RT-PCR problems on mouse ear RNA - (Aug/29/2006 )
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 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 firstname.lastname@example.org. Thanks a lot.
I also have problems with my cartilage samples. My problems are mainly that I cannot measure RNA with a spectrofotometer. However, if I just take 10 microliter RNA for cDNA synthesis and than go for 18s on RT-PCR, I can measure it. But I always have a few samples where nothing happens, and since I cannot measure the concentration and the quality, I don't know where it goes wrong.
I think that the trizol protocol works the best, put it's far from perfect.
I think that there are still proteoglycans left which are giving the problems.