Absolute Quantification of mRNAs - Ask TaqMan #26

Relative quantitation is the most common application with real-time PCR, but sometimes fold change data is just not enough. For instance, let's say I'm looking at samples infected with HIV and I need to know exactly how many copies of virus are present in the sample. What other options are there, when you need more concrete answers? That brings us to this great question from Jamsai Duangporn at Monash University who asks "Can TaqMan assays to be used to determine the absolute quantity of the mRNAs?"
TaqMan assays can be used for a technique called Absolute Quantitation, sometimes also known as Standard Curve analysis. ABSOLUTE QUANTIFICATION involves the precise molecular measure of a target concentration. In an ABSOLUTE QUANTIFICATION experiment, samples of a known quantity are serially diluted and then amplified to generate a standard curve. The unknown samples can then be extrapolated into quantities based on the slope of this curve.
The main hurdle in an ABSOLUTE QUANTIFICATION experiment is the generation of this standard curve. Although it seems simple in principle, there are a lot of things to consider! Your standard needs to meet the following criteria:
First, the quantity of a sample must be known by some independent means. For this step, the concentration can be measured by with a spectrophotometer and converted to number of copies using the molecular weight of the DNA or RNA. You can also refer to our handy guide called "Creating Standard Curves" for more details on how to do this.
Second, the standard should closely resemble the target from a biological standpoint, and it is very important that the DNA or RNA be a single, pure species. For example, when measuring gene expression of RNA transcripts, you would want to use in vitro transcribed RNA. Take care here because purity will be an important factor in the accuracy of your measurement.
Third and finally, don't forget that your excellent pipetting skills can be put to good use here! One of the major pitfalls for scientists setting up standard curves is that they do not pay enough attention accuracy of pipetting of technical replicates. For the best results from your standard curve, ensure that your pipets have been recently calibrated. Be very careful when making dilutions and pipetting into the plate, and ideally make use of low retention tips.
Now that we have our standards, let's setup a dilution curve in our plate. We recommend to run in triplicate, with 10 fold dilutions and at least 5 points.
When set up correctly, all ABI Real-Time PCR instrument software will generate a standard curve for you from these points. The equation of the linear regression line through those points is then used to automatically calculate the quantities of any unknowns on the plate, in the same units.
For example, in this curve I have 5 points, starting with 20,000 copies of my target as the highest concentration going down to 1250 copies. The standard curve plot is showing the input on the x-axis as [log X] and the Ct values on the y-axis. Quantities are then determined from this equation: We'll remove any outlying replicates or points when necessary.
Solving for X using the Ct values of our unknown samples will give us the missing quantities. Notice that they will be in the same units as our standards; copies for copies, ng for ng, and so on. So now we have determined the absolute quantities of our unknown samples!

[yanwang]

hi, could you please tell me if my target RNA from Multi-species in soil could use this Absolute Quantification method?

[quratulainaini]

How can we quantify without serial dilutions and if we want to quantify from ct value then how can be

[ScholarCAN]

Hey. So if we want to measure 1 gene in 2 different cell lines should we use standard curve approach or relative approach. If we use standard curve approach what will be our unknown concentration?

[lucamarchetti]

Hi, I need to determinate the absolute quantity of non human replicative RNA virus in mouse brain samples so i was wondering if the best way sould be create a standard curve with my target cloned into a vector or others ways could be better

[masrooralam]

Hi, Could you please help and demonstrate on how to setup standard curve experiment using ABI7500 machine for measuring three targets in a single sample simultaneously?

[DA-sjgw]

0 min: ct = 34, stimulated 30 min: ct = 35, stimulated 60 min: ct = 32, RT- = 33 how do I calculate ?