How can I extract intact total RNA from frozen HUman Skin? - This cumbersome work really kills me! (Jul/19/2006 )
Hi ladies and gentlemen:
Recently I encounter a problem that, I try to extract total RNA from frozen human skin, but I cannot get intact total RNA, plasticware were treated with 0.2% DEPC H2O, then autoclaved at 121 celsius degrees, for 45min, glassware and pestle and mortor were baked at 195 celsius degrees for more than 5hrs. Then work bench was treated with 5% commercial available bleach, then wiped by 75% ethanol (I wonder whether this can inactivate the residual RNase on the bench top) the centrifudge is treated with just 75% ethanol , I abide with the RNA protocol strictly. I pulverize the human skin ( stored in liquid nitrogen) is liquid nitrogen. I add 1ml Trizol per 30mg skin powder before tissue thawed. in the subsequent steps I change my plastic glove once after I touch others besides the microcentrifuge tube.
So, one problem is whether my Workbench is treated correctly, If not , How should I treat the workbench?
the other is 1ml Trizol per 30mg skin powder is enough?!
Any one can give me some suggestion to optimize total RNA extraction. I sincerely expect anybody abundent in human Skin RNA extraction experience give me some valuable suggestion.
BTW, Anyone has this article:"Solubilzation in formamide protects RNA from degradation" by Chomczynski, Nucl. Acids Res.1992; 20: 3791-3792
Many thanks in advance!
Albert
Albert,
I don't use Trizol, so I am of no help there, but I use the Qiagen RNeasy kit, and the name does not disappoint. If you continue to have troubles, consider using this method. You might want to check out the Qiagen website-they have pdf's of their protocols so you can look at it before you buy and decide if it is a good method for you. Good luck!
I don't use Trizol, so I am of no help there, but I use the Qiagen RNeasy kit, and the name does not disappoint. If you continue to have troubles, consider using this method. You might want to check out the Qiagen website-they have pdf's of their protocols so you can look at it before you buy and decide if it is a good method for you. Good luck!
Hi LabGirl:
Thanks for your kind suggestion!
Today I again repeat the failure of isolating Total RNA from human skin using Trizol reagent.
I don't know why? I am deeply dicouraged now! When I precipitate the RNA form the aqueous phase using isopropanol, A strange phenomenon appears: I see white floccule forms. I remove the aqueous phase with disturbing the interphase. At last, The Agarose gel eletrophoresis shows no RNA bands but in the loading wells, very bright bands appears. I guess genomic DNA was mixed into the aqueous phase. I cannot understand that since I pippet the Aqeous phase without disturbing Interphase, where does genomic DNA come?
More suggestion is appreciated!
Many thanks
Albert
hi
you should add more trizol to your sample.
It's supposed that the volume of your sample = 1/10 of volume of trizol to be used.
Moreover, keep your samples frozen and add 56°-pre-heated trizol to them
you should add more trizol to your sample.
It's supposed that the volume of your sample = 1/10 of volume of trizol to be used.
Moreover, keep your samples frozen and add 56°-pre-heated trizol to them
Hi fred_33:
Thanks for your kind suggestion!
Today, I assess the PH of my Trizol, Trizol PH is 5.9 at RT, I wonder whether this will affect my isolation of RNA? Besides, you say add 56°-pre-heated trizol to my tissue, why? and the Trizol using protocol does not say.
Before, I add 1ml Trizol per 30mg skin powder, I found that the RNA Recovery is too bad. When I loading 10ul (1/3 of the resolution volume) RNA in electropherosis, the band is rather weak. Degradation also appears!
Any suggestion is appreciated!
I really need your help!
BTW, Many protocols say that the sample volume should not exceed 10% of the volume of TRIzol reagent used. the sample volume means tissue powder pulverized by pestle?? or tissue pellet before pulverizing?