I have carried out my first run in QPCR trying to relatively quantificate specific gene. In this regard, I run my experiment in triplicate for NQO1 gene and reference gene (GADPH). The first result gives my acceptable amplification curve as the photo in top, but then I continue the same reaction condition with another samples, I got bad amplification curve as the down photo. What was the cause?
Firstly, to address your question: are the Y axis scales the same? The lower image has very low relative fluorescence unit (RFU) values. Possibly the scale has been changed such that in the lower image we are effectively looking at a very magnified image, from only a small section of the plot. You should be able to find some way of changing the scale.
Secondly, and perhaps more importantly, the amplification plots even in the top image are not good. Looks like primer dimer or non specific product being amplified. And whatever is being amplified it's very inefficient reaction i.e. it is not doubling in quantity every cycle. The results you get will not be reproducible or accurate. I suggest you pick one set of conditions e.g. manufacturers recommendations, design and synthesize a whole lot of primers, and pick the ones that work the best.
Some criteria for a good primer set:
A single tall, sharp narrow peak on the melt curve.
No primer dimers or non-specific products in negative controls or no template controls.
A smooth amplification plot which rises suddenly then eventually plateaus.
Early Cts, which are indicative of efficient reactions, and give you more dynamic range.
You can go ahead and do standard curves, but for screening purposes early Cts are a very good guide.
agree with Roger. The fact that you don't have any increase in relative fluorescence up to 20 cycles shows that something is seriously affecting the amplification of your desired target. In the picture below, from the way the X-axis is represented, I believe too that it is a maginification of a small portion. you should probably think about designing new primers, BLAST them for specificity, run an end-point PCR and check the amplicons on an agarose gel and then proceed to RT-PCR.
I agree on testing at least a few primer sets for your genes, just basic unlabelled primers, have a go at optimising the pcr conditions in end-point pcr and run them on agarose gels (or bioanalyser if you have access) to see if you are getting the fragment sizes you expect. Sequencing the amplification product is also an important step to prove you are amplifying your target gene and not some non-specific product.
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