# qPCR analysis - (Dec/22/2009 )

Hi,

Can anyone help with qPCR analysis... I have tried using download3ed software but for one reason or another it wont work . I tried using the Pfaffl equation, but I'm not sure I am calculating it correctly. I am using:

2 to the power of (Cp untreated sample - Cp treated sample) Target gene.

Divided by:

2 to the power of (Cp untreated sample - Cp treated sample) Reference gene.

I have the efficiencies for my primer/probe paris so I use this rather than 2.

Is this correct? Someone from another lab uses a differet method using a 'pooled calibrator' which is an average of ALL Cp values for target and ref gene, however I dont expect my target gene to be similar so this is giving a big number....

I'm just trying to pin down the correct equation so I can put it in excell and do it myslef.

Any help would be much appriciated!!

Thanks!

Sci!

which qPCR-apparatus are you using?

scistudent on Dec 22 2009, 05:47 AM said:

Can anyone help with qPCR analysis... I have tried using download3ed software but for one reason or another it wont work . I tried using the Pfaffl equation, but I'm not sure I am calculating it correctly. I am using:

2 to the power of (Cp untreated sample - Cp treated sample) Target gene.

Divided by:

2 to the power of (Cp untreated sample - Cp treated sample) Reference gene.

I have the efficiencies for my primer/probe paris so I use this rather than 2.

Is this correct? Someone from another lab uses a differet method using a 'pooled calibrator' which is an average of ALL Cp values for target and ref gene, however I dont expect my target gene to be similar so this is giving a big number....

I'm just trying to pin down the correct equation so I can put it in excell and do it myslef.

Any help would be much appriciated!!

Thanks!

Sci!

REST 2009 software is easy to use - plug in the Ct values and it does the rest! Even gives statistical probabilities

(just make sure you have good efficiencies)...

Mullet on Feb 19 2010, 04:36 AM said:

(just make sure you have good efficiencies)...

Hi,

I tired to use rest but found it difficult to input my data. Also I would like to just use a calculation so I can see where the results are coming from. Im using a roche light 480 cycler. But it was suggested that exporting the Cp values was the best plan and then do my own clculations...

Also I was wondering when I use the pfaffl equation, it is my ratio i.e control 1 treatment a) 0.2 that I graph ??

Many thanks for any help!!

We're also using Roche Lightcycler 480 and have been using the demo version of qBASE Plus (Biogazelle) for analysis. It's been working great for us - especially if you are also normalizing against various reference genes.

As far as I know the demo version is downloadable from their site and free for use. It is fully functional - it just limits your results to one experiment of 5 x (384 well) plates.

scistudent on Feb 19 2010, 07:44 AM said:

Mullet on Feb 19 2010, 04:36 AM said:

(just make sure you have good efficiencies)...

Hi,

I tired to use rest but found it difficult to input my data. Also I would like to just use a calculation so I can see where the results are coming from. Im using a roche light 480 cycler. But it was suggested that exporting the Cp values was the best plan and then do my own clculations...

Also I was wondering when I use the pfaffl equation, it is my ratio i.e control 1 treatment a) 0.2 that I graph ??

Many thanks for any help!!

Use the following calculation:

Efficiency =(<10(-1/slope)>-1) * 100

Relative Expression = ((1+amplification efficiency%)^(Ct treatment- Ct control)

Example: Amplification Efficiency = 96.250%

Ct Gene X following treatment = 27.44

Ct Gene X control = 25.84

Expression = (1.9625)^(25.84-27.44) = 0.34

Treatment reduces gene X expression by 66%

hi,

You can do relative quantification, testing one or more stable genes, on the same sample.

Furthermore, you can also use qbaseplus

scistudent on Dec 22 2009, 05:47 AM said:

Can anyone help with qPCR analysis... I have tried using download3ed software but for one reason or another it wont work . I tried using the Pfaffl equation, but I'm not sure I am calculating it correctly. I am using:

2 to the power of (Cp untreated sample - Cp treated sample) Target gene.

Divided by:

2 to the power of (Cp untreated sample - Cp treated sample) Reference gene.

I have the efficiencies for my primer/probe paris so I use this rather than 2.

Is this correct? Someone from another lab uses a differet method using a 'pooled calibrator' which is an average of ALL Cp values for target and ref gene, however I dont expect my target gene to be similar so this is giving a big number....

I'm just trying to pin down the correct equation so I can put it in excell and do it myslef.

Any help would be much appriciated!!

Thanks!

Sci!