I've found in the literature both kinds of quantifications. The "absolute papers" say that absolute is better because there are many fluctuations among single-cell genes, and the "relative papers" says that the process of extracting RNA by LMD is stressful to both references and targets, and that is why they used it. I was planning to do the relative quantification, but I am a little confused. I will compare mRNA expression of many different genes in treated and control samples. Any suggestions?
Hi, Larissa, this directly dependent from your aim and system. In the case if you homogenous cell population and isolate equal amount of RNA (per cell) form both, absolute value is OK. From the other side, reference gene must be constant for both cell population, so, your final results will be similar as in the absolute calculation.
However, if you reference gene are variable among population (let say number of copy per cell), relatively calculation will reflected rather relative amount of RNA to compare with reference.
In this case you have to use different reference genes and compare results.
In some case different reference gene can give you different results..
So, you have to consider both approach and different reference genes to get correct conclusions.
Good luck!
Hi, Larissa, this directly dependent from your aim and system. In the case if you homogenous cell population and isolate equal amount of RNA (per cell) form both, absolute value is OK. From the other side, reference gene must be constant for both cell population, so, your final results will be similar as in the absolute calculation.
However, if you reference gene are variable among population (let say number of copy per cell), relatively calculation will reflected rather relative amount of RNA to compare with reference.
In this case you have to use different reference genes and compare results.
In some case different reference gene can give you different results..
So, you have to consider both approach and different reference genes to get correct conclusions.
Good luck!
I think that single-cell PCR is to be considered relative. In many reports it is indicated that results from cells that failed to give any signals were eliminated, which indicates this. But, I only know of papers based on single-cell sorting by flow cytometry. In many cases, data are generated using the Fluidigm platform, where you can test in a single run, 96 different genes in each of 96 individual cells. I think this type of approach is close to what you intend to do ...
Absolute or relative quantification has noting to do with single cells or the experimental design (eg. treated vs. control). To give a more everyday life analogy:
The absolute quantification of, say, the familiy income would be given in a currency (e.g. in dollars, yen or euro) or some other measure (grams of gold, CO2 production, ...). This might or might not be interesting, but it is an absolute value. In contrast, a relative quantification is always relative to something else, for instance to the the income of the nation's president, or to the average income (of the city, region, nation, world...), or to the income of the worlds richest (or poorest) person or to the GDP or to the export surplus, or or or... again thsi might or might not be interesting, but these are all valid relative quantifications of the family income.
Coming back to qPCR: an absolute quantification of the mRNA is given as a number of molecules. This is it. Knowing some experimental details this number can very quickly be related to many other things, like the number of cells used for RNA isolation, or the amount of RNA obtained, or the amount of cDNA inserted to the PCR, and and and. This would already be a relative measure. To my opinion, measures like "number of mRNA target molecules per cell" may be biologically meaningful. However, these relative measures still depend on an absolute quantification (I do quantify the number of molecules).
If the number of molecules is never quantified, we talk about a relative quentification. The result of a relative quantification in one sample is meaningless, it can't be interpreted. However, such velaued from different samples can be compared. It is possible to determine the differences or fold-changes between samples. In the simplest case, the measurements are related to the amount of input. As long as the input is similar in all compared samples, the comparisons are valid. If this cannot be assured, one might normalize the measurements by some "loading control" (typically measurements from one or more reference genes). Such normalized quantities again refer to similar amounts of "input" - where input means "amount of reference gene transcripts". When the number of reference gene transcripts is constant per cell, the comparison is about "target mRNA copies per cell", what might or might not be interesting. For instance, it could also be interesting to compare the amounts of target mRNA copies per RNA content. Or per number of mitochondria. Or per cell volume. Or or...
Again: absolute quantification is based on the determination of a number of molecules, whereas relative quantification at no place invokes the quantification of the number of molecules. Absolute quantifications allow interpretable statements about single samples, whereas relative quantifications can only be comparatively interpreted (as difference or fold-change between samples).
The question for a biological meaning is not related to this kind of quantification. Amounts as well as changes of transcripts may be biologically relevant in relation to the cell number, content of a cell compound (RNA or DNA or protein or some other compount, e.g. specific proteins, fatty acids, homons, organelles or whatsoever), the cell volume, some cell activity and many more. This depends on the biological question and the biological background. Such "related" quantities are also, per definition, relative measures, but this has nothing to do with the kind of qPCR-quantification (absolute or relative [see previous paragraph]).
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