6 replies to this topic

[scistudent]

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!

[susanna]

which qPCR-apparatus are you using?

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

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!

[Mullet]

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)...

[scistudent]

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

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)...

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!!

[flammaefata]

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.

[Dr Teeth]

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

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

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)...

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%

[susanna]

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:

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!