1.When I detected some miRNAs which were shown to exist only in human (by searching the database such as miRbase，NCBI gene, etc.) in mouse neurons using RT-qPCR, I found that the these several miRNAs could be amplified and detected. The Ct value was not more than 25. Is it enough to prove these miRNAs may exist in mouse? What else can I do to identify these miRNAs?(PS:I used polyA polymerase method to get the reversely transcriptional cDNA）
2. Is polyA polymerase only specific to the miRNA in this RT method? How does it work specifically?
First, I am going to answer your second question. The Poly-A polymerase is a template independent enzyme, which means it can bind to 3' end of any RNA present in the sample. Therefore, you are not specifically reverse transcribing only miRNAs; instead you are reverse transcribing all the RNAs. In a nutshell, the RNA binding domain of Poly-A polymerase binds to RNA and add poly-A tail at the 3' end of the RNA. For this, it needs ATPs.
Now the second question, as you are saying the threshold cycle (Ct) is less than 25; in my opinion, those miRNAs are expressed in Mouse neurons too. But one thing I would like to suggest is that you should use mature miRNA sequence (Taqman probes for mature miRNAs) for the realtime PCR to be specific, as pre-miRNAs may contain similar sequences in two distinct animals. Also, note that your sample have cDNAs from all RNAs, therefore non specific binding of the primer/probe is possible, although very rare when you use Taqman. I suggest you to check the sequence of the mouse probe/primer you use for realtime PCR if they are similar with humans.
RT-qPCR is not the tradition way to identify a novel miRNA.
You should use Northern Blotting to detect miRNAs, design probes for the mature miRNA sequence and once blotted that should identify your pri-miRNA, pre-miRNA and mature miRNA sequence. It will also give you a brief insight into the biogenesis efficancy, meaning that the detection may show you a thick band of pre-miRNA but a light band of mature miRNA sequence. Which would identify whether the miRNA is favored and continues to a mature form or not.
But northern blotting is the best detection platform to identify all stages of the biogenesis of your miRNA.
Good luck
Swaroop Kumar Pandey Prabin Upadhyaya Matthew de Cruz Thanks a lot for your suggestions! It's more accurate to use a probe to detect it. Although the costs of taqman and Northern blot are needed to be considered, I wil try them after I totally confirm the biological function.
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