I wonder if I can heat Trizol reagent for 30 min 65C. The goal is to disrupt protein-RNA complex while inhibiting nucleases. (I can't use RNasin cause it's inactivated in 65C, and can't use RVC cause reaction contains EDTA). I realize that heating Trizol is not safe and its vapors are hazardous, but i can perform it in the hood.
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1$\begingroup$ As far as I know, Trizol disrupts protein-RNA complexes on its own - check the protocol. Also, phenol, a major component, is flammable. I don't know what the flash point of Trizol is, but I would be extremely careful. This protocol claims you can heat it. Does your experiment not work at RT? $\endgroup$– MattDMoCommented Apr 8, 2013 at 15:46
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$\begingroup$ Heating wont be a nice idea. But if your cells are too resistant to lysis then you can keep the tubes in a warm water bath. However, bring it back to RT before adding chloroform. BTW what sample is this? $\endgroup$– WYSIWYGCommented Apr 8, 2013 at 16:08
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$\begingroup$ If you are worried about safety, contact your university's Environmental Health and Safety office (or the equivalent). They can (should, and will) advise you on safety issues. $\endgroup$– kmmCommented Apr 9, 2013 at 0:46
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$\begingroup$ What kind of cells or complexes do you have? I have worked a lot with Phenol based reagents but never found problems to work with it at room temperature. Besides that Phenol is volatile, especially at higher temperatures. You will have to do that in a good fume hood, otherwise this is a serious health issue. And definitely cool down before adding Chloroform. $\endgroup$– Chris ♦Commented Jan 18, 2014 at 9:56
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Working with hot phenolic solutions is rarely been done due to the dangers of this. Phenol needs to be handled in the fume hood anyway, at higher temperatures it gets even more volatile. So if you plan to do this, do it with extra care to avoid injury.
There is one paper that describes this method for bacteria, but this should work for other samples as well: "Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation". However, there is another method, which was developed for extracting RNA from plant material infected by bacteria which is also worth a try (although it might need some adaptation), which works without hot phenol: "An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7".
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$\begingroup$ So what happens if you don't keep your phenol:chloroform in the fume hood? Because I haven't. Am I going to die? I also occasionally touch a gel that contains ethidium. Should I be writing my will? $\endgroup$– user137Commented Aug 5, 2014 at 19:03
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$\begingroup$ You should be very careful with Phenol as this causes nasty and long lasting chemical burns. It basically desintegrates the cells, thats why we use it for setting the nucleic acids free. Inhaling is a bad idea. EtBr on the other hand should be handled with care, but most guides about the dangers are overrated. It is mutagen (can cause mutations) and teratogen (can cause damages to the developing embryo) but I haven't seen any proof yet, that it is cancerogen (except when causing mutations). This is different for benzene for example, which is directly cancerogen. $\endgroup$– Chris ♦Commented Aug 5, 2014 at 20:01
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$\begingroup$ Also: EtBr has a very good water solubility (which means it doesn't cross the skin barrier very easy, especially not as a charged molecule) and it is light sensitive. So use it with care, but don'T overreact. $\endgroup$– Chris ♦Commented Aug 5, 2014 at 20:02