Real time PCR
Reagent:
- Lightcycler 480 SYBR green I master from Roche #04707516001 (5 X 1 ml).
- Lightcycler 480 machine from Roche.
- Keep the real time PCR reagent frozen at -20C.
- When use, take one vial of 1 and 2 and thaw out quickly. Store the unused at 4C (no re-freezing) for up to a week.
Real-time PCR method has been widely used in biology to determine either absolute or relative gene expression. It is convenient, quick, accurate and safe compared to conventional methods, such as Northern blotting.
In most applications, relative quantification is sufficient to answer a particular question regarding a biological phenomenon. Based on the mathematics of real time PCR, DeltaDelta Ct method has been adopted and applied to calculate relative quantity of a favorite gene. Here is the application of this method for relative quantification. Detailed mathematical equations can be found in Livak KJ and Schmittgen TD, Methods, 2001 and in www.gene-quantification.de.
There are at least two ways to perform an experiment.
1. Amplify target gene and reference gene in two independent tubes.
2. Amplify target gene and reference gene in a same tube or dependent tubes.
Usually, what I selected is the second approach, which is also more accurate and less error-prone. Here is the procedure to quantify your favorite gene. As an example, I will compare adiponectin (our favorite gene) expression level in mesenteric fat pads after vehicle and drug treatments. n = 5 per group.
1. Copy the threshold cycle (Ct) for each sample in an Excel spreadsheet.
2. Calculate the first Delta Ct: adiponectin (Ct) minus actin (Ct) for each sample accordingly. Do not average for treatment group.
3. Calculate the DeltaDelta Ct: subtract each Delta Ct with the biggest Delta Ct from step 2. In the following example, it is 4.71.
4. Calculate the relative expression level by 2^(-(DeltaDealta Ct)).
5. Average the relative results for each group and calculate the standard deviation. In this example, the relative expression of adiponectin in vehicle and drug groups is 2.03 : 14.48.
The example is shown in the next table:
An example of relative quantification with DeltaDelta Ct method.
| Treatment | Mouse | adiponectin | actin | Delta Ct | DD Ct | Relative level |
| vehicle | 1 | 25.48 | 21.07 | 4.41 | -0.3 | 1.23 |
| vehicle | 2 | 23.67 | 20.98 | 2.69 | -2.02 | 4.06 |
| vehicle | 3 | 24.91 | 21 | 3.91 | -0.8 | 1.74 |
| vehicle | 4 | 24.12 | 20.5 | 3.62 | -1.09 | 2.13 |
| vehicle | 5 | 25.01 | 20.3 | 4.71 | 0 | 1 |
| drug | 6 | 22.1 | 20.9 | 1.2 | -3.51 | 11.4 |
| drug | 7 | 21.98 | 21.12 | 0.86 | -3.85 | 14.42 |
| drug | 8 | 21.15 | 20.34 | 0.81 | -3.9 | 14.93 |
| drug | 9 | 20.97 | 20.14 | 0.83 | -3.88 | 14.72 |
| drug | 10 | 21.54 | 20.91 | 0.63 | -4.08 | 16.91 |
Comments
Add a comment| Posted by Mariana Silveira, Brazil | Tue, 30 Mar 2010 14:05:31 | |
| Subject: doubts | ||
|
In the real time protocol (http://www.ebiomethods.com/methods/realtimepcr), I am curious to understand what do you mean by dependent tubes in 2. Amplify target gene and reference gene in a same tube or dependent tubes.
And how you could amplify two target genes in the same tube with SYBR green method? I think it does not apply. Do you suggest any way to obtain standard deviation values when real time is performed in independent tubes for target and endogenous control? Thank you very much for the attention. Mariana |
Posted by guest | Sat, 14 Jan 2012 08:24:35 |
| Subject: Error analysis | ||
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How do you go about calculating the error and graphing error bars? How do you evaluate statistical significance?
Thanks |