In one experiment that I am planning, I need to visualize the morphology of arteries in mice using methylene blue. I however would like to be able to use some of the tissues for real time quantitative PCR. Does any one know if methylene blue affects PCR results? References will be greatly appreciated.
Wikipedia: In biology methylene blue is used as a dye for a number of different staining procedures, such as Wright's stain and Jenner's stain. Since it is a temporary staining technique, methylene blue can also be used to examine RNA or DNA under the microscope or in a gel: as an example, a solution of methylene blue can be used to stain RNA on hybridization membranes in northern blotting to verify the amount of nucleic acid present. While methylene blue is not as sensitive as ethidium bromide, it is less toxic and it does not intercalate in nucleic acid chains, thus avoiding interference with nucleic acid retention on hybridization membranes or with the hybridization process itself.
It can also be used as an indicator to determine if a cell such as yeast is alive or not. The blue indicator turns colorless indicating living cells. However, if it stays blue it doesn't mean that the cell is dead or there are no cells. Methylene blue can inhibit the respiration of the yeast as it picks up hydrogen ions made during the process and the yeast cell cannot then use those ions to release energy
Thus methylene blue binds to RNA/DNA: http://www.ncbi.nlm.nih.gov/pubmed/19823747
Thus quantitative PCR may be influenced depending on your technique/read out.
Methylene blue has very limited fluorescence, neglegible stokes shift
And Methyl green is a completely different dye! Methylene blue is a redox active compound, methyl green ...
Wikipedia: In biology methylene blue is used as a dye for a number of different staining procedures, such as Wright's stain and Jenner's stain. Since it is a temporary staining technique, methylene blue can also be used to examine RNA or DNA under the microscope or in a gel: as an example, a solution of methylene blue can be used to stain RNA on hybridization membranes in northern blotting to verify the amount of nucleic acid present. While methylene blue is not as sensitive as ethidium bromide, it is less toxic and it does not intercalate in nucleic acid chains, thus avoiding interference with nucleic acid retention on hybridization membranes or with the hybridization process itself.
It can also be used as an indicator to determine if a cell such as yeast is alive or not. The blue indicator turns colorless indicating living cells. However, if it stays blue it doesn't mean that the cell is dead or there are no cells. Methylene blue can inhibit the respiration of the yeast as it picks up hydrogen ions made during the process and the yeast cell cannot then use those ions to release energy
Thus methylene blue binds to RNA/DNA: http://www.ncbi.nlm.nih.gov/pubmed/19823747
Thus quantitative PCR may be influenced depending on your technique/read out.
Methylene blue has very limited fluorescence, neglegible stokes shift
And Methyl green is a completely different dye! Methylene blue is a redox active compound, methyl green ...
I agree with Boris Schmidt, it affects your qPCR results only when you read your dye´s fluorescence within the same wavelenght of fluorescence of methylene blue; if it is the case you just have to change the stain or increase the time for your methylene blue washing process
- Badges
- Science method