my results of the expression of two genes by real time PCR is too high or too low after calculating by the equation 2- delta delta Ct , all the results are expressed as (n.n)E(+/- n) for facilitation of reading, but still not like many papers talking about gene expression (most papers express it as for example n.n ..without E(+/-n)). what can I do?may any one help?
I think you need to add more information to your question.
Gene expression determined in this way is relative within an experiment, both to endogenous (housekeeping) genes and a standard. So saying your results are too high or too low doesn't mean much.
Once you work out your 2^-ddCT you should be given a number. From memory, if the answer is above 1, it is upregulated. Below 1 it is downregulated. You can barplot all your values on a y-axis log scale to get a good idea of a comparison of the gene expression between your samples.
You can also explain your gene expression as fold change, which is possibly what the other papers you are referring to are doing. So fold change in relation to a known standard (for human, we use stratagene - you can google it).
This article may help you: http://www.nature.com/nprot/journal/v3/n6/full/nprot.2008.73.html
Can you give a concrete example?
What are ddCt values in your case?
And for explanation of the notation:
0.00000000123 = 1.23*10-6 = 1.23E-9,
1230000000 = 1.23*108 = 1.23E9
The notation using factors of a power (of 10) iscalled the "scientific notation". It allows to give the relevant digits of numbers in a short format when these digits are many decimal paces before or after the decimal point. Old pocket calculators could not display the exponents correctly. To indicate that the last digits of numbers shown in the scientific notation refer to the exponent (of the base 10), the manufactures of these pocket calulator displays decided to place the letter "E" in front of the exponent. Thi is called the "E notation". This notation is a historical artefact. Today, computers can show and print the correct scientific notation with superscript digits for the exponent.
https://en.wikipedia.org/wiki/Scientific_notation
Hi Asmaa,
I fully agree with Sophie. Saying that a qPCR result is too high/low doesn't mean much. Actually using the 2^-ddCt you first calculate the delta-Ct of Case vs housekeeping, then the delta-Ct of Control vs its housekeeping and then you 'convert' the results in a real ratio assuming that the overall efficiency of the q-PCR is 2. The 'meaning' of this analysis is that you measure the ratio between the relative expression (relative means that expression is normalized against the housekeeping, so that a value of, say, '3' means that your goi is expressed 3x the housekeeping), assuming same PCR efficiency) of your goi in Case vs Control. Therefore it's entirely possible to end-up with a very high (e.g. goi expression is nearly absent in control and gets activated in case) or very low (vice versa) result.
Can you provide the Ct values you got from your exps?
Use qGene or similar software to analyze qPCR data.
In order to be sure about the results use more than one housekeeping gene and create a virtual HKG using Bestkeeper, eventually analyze stability of the HKGs in relevant microarray datasets with many samples. Measure the efficiency of your system and use the efficiency in the calculation (easy in qGene).
http://www.gene-quantification.com/download.html#qgene
http://www.gene-quantification.com/download.html
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