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Degradation of mRNA after cell lysis?

Degradation of mRNA after cell lysis? - Biology Forum

Degradation of mRNA after cell lysis? - Biology Forums. Ask questions and discuss the study of Biology. If you have Biology questions from your homework ask them here!


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Old 11-17-2010, 05:52 PM
Pipette Filler
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Default Degradation of mRNA after cell lysis?



I extract mRNA from a sample by lyseing the cells and using a magmax RNA extraction kit. I then RT the samples to get cRNA and run a qPCR. Recently I received samples of cells lysed by virus in media. I extracted 50ul, RT’d and ran a qPCR for detection of a target mRNA (cDNA now). I was told that I was expected to measure viral particles (oops!) and that there wouldn’t be any cellular DNA and RNA because ‘once the cells lyse there is destruction of the cellular DNA and RNA by nucleases’. The cell’s nucleases? I can’t see how this is true since I lyse my cells for mRNA extraction all the time. What am I not understanding? (also, I did see target detection in my qPCR)

Somebody please help! Thanks!

-DZ
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Old 11-17-2010, 07:21 PM
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Default Re: Degradation of mRNA after cell lysis?

First, there are nucleases in the extracellular matrix, too. For instance, skin has nucleases (skin is formed by death keratinized cells, well its outer part is). Those nucleases will stay even after the cells die.

Second, the first step of RNA extraction involves destruction or denaturing of nucleases by strong denaturing agents like guanidine isotiocyanate, which is added prior to cell lysis. Reagents like TRIzol do both things at the same time (get rid of nucleases and lyse the cells). Even if your cells are death, nucleases will be active, so the same first step must be given to extract viral RNA.
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Old 12-08-2010, 06:32 PM
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Default Re: Degradation of mRNA after cell lysis?

Remember, nucleases are enzymes. Enzymes doesn't need to be inside a living complex (cell, tissue, organism) to be active. As long as there are substrate and the necessary cofactors (metal ions, i.e. Mg, Mn...) they can work even in sterile water.

By the way, greh there is a quote option which you can use to quote other peoples posts (or part of them).
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