2 replies to this topic

[katenkak]

Hi all!
Does quantity of RNA matters for the qPCR when I want to determine a relative changes in certain gene expression (knockout is compared to wild type) using 2-ΔΔCT method and normalize to Gapdh?
For example, can I run qPCR if for cDNA synthesis I use samples with 3 µg (knockout) and 2 µg (wild type)? It will be finally normalized to Gapdh, isn't it?
Or I should use excatly the same quantity of RNA for cDNA synthesis.
P.S. I neither purify or measure cDNA, but directly use the reaction mix for qPCR.
Many thanks for you answer!  

[cellcounter]

katenkak, on Aug 11 2009, 12:58 AM, said:

Hi all!
Does quantity of RNA matters for the qPCR when I want to determine a relative changes in certain gene expression (knockout is compared to wild type) using 2-ΔΔCT method and normalize to Gapdh?
For example, can I run qPCR if for cDNA synthesis I use samples with 3 µg (knockout) and 2 µg (wild type)? It will be finally normalized to Gapdh, isn't it?
Or I should use excatly the same quantity of RNA for cDNA synthesis.
P.S. I neither purify or measure cDNA, but directly use the reaction mix for qPCR.
Many thanks for you answer!  

1. As long as none of the (KO and Wt) RNA is too low, and

2. as long as the conc. difference is not too big (100ng, 2ug),

you should be fine.

Additional considerations come into play too. If your PCR reaction efficiency, the slope R^2 values are not too good (non-linear amplification), you may face troubles if you use different concentrations of template cDNA, even if you normalize against best house-keeping genes. The best thing to do is to try to get equal amount of mRNA -> cDNA to start with.

[katenkak]

Thanks A LOT!!! Good luck in YOUR experiments.

cellcounter, on Aug 13 2009, 11:00 PM, said:

katenkak, on Aug 11 2009, 12:58 AM, said:

Hi all!
Does quantity of RNA matters for the qPCR when I want to determine a relative changes in certain gene expression (knockout is compared to wild type) using 2-ΔΔCT method and normalize to Gapdh?
For example, can I run qPCR if for cDNA synthesis I use samples with 3 µg (knockout) and 2 µg (wild type)? It will be finally normalized to Gapdh, isn't it?
Or I should use excatly the same quantity of RNA for cDNA synthesis.
P.S. I neither purify or measure cDNA, but directly use the reaction mix for qPCR.
Many thanks for you answer!  

1. As long as none of the (KO and Wt) RNA is too low, and

2. as long as the conc. difference is not too big (100ng, 2ug),

you should be fine.

Additional considerations come into play too. If your PCR reaction efficiency, the slope R^2 values are not too good (non-linear amplification), you may face troubles if you use different concentrations of template cDNA, even if you normalize against best house-keeping genes. The best thing to do is to try to get equal amount of mRNA -> cDNA to start with.