RT-PCR problems on skin total RNA

Zhenghui Liu Zhenghui-Liu at omrf.ouhsc.edu
Tue Aug 29 11:16:09 EST 2006


Hi,
I am experiencing problems of RT-PCR on mouse ear RNA using Trizol 
miniprep protocol and a 2 step real-time RT-PCR protocol to examine 
several gene expressions.

I am isolating mouse ear total RNA from wild-type and contact 
hypersensitivity model mice.  I believe I got enough amount and good 
quality of RNA from ear by measuring the concentration, ratio of 
A260/A280 and running samples in the gel.  But it is very strange 
because the subsequent RT-PCR only work with RNA from tissues such as 
heart, liver, lung, but not with RNA from ear, even with primers for 
mouse house-keeping gene, beta2-microglobin (I have worked on mouse RNA 
from other tissues using regular PCR and real-time PCR for several 
years.  But this is my first time working with mouse ear RNA).

At the very beginning, I thought RNA samples from ear are degraded 
because of dirty ear tissues with hair.  My reasoning is confirmed 
right by running the samples in the gel.  Then more careful extraction 
is performed and intact good quality of RNAs are got by running them in 
the gel.  However, no amplifications occur again on these samples with 
all my primers which have been used for several years (good PCR 
amplification is observed from simultaneous RT samples on RNA of other 
tissues in the same plate.)

I suspect that there are special inhibitors of RT reaction existing in 
mouse ear RNA.

1. Is there any special considerations during isolation of ear RNA than 
other tissues?
2. How much RNA could you get from one mouse ear generally?
3. Does anybody performing RT-PCR on ear RNA have good amplification as 
on RNA from other tissues in your practice?
4. Mouse ear is rich in cartilage.  Does it have any affections on RNA 
production, RT, etc?

I would appreciate any suggestions.  You can directly email me at 
liuz at omrf.ouhsc.edu.  Thanks a lot.

ZL


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