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A rapid, accurate, nonradioactive method for quantitating RNA on agarose gels.
1Department of Molecular and Cellular Biochemistry, Loyola University of Chicago, Maywood, IL 60153.
Biotechniques
|December 1, 1991
Summary
This study demonstrates a radiation-free method for standardizing Northern blots. Ethidium bromide-stained 28S ribosomal RNA (rRNA) fluorescence can be quantified to ensure accurate gene expression analysis.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Quantitative gene expression analysis using Northern blots requires reliable internal standards.
- Accurate loading controls are crucial for verifying even sample loading and correcting for variations between lanes.
Purpose of the Study:
- To establish a non-radioactive method for internal standardization in Northern blot analysis.
- To validate the use of 28S ribosomal RNA (rRNA) fluorescence for quantitative standardization.
Main Methods:
- Two-dimensional quantitation of ethidium bromide-intercalated 28S rRNA fluorescence.
- Assessing the linearity of film response with respect to loaded RNA amounts.
Main Results:
- Ethidium bromide-stained 28S rRNA fluorescence can be effectively quantified for Northern blot standardization.
- A linear relationship was observed between fluorescence and loaded RNA (2.5-12.5 micrograms) under the tested conditions.
- The linear range for standardization was identified, beyond which the relationship deviates.
Conclusions:
- Two-dimensional quantitation of 28S rRNA fluorescence provides a viable, radiation-free alternative for Northern blot standardization.
- This method enhances the accuracy and safety of quantitative gene expression studies.