1st time real-time PCR questions - (Dec/15/2005 )
Hi,
I am trying to do real-time PCR for the first time. I have received a protocol from someone and they do not seem to be able to answer my questions. I thought someone how knows about this topic might be able to help me.
I work in Arabidopsis. Since this is my first time doing this, I am looking at one gene that I know should have high expression in one sample and low in the other. I am using ubiquitin as a control.
1. This protocol, like others I have read, says to use a 2-step cycle for the PCR. I was wondering if 3-step could be used and what the advantages/disadvantages are of each.
2. This protocol uses a standard curve. I understand using a cDNA of known concentration for serial dilutions in order to have something to compare your unknowns to. However, the first step of this protocol is to mix the cDNAs together (10 microL each) and use this for the dilutions. I do not understand what this will achieve.
3. What could be the problem if the threshold is too high?
Thank you very much!
[quote name='Kira' date='Dec 16 2005, 07:24 AM' post='34644']
Hi,
I am trying to do real-time PCR for the first time. I have received a protocol from someone and they do not seem to be able to answer my questions. I thought someone how knows about this topic might be able to help me.
I work in Arabidopsis. Since this is my first time doing this, I am looking at one gene that I know should have high expression in one sample and low in the other. I am using ubiquitin as a control.
1. This protocol, like others I have read, says to use a 2-step cycle for the PCR. I was wondering if 3-step could be used and what the advantages/disadvantages are of each.
depends on the anneal temp and the length of the product. If the product is short and the anneal temp is around 60-65oC, the extension usually happens during the anneal step hence its only necessary to have 2 step. This is common for Taqman but sybr green assay tend to use 3 step.
2. This protocol uses a standard curve. I understand using a cDNA of known concentration for serial dilutions in order to have something to compare your unknowns to. However, the first step of this protocol is to mix the cDNAs together (10 microL each) and use this for the dilutions. I do not understand what this will achieve.
By pooling the sample DNA there should be a range similar to the sample concentrations, however i would not expect this pool to be concentrated enough for the high range samples? The standard curve should span the samples. It is more common to use either cDNA, Plasmid or previously amplified product in a serial dilution. In this case i would suggest a high concentration previous product serially diluted.
3. What could be the problem if the threshold is too high?
The threshold needs to be through the exponential region of the amplification curve if not the efficiencies will not be optimal and the result inaccurate.
there are many different way the data can be analysed, LinREG (Ramakers 2003) is popular for
such analysis, however i would recommend Rest 2005 (Corbett Research and Pfaffl 2005) which allows you to use different efficiencies for each reaction and take them into account.
Regards