Quantitative reverse transcription-PCR-HPLC for nerve growth factor mRNA using a deletion RNA as an internal standard

Area of Science:

• Molecular Biology
• Biochemistry
• Neuroscience

Background:

• Accurate quantification of mRNA is crucial for understanding gene expression.
• Traditional methods like Northern blotting struggle with low-abundance targets.
• Nerve Growth Factor (NGF) mRNA is vital for neuronal development and function but often present at low levels.

Purpose of the Study:

• To develop a reliable and reproducible method for measuring absolute amounts of nerve growth factor (NGF) mRNA in tissue samples.
• To enable routine quantification of low-abundance mRNAs, overcoming limitations of existing techniques.

Main Methods:

• A novel approach combining RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR), and high-performance liquid chromatography (HPLC).
• Incorporation of a deletion mutant RNA as an internal standard for accurate RNA recovery correction.
• Optimization of PCR conditions for linear amplification and high reproducibility.

Main Results:

• The method successfully quantified NGF mRNA in skin (14.1 ± 4.6 fg/mg) and skeletal muscle (11.0 ± 2.2 fg/mg).
• Achieved high precision with a coefficient of variation below 2.8%.
• Demonstrated applicability to tissues with low mRNA levels, undetectable by Northern blotting.

Conclusions:

• The developed method provides a convenient and reliable assay for absolute NGF mRNA quantification in tissues.
• This technique is adaptable for the routine measurement of other low-abundance mRNAs.
• Offers a significant advancement for molecular biology research involving gene expression analysis.