I aim to compare the transcription level of two genes that encode two enzymes with the same function. I extracted mRNA and compared their transcription using RT-PCR. The Ct (also called Cq) values of two genes from RT-PCR analysis are obvious different. However, I don't how to present this result properly ? Or, are there other methods to achieve this purpose ? By the way, I already used the method of lacZ reporter to compare their expression indirectly.
If The Ct values are very different (say, about 5 cycles or more), this can be taken as indication for unequal expression (lower Ct -> higher expression). Note that this is a "semi-quantitative" interpretation: you can't say how much different the expression is; the large difference in Ct values only makes it likely that the gene with the lower Ct is expressed at (somewhat) higher levels. This is because the Ct is not an absolue measure of the concentration but also depends on many other things that are assay (and primer!) specific, like signal generation for the PCR product, characteristics of the amplification curve, and the choice of threshold value.
If the difference in Ct values is less (than about 5 cycles), it becomes increasingly difficult to attribute these differences to the expression levels, as the assay-specific differences may also be responsible for the observed Ct difference. In that case you will need external standards to quantify and compare the observed concentrations of the target sequences.
You have to compare the Ct values of your gene-of-interest to the Ct values of a "housekeeping" gene from your samples to account for differences in sample preparation. You also must include a standard curve to calculate your primer efficiencies otherwise you don't know if your data are reliable
Here's a link to the MIQE standards for publishing qPCR results.
Article The MIQE Guidelines: minimum Information for Publication of ...
Katie A Burnette , I think that here aliquots of the very same cDNA is used to measure the two genes. So both genes are measured always in the same biological sample (just like "gene of interest" and "reference gene"). There is no need to correct for loading differences. But what is needed for a quantitative comparison between genes (within samples) are external calibrators (standards with known concentration), for both genes (and this may even not be neccessary if the differences are quite large and a semi-quantitative conclusion is sufficient).
You need external calibration (a "standard curve") to measure the efficiencies of your primers. Reliable primers have efficiencies of 95-105%. You also need to run samples in at least triplicate and calculate the R^2 values to show that you can minimize technical (i.e. pipetting) errors.
Not to be negative, but a large difference in Ct values can be explained by poor primer efficiency.
It's best to be thorough and proceed as if you are trying to produce publication quality data.
Jochen Wilhelm
Hi Jochen,
Thanks for answer. It's helpful. Could you explain more about "external standards" ? Or, relative references ?
There are enough free sources around in the internet. Try to inform yourself first and come back with a more specific question.
Jochen Wilhelm, Katie A Burnette Thanks everyone. The messages you provided are very helpful cause I need some hints (key words) to search what I should learn and make sure I get qualified data finally. Forget to mention that I already checked efficiency of every primer and make sure they are between 95-105%.
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