Hello,
I've been recently doing qPCRs to compare how a certain stimulation can increase the gene expression for a particular trannscription factor. I have some issues as this technique is not commonly used in my lab. I wonder if I should run a negative control (not NTC a non-stimulated sample) for every batch of qPCR samples I run. Or should i have a standard value that I always compare them to. I need my negative to calculate the fold change of my GOI.
Another question is also how to represent these values better. I've been showing them in a bar graph with each dot representing a single sample. Each sample value is obtained from 3-4 replicates (cDNA replicates) that I run in a qPCR and then average the CT values obtained.
Please anyone that works with qPCRs or has published some data as such, if you could help me it would mean a lot.
Hello, in order to check for and avoid primer dimer formations it is necessary to run a negative control sample (H2O) within a qPCR run and a positiv control sample (standard) for quantification.
Further informations:
https://publikationen.sulb.uni-saarland.de/bitstream/20.500.11880/22476/1/Dissertation_Roland_Kindich.pdf
Read the MIQE guidelines so you'll know what you need to have publishable data. You should know exactly how you'll analyze your data BEFORE you start the qPCR.
A bar graph of raw Ct values is not correct, you need to show analyzed data of either relative or absolute expression.
Definitely yes, best to run a water sample to check for contamination.
a known negative to compare fold change expression.
a known positive, if available, in the likely chance the assay does not work as intended.
It's important you also include primers (+ probe if taqman) for a housekeeping gene.
You'll want to do this each batch to account for batch-to-batch variation. Small differences in Ct can have a large impact.
To display data you can use a column graph of fold change expression (Ct values mean very little). Theres lots of information out there to calculate fold change from Ct but you will need housekeeping.
Happy to help further,
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