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Related Concept Videos

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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Other Nuclides: 31P, 19F, 15N NMR01:16

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Many organic, inorganic, and biological molecules contain spin-half nuclei such as nitrogen-15, fluorine-19, and phosphorus-31. As a result, NMR studies of these nuclei have found extensive applications in chemical and biological research.
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Quantification of mRNA Using 31P NMR Spectroscopy and CRAFT.

Gennady Khirich1,2, Vanessa A Noreika3, Kaitlyn Doolittle Catlin1

  • 1Protein Analytical Chemistry, Genentech, Inc., South San Francisco, California, USA.

Magnetic Resonance in Chemistry : MRC
|February 20, 2025
PubMed
Summary
This summary is machine-generated.

Quantitative 31P NMR spectroscopy offers a reliable method for measuring messenger RNA (mRNA) concentration. This technique accurately calibrates mRNA extinction coefficients, overcoming limitations of traditional UV spectroscopy for vaccine development.

Keywords:
31P qNMRCRAFTNMRUV spectroscopymRNAnucleotidesround‐robintime domain analysis

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Molecular Biology

Background:

  • Messenger RNA (mRNA) is a key therapeutic in vaccine development, requiring precise quantification.
  • Traditional UV spectroscopy for mRNA measurement is susceptible to solution condition variability.
  • Accurate calibration of mRNA extinction coefficients is essential for reliable concentration determination.

Purpose of the Study:

  • To establish a quantitative 31P NMR spectroscopy method for accurate mRNA concentration measurement.
  • To address the limitations of UV spectroscopy in quantifying mRNA across diverse chemical environments.
  • To enable precise calibration of mRNA extinction coefficients for research and manufacturing.

Main Methods:

  • Utilized quantitative 31P NMR spectroscopy as an orthogonal method for mRNA quantification.
  • Developed a quantitative treatment for mRNA samples with multiple polyadenylated sequences.
  • Investigated limitations of frequency-domain integration for broad mRNA signals via a round-robin study.
  • Employed time-domain CRAFT quantification for superior accuracy over integration.

Main Results:

  • The developed 31P NMR method introduces a bias of ≤ 4% in measured mRNA concentrations.
  • Frequency-domain integration of broad mRNA resonances presents limitations and subjectivity.
  • Time-domain CRAFT quantification using 31P NMR proved superior to integration.
  • Accurate total mRNA quantification was achieved, enabling precise extinction coefficient calibration.

Conclusions:

  • Quantitative 31P NMR spectroscopy provides an accurate and reliable method for total mRNA quantification.
  • This technique overcomes the limitations of UV spectroscopy for mRNA measurement.
  • Accurate calibration of mRNA extinction coefficients is now feasible, supporting vaccine development and manufacturing.