify modified mRNA against a standard curve of the same modified transcript rather than against a sequence calculation.
Does the calculator include the cap and the poly-A tail?
Only what you paste. Add the poly-A tail to the sequence if it is part of the transcript, since a 120 nt tail is a real part of the mass and the absorbance. The molecular weight is for a strand with a free 5-prime hydroxyl; an uncapped in vitro transcript carries a 5-prime triphosphate, which adds 159.96 g/mol, and a cap adds one more nucleotide. On a transcript of a thousand bases or more both are well under half a percent.