I ran an RT-PCR for monoamine oxidase A in trigeminal ganglion, after migraine induction. However, I could not detect any band for the gene. The primers are correct, all the running conditions are also fine. Is it possible to get a blank gene expression for a constitutively active enzyme?
Check your target gene for splicing variants. You may get enzyme activity of different isoform than mRNA you target is to generate. The other issue can be SNP in case one is located at 3'-end of your primers.
You have to check the quality of your sample first of all, if all the other parameters are ok. Did you check the quality of RNA, which you used? The absorbance of A260/A280 is a first step, and if you can you can run a denaturing-agarose gel. If the gene is active, and you are sure about it, you must get a product, even if the expression is too low. An other simple method is to increase the cycles of your reaction.
If the gene really is constitutively active, you would expect there to be expression of the gene to some extent. Even in genes expressed at very low levels you would expect to see a PCR product after 40 cycles. How do you know the running conditions are fine, have you ran a positive control? What you need is a sample that you know has some expression of this gene as a positive control, to test if there is a technical problem. To test if your RT reaction has worked, you should try to find some primers from your or another lab for a gene that you know will be expressed in your tissue to test that there is cDNA there to be amplified.
Check your target gene for splicing variants. You may get enzyme activity of different isoform than mRNA you target is to generate. The other issue can be SNP in case one is located at 3'-end of your primers.
I agree with Oliver. You can check the expression of a house keeping like beta actin or GAPDH in your RNA samples to rule out the quality of RNA also you can use a bioanalyzer to check the RNA integrity of your sample.
Thank you all. GAPDH is indeed giving a good signal. I will check another gene of interest and also check for splice variants. Thank you once again.
I use more than three housekeeping genes for test my RNA samples.For each gene I have designed two different primer pairs, one for qPCR (100-150bp), and another for amplifying the whole gene (1400-1500bp). I have noticed, that in some cases, I get good signal in qPCR, but there is no signal for the whole gene. Maybe the degradation is a reason, why you don't ge signal.
You may consider ordering several pairs, say for 5', middle, and 3' end. Also, at least theoretically, it might be a case when RNA does not present but protein is there.
Anyway, I would not bet on it, and proceed with the experiments that other guys recommended already. In case this particular protein is very important, you may try the alternative RNA detection approach, for instance RNA-FISH, It will add the credibility to your observation.
Thank you Dr. Suslov, Dr.Apostolou for your valuable suggestions. I will check them out.
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