This is the best set up for the primer I have for the miRNA I am working with. It is same for all 40 samples. I myself think it is not applicable but, I need your comments. Thank you all.
https://arashazimzadeh.shutterfly.com/
When you test new primers, ALWAYS have a look at the product in a gel, best is a 6%-10% PAGE (high resolution, especially for small products of ~60-100 bp). A melt peak is a melt peak, it can be a very specific peak from the very wrong product! Further, many different product can have quite similar Tms, so a single melt peak can underly two or more different products.
Hardliners say that you can only believe to have a specific PCR product when you sequence the product. However, I find it sufficient when both, melting curve has a single peak (at a reasonable Tm) AND gel analysis has a single band on the correct position. I don't find it sufficient to look only at the melt curve, no matter how the curve looks like.
What do the amplification curves look like?... Your melt curves do seem 'sleepy' and broad... but are almost too high of a melt temperature to indicate primer dimer - but I am not absolutely certain about that without knowing the length and composition of your primers. Also,,, miRNA qPCR is riddled with problems like this. You could very well be getting a sympathetic amplification of something that is not your miRNA target at all.
When you test new primers, ALWAYS have a look at the product in a gel, best is a 6%-10% PAGE (high resolution, especially for small products of ~60-100 bp). A melt peak is a melt peak, it can be a very specific peak from the very wrong product! Further, many different product can have quite similar Tms, so a single melt peak can underly two or more different products.
Hardliners say that you can only believe to have a specific PCR product when you sequence the product. However, I find it sufficient when both, melting curve has a single peak (at a reasonable Tm) AND gel analysis has a single band on the correct position. I don't find it sufficient to look only at the melt curve, no matter how the curve looks like.
Thank for your answers.
I am currently working on four different miRNAs. Three of them have single pick around 76 like this one,but this miRNA shows another larger product that in the picture is flat line between 80-82. Gel analysis shows two band although the larger one is not sharp. But what's the problem?
As this larger product happens in all samples I am not sure if the data for ct are correct for analysis.
This seem to be some amplification of one or several unspecific products. I would not trust the ct values in this case. Try different primers if possible. If not, you can start fiddeling around with annealing temp and times, extension times, primer concentrations, Mg++ concentration ...
Which system are you using to detect your miRNA? E.g., the Qiagen miScript kits enzymatically attach a poly-A tail to the miRNA before reverse transcription. The PCR is then done with a miRNA-specific primer + a universal primer. The poly-A tail varies in length, resulting in a smear if you put the result of the qPCR on gel. We got problems with specificity in this system when closely related miRNAs exist, and at low expression levels, resulting in a messy (multiple small peaks) melting curve.
In contrast to Berlinda Verdoodt's comment on the miScript system, we particularly like this technology. It is sensitive (in combination with preamp), specific and flexible. Qiagen has the largest content of validated miRNA assays available. In August, our paper in Nature Methods will come out that compared 12 commercial microRNA gene expression platforms (Mestdagh et al., "Evaluation of quantitative miRNA expression platforms in the microRNA Quality Control (miRQC) study"). The miScript system is our preferred qPCR technology at Ghent University and Biogazelle (see link).
http://www.biogazelle.com/lab-services/applications/mirna
It seems to depend on the miRNA whether miscript works well. I've had myself good results with it, but we also ran into a couple, including miR-214, where the qPCR was really unreliable. An advantage of this system is that one cDNA synthesis can be used for a large number of different qPCR targets. Saves time and money.
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