Today I was isolating RNA from human atherosclerotic plaque tissue. During the process, I found the interphase extremely thick. Does anyone know of a reason what could cause this? Does it have further effects on the RNA isolation?
The interphase of a Trizol-based RNA extraction typically captures the DNA while proteins are trapped in the lower organic layer. The interphase thickness will generally correlate to the amount of cells/tissue that are used. You should do your best to avoid collecting any interphase or organic phase as these will reduce the purity of RNA due to DNA or phenol contamination. However, a substantial interphase is good to have as it will minimize the chance of phenol contamination.
Since you are doing a high lipid content tissue, it may also be beneficial to remove any visible fat layer present at the top of the phenol after tissue homogenization. (see Article Optimising total RNA quality and quantity by phenol-chlorofo...
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Hi Imke Jansen
I guess amount of your sample is alot or have much more lipid and protein that interphase become very thick. You can reduce your primery sample or increase amount of Trizol to have pure RNA.
Be successful
I agree with Omid Rezaie and John Hardy Lockhart the interphase usually consists of the genomic DNA and in the case of tissues, I too have observed higher levels of lipids. Maybe the fact that your atherosclerotic plaques themselves are rich in lipids may have contributed to a thicker interphase level. I do not feel it would affect your RNA quality as long as you do not disturb the interphase.
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