Reference gene qPCR - (Oct/25/2009 )
Hi everyone
I have a few questions regarding the use of reference genes for qPCR that I was hoping you could help me with.
Both reference genes I have tried vary with treatment. I am not sure how much variability to accept. If I compare for example the changes in one of the reference genes between the CTRL and PE-treated sample I get 1.4 fold change. Is 1.4 too much of a change?
After isolating the RNA, i measure their concentration with the nanodrop machine. I use 2micrograms to synthesize the cDNA and then I use 20ng of the cDNA per well for my qPCR reaction. So would it be accurate to say that most likely the changes that I see in my reference gene across the treatments are due to a responsive reference gene and not to differences introduced by me?
And lastly should I exclude experiments where the reference gene changes more than the target gene?
Most appreciated
latourde on Oct 25 2009, 11:43 PM said:
I have a few questions regarding the use of reference genes for qPCR that I was hoping you could help me with.
Both reference genes I have tried vary with treatment. I am not sure how much variability to accept. If I compare for example the changes in one of the reference genes between the CTRL and PE-treated sample I get 1.4 fold change. Is 1.4 too much of a change?
After isolating the RNA, i measure their concentration with the nanodrop machine. I use 2micrograms to synthesize the cDNA and then I use 20ng of the cDNA per well for my qPCR reaction. So would it be accurate to say that most likely the changes that I see in my reference gene across the treatments are due to a responsive reference gene and not to differences introduced by me?
And lastly should I exclude experiments where the reference gene changes more than the target gene?
Most appreciated
if you mean 1.4 Ct value difference then tat is a lot as a Ct value of 1 means doubling!!!
I understand 1.4 means that you observe 1.4 fold of change in your gene expression after treatment, am I correct? I would try to find another reference gene, I think Sigma ofers panels of reference genes you can validate under your conditions
linainwerse on Oct 27 2009, 04:35 AM said:
Yes, you are correct. Thank you for the suggestion. I will look that up but as far as I have seen, people working with neonatal myocytes have a preference for using GAPDH or 18srRNA.
latourde on Oct 27 2009, 04:45 PM said:
linainwerse on Oct 27 2009, 04:35 AM said:
Yes, you are correct. Thank you for the suggestion. I will look that up but as far as I have seen, people working with neonatal myocytes have a preference for using GAPDH or 18srRNA.
i would be careful with GAPDH because I've seen some publications indicating it is involved in oxidative stres response. 18SrRNA is highly expressed and if your target is low expressed it may influence your results especially when you do multiplexing. Good luck!