i'm planning to do a relative quantification and from what i know, i have to perform standard curve for each genes that i used in qPCR. actually, i have about 25 genes of interest. so, do i really need to perform the standard curve for 25 genes??
0
3 replies to this topic
#2
Trof
-
- Global Moderators
-
- 946 posts
Brain on a stick
66
Excellent
Excellent
Posted 21 February 2011 - 02:22 AM
Theoretically you don't. If you use simple delta-delta Ct, you don't need standard curve or efficiency (which is calculated from standard curve). But it's usually better to calculate the efficiency and do the calibration and normalization with it and use the Pfaffl equation (if you don't know it, search the forums).
Other way is to calculate efficiency right from the reaction itself using algorithms like PCR Miner, but I'm not sure how it corresponds with reality. Maybe someone has an opinion in this?
Other way is to calculate efficiency right from the reaction itself using algorithms like PCR Miner, but I'm not sure how it corresponds with reality. Maybe someone has an opinion in this?
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
I never trust anything that can't be doubted.
I never trust anything that can't be doubted.
#3
ivanbio
-
- Active Members
-
- 116 posts
Veteran
6
Neutral
Neutral
Posted 21 February 2011 - 02:42 PM
My recommendation is to run a standard curve for all 25 genes. I know it sounds like a lot, but when you think about it you are making sure that each assay is working the way it should before running your experiments.
I would disagree with Trof that you do not need to know the efficiency of your assays if you are analyzing by the delta-delta Ct method. If the two assays you are comparing using delta-delta Ct have significantly different efficiencies, you cannot compare them. It would be like saying that it is ok to compare and assay that needs 3.7 PCR cycles to double your amplicon to an assay that needs 3.2 PCR cycles to double your amplicon. Clearly one will have much less DNA amplified than the other at any given Ct.
I would disagree with Trof that you do not need to know the efficiency of your assays if you are analyzing by the delta-delta Ct method. If the two assays you are comparing using delta-delta Ct have significantly different efficiencies, you cannot compare them. It would be like saying that it is ok to compare and assay that needs 3.7 PCR cycles to double your amplicon to an assay that needs 3.2 PCR cycles to double your amplicon. Clearly one will have much less DNA amplified than the other at any given Ct.
Ivan
Carlsbad, CA
#4
Trof
-
- Global Moderators
-
- 946 posts
Brain on a stick
66
Excellent
Excellent
Posted 22 February 2011 - 03:09 AM
ivanbio, on 21 February 2011 - 02:42 PM, said:
I would disagree with Trof that you do not need to know the efficiency of your assays if you are analyzing by the delta-delta Ct method. If the two assays you are comparing using delta-delta Ct have significantly different efficiencies, you cannot compare them. It would be like saying that it is ok to compare and assay that needs 3.7 PCR cycles to double your amplicon to an assay that needs 3.2 PCR cycles to double your amplicon. Clearly one will have much less DNA amplified than the other at any given Ct.
Yes, you're right. You don't need it directly for the equation, but you should know it for using it in the first place.
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
I never trust anything that can't be doubted.
I never trust anything that can't be doubted.
