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My RNA extraction failed (From a newbie )

Trizol RNA

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2 replies to this topic

#1 Angie.kaoru

Angie.kaoru

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Posted 13 June 2012 - 07:01 PM

Dear all,

I am quite newbie in molecular biology, and i'm just learning how to extract RNA from my tissue samples (mice dental pulp) in order to , in future, can do PCR analysis.
i followed all the steps of the TRIZOL protocol for RNA extraction but when i did the reading of the concentration using the spectrophotometer those values were really low! 69 ug/ml or so...quite different to those obtained by my supervisor, up to 2000 ug/ml
After I took the dental pulp of mice (more than one week ago) I storaged them in trizol *1ml* at -80C , then I defrost at room temperature and homogenized by pippeting and normal vortex...I recognize I have probably taken some of the interphase together with the aquous phase after adding chloroform ...but according to one senior it should'nt be the reason for such low values. I could observe the precipitation even after 70% ethanol washing...Is this problem related to the sample?
Any suggestion would be appreciated Posted Image

Thanks,


Angie

#2 goeselen

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Posted 13 June 2012 - 08:01 PM

Hi Angie,

Which TRIZOL extraction are you using? Is this a TRIZOL LS method from Invitrogen? In my experience, TRIZOL gives me high reading yield, but the RNA is not so clean. However, there is a chloroform step involved. Also, you have to use some salt carrier or glycogen in order to precipitate the RNA. Are you doing this? That may increase your yield.

#3 elader

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Posted 14 June 2012 - 04:13 AM

You cant store undisrupted tissue in Trizol, the RNA will degrade. Either homogenize it in trizol and store it or snap freeze it or store it in RNAlater - dropping a piece of tissue in trizol and NOT immediately disrupting it is a very bad thing - slow lysis and release of nucleases. That is absolutely your problem. If you are having lengthy dissections and accumulating sample, RNAlater is probably the safest way.





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