I run qPCR with cDNA and got nice amplification curve but melt curve is really horrible.
I extracted RNA from bacteria using Trizol and digest RNA with DNase and synthesized cDNA with Takara 1st strand cDNA sythesis kit using hexamers. On agaraose gel, got a clear and single band from qPCR product without primer dimer or nonspecific amplification. I am using Step one real time PCR system with syber green mastermix with high ROX.
Please have a look here and give valuable suggestions.
Hi Swati,
Do I see correctly that the recording of your melt curve stops around 70°C? How high do you go in temperature for the melt curve and how large is your amplicon? In most cases it´s around 80°C and up where things become really interesting.
My guess would be what you see is just the noise at the beginning?
Carla
I share Carla's concern, the melt curve needs to go to completion.
The data for your melt curve and your deritvative melt curve show evidence of primer-dimer dissociation at low TMs, but I expect there would be a larger change in flourescence indicative of specificia amplification if the melt curve were run longer.
If the agarose gel showed a band, and if that band was at the expected mass, then I'd say it's reasonable to assume you've got specific amplification. The gel trumps the melt curve as an indicator of specific amplification.
Your amplification plot looks a bit odd. What CT value did the assay report (if any)?
@ Hi Brain... Thanks for your replying.
See the gel picture
and ct value is 30.01
@Carla
yes you are right it stops around 70 and amplicon size is about 110 bp
I am also surprised that why it melt curve is not going at compilation?
What do you think can i use ct values from such experiment for expression studies (as on gel no primer dimer or non specific product is seen)???
I strongly recommend testing the linearity of a qPCR assay
(e.g. via a serial 10-fold dilution of a highly expressing sample). In my experience, few assays are very linear to a CT above 30. Only if you can demonstrate that the assay is linear up to a CT of 30 should the data be usable.
A cheaper, easier, alternative would be to determine the limit of detection for the assay by including a non-amplified control (NAC, e.g. a cDNA reaction prepated without reverse transcriptase). such a control should have either a very high CT, or no determinable CT value at all. If the NAC's CT value is 30, however, it means that your result of 30 cannot be interpreted since that expression level is below the limit of detection.
This looks like an issue I've had with some samples. You can see the curve start at cycle 1 and dips below the x-axis by cycle 2. In my experience this is one of two thing. 1) Starting DNA concentration is too high or 2) there is some inhibitor/interference of fluorescence from the RT-PCR. Either way, I would do a 1:5 dilution series with a single sample and run as a standard. You may see a big drop in the Ct within your first couple dilutions.
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