RT-PCR problems on skin total RNA
Zhenghui-Liu at omrf.ouhsc.edu
Tue Aug 29 11:16:09 EST 2006
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
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.
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