I'm working on gene expression in different stages of mouse embryo development
I found that I need a calibrator to make the statistics of RT-qPCR data and in all the papers I read the calibrator is the data of a control group, but I don't have neither control nor treated groups
I only have the normal mouse embryo and I want to compare the levels of gene expression in different stages (10.5 E, 12.5 E, 14.5 E and 18.5 E) of mouse embryo development
so I want to know what is the calibrator for my data?
You can choose any one of the stages to be your calibrator; just state which group it is: either the earliest stage or the latest stage (whichever stage gives non-zero expression for all targets).
Heba as per your attached PDF, your calibrator would be sample with lowest Ct value.
please correct me if i am wrong,, i too had similar queries, my appreciation.
Below lines are from your PDF. page 9
Comparing Samples with a Calibrator
The normalized amount of target (c-mycN) is a unitless number that can be used to
compare the relative amount of target in different samples. One way to make this
comparison is to designate one of the samples as a calibrator. In Table 1 on page 10,
brain is designated as the calibrator; brain is arbitrarily chosen because it has the
lowest expression level of the target.
Ref: User Bulletin #2: ABI PRISM 7700 Sequence Detection System Page 9 of 36
Dear Heba,
Suresh is right. your calibrator should be had lowest Ct. in this way, you can use comparative method using 2-deltaCT method.
Lowest expression level = highest Ct value. Expression level and Ct value are inversely correlated.
Dear Mr. Suresh Oncosuresh
Sorry for being late in answer
please return to the attached pdf page number 18 you will find the following statement:
Calibrator cb may be a control treatment, or the sample with the highest Ct value.
The sample with the highest Ct value means that it has the lowest expression level since Ct value and expression levels are inversely correlated
Dear all
Because I do real time PCR first time, I dont know too much about this.
How do we know which one will be highest Ct value or lowest expression level before we do real time PCR?
The machine that I use from Appied Biosystems 7500, They require to detail reference sample or calibrator and reference gene before do real time PCR but because of I dont know the expression of them how I can detail them?
And I would like to ask about this machine, Is it impossible to do real time PCR saparately between target gene and reference gene? Because of I have too many samples if I do reference gene same plate with target gene, I afraid that well it is not enough for all stage.
Thank you
Dear Karen,
In the case of your first question, you have to select reference sample (arbitrarily) and reference gene before starting qPCR. It is suggested that the calibrator can be sum of your control samples or cell line that expresses your gene of interest. In the case of reference gene, you must also find the best housekeeping gene with stable expression in your samples (controls and treatment samples) by using genorm/Normfinder or bestkeeper algorithms before starting analysis of your gene of interest.
To get answer for the last question, you can follow below link which supported by Dr. Joe Vandesompele:
https://blog.qbaseplus.com/four-tips-for-rt-qpcr-data-normalization-using-reference-genes
I hope that this explanation will be useful.
best
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