# Calculating relative efficiency of genes - Standard curve (May/25/2006 )

Hi all,

I just ran a standard curve with a series of dilutions of my RNA for several genes and I read that it was important to make sure the efficiency for these genes be around the same. My question is how do you calculate the relative efficiency of these genes after running the standard curve? How far off can the efficiency be? I use the equation E= 10^(-1/slope) to calculate PCR efficiency, is this the equation to use? So say my effiency(E) for one gene is 2.00 and the other is 1.8, would I calculate by using (2.00-1.8)/2.00 which means they are about 0.1 difference in efficieny...is that how I compare? and is 0.1 the acceptable range? I see people saying their efficieny is <0.1 or within 2% etc., but what does that mean? how do you know? What equation do you use to obtain those figures? Please advise!

-newb-

Hi newb,

I am doing this at the moment too. Another equation I have seen used is E= 10^(-1/slope) -1
which would mean that your E = 2 = 1= 100%, and an E = 1.8 = 0.8 = 80%.

When people discuss % difference in E, I assume this is what they mean. ie in the example above, there would be a 20% difference in E. If the difference is within 10%, so I've been told, its ok to use the delta-delta Ct method. If the difference is larger, its better to use the Pfaffl equation method.

Of course both methods would give more reliable results with E values closer to 100%.

-smurray-

hi Smurray,

I see, thanks so much! I am also facing another odd phenomenon with my standard curve lately. I test my RNA at dilutions across the range of 0.1ng, 1ng, 10ng, 100ng, and 200ng on 3 associated genes and using actin as my reference gene. I find that while my actin gene gives me a beautiful curve and decreases as template concentration increases as it should, however, my other 3 genes start increasing in CT after 10ng. Initially it starts out decreasing from the 0.1ng - 10ng stage and later on it starts to increase. At first I thought it could be experimental error but how could all 3 show the same pattern while my reference was perfect? I also ran it at again from the 10ng to 200ng range and found the same pattern...the CT increases throughout this range. Anyone have any thoughts on this? Perhaps I should just avoid using this template concentration and use 10ng or less of my RNA, but I was just curious to know why this happens.

-newb-

Hi,

So high concentrations seem to be inhibiting the PCR at this point? It could be that there is some sort of inhibitor present in your cDNA. I would say, just use the lower concentrations and disregard the higher ones that don't seem to give valid results. Are these lower concentrations within the range that you will use in your assays?

QUOTE (newb @ May 27 2006, 04:08 AM)
hi Smurray,

I see, thanks so much! I am also facing another odd phenomenon with my standard curve lately. I test my RNA at dilutions across the range of 0.1ng, 1ng, 10ng, 100ng, and 200ng on 3 associated genes and using actin as my reference gene. I find that while my actin gene gives me a beautiful curve and decreases as template concentration increases as it should, however, my other 3 genes start increasing in CT after 10ng. Initially it starts out decreasing from the 0.1ng - 10ng stage and later on it starts to increase. At first I thought it could be experimental error but how could all 3 show the same pattern while my reference was perfect? I also ran it at again from the 10ng to 200ng range and found the same pattern...the CT increases throughout this range. Anyone have any thoughts on this? Perhaps I should just avoid using this template concentration and use 10ng or less of my RNA, but I was just curious to know why this happens.

-smurray-

[quote name='smurray' date='May 28 2006, 04:38 PM' post='53361']
Hi,

So high concentrations seem to be inhibiting the PCR at this point? It could be that there is some sort of inhibitor present in your cDNA. I would say, just use the lower concentrations and disregard the higher ones that don't seem to give valid results. Are these lower concentrations within the range that you will use in your assays?

[quote name='newb' post='53224' date='May 27 2006, 04:08 AM']

The lab usually uses around 50ng RNA which is within the range where my CT is misbehaving, it would make sense to disregard the higher concentrations except for the fact that my disassociation curves look off for the lower concentrations, seems like there are primer dimers so the CT readings I am getting for the lower concentrations may not even be accurate. The curves for the higher concentrations actually look normal so my boss is telling me to run them again at 50ng and 100ng but again, the CT's at this range are completely opposite of what it should be! Seems like a lose lose situation, either the disassociation curve looks right and the CT's look wrong or vice versa.

-newb-