help!real-time PCR to see gene's expression after treatment with cytokin - (Feb/13/2008 )
Hi,everyone,could you give me some advice for planning my real-time PCR experiment?
my aim is to see if target gene's expression level can be induced or enhanced by treatment of some cell cytokines.as to one kind of cell line and one kind of treatment,i will leave the cell serum-starved for 24h,and then add the culture medium containing the cytokine as the treatment ,for each treatment,i'll collect cell lysates at 4 time points,e.g 0h 12h 24h and 48h.i want to use the gene's expression level at 0h(just starved for 24h) as control and quantify the same gene's expression at different treatment time points.
my question is: 1.I want to apply the deltadeltaCt method and it's necessary to compare the amplification efficiency of target and reference genes at first,as to my experiment,have i to do this validation in sample of every time point?if the efficiencies are unequal at one or two time points,what should i do to optimize the experiment?to dilute the template,the primers,or just redesign the primers?
2.dose anyone has experience in similar experiment(cytokine or serum treatment for different time period),which gene do you choose as the internal control?
3.a question to discuss with all:i have read a paper( Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR )which applied a modification of the DDCt method to quantify serum treatment on different housekeeping genes,where dCT=CtTimeX- CtTIME0 and time 0 represents the 1X expression of each gene.another paper said that "this example demonstrates how the method may be used to analyze relative gene expression data when only one gene is being studied."i think if use this method i could just compare the target gene at different time points, regardless of the internal control,am i right in that?
i don't know if i expatiated my question clearly,please tell me if anything confused and thank u very much for suggestions!
the paper mentioned was attached:
Okay, let's go point by point !
1. The efficiency of the PCR reaction can be done only once. There are no reasons for the reaction to be different from a timepoint to another since the amplification of your gene and the housekeeping gene will be done with the same primers.
2. I never did such experiments as treatment and time points with a cytokine, but all the method is the same. I use 36B4 as my housekeeping gene.
3. I personnaly doubt that the method they propose is the right way to go. If you do that, you can't be sure that the difference in expression you see is caused by a different expression level of you gene.
Ex : The ratio of expression seen with the method they propose gives you 4times less expression of your gene at 12hres. If you do not check a HKG, the difference you see may only be due to a pipetting error, or a less concentrated sample of cDNA.
The beauty of the delta-delta ct method is that it corrects the pipetting/concentration errors by calculating the amount of your gene/ amount of a stably expressed gene. If you omit this test, you may well have false results.
The thing I would do, is to do the delta-delta ct method as usual. You only have to set the expression ratio of your gene/HKG before treatment to 1.
Hope I'm clear!
1. The efficiency of the PCR reaction can be done only once. There are no reasons for the reaction to be different from a timepoint to another since the amplification of your gene and the housekeeping gene will be done with the same primers.
2. I never did such experiments as treatment and time points with a cytokine, but all the method is the same. I use 36B4 as my housekeeping gene.
3. I personnaly doubt that the method they propose is the right way to go. If you do that, you can't be sure that the difference in expression you see is caused by a different expression level of you gene.
Ex : The ratio of expression seen with the method they propose gives you 4times less expression of your gene at 12hres. If you do not check a HKG, the difference you see may only be due to a pipetting error, or a less concentrated sample of cDNA.
The beauty of the delta-delta ct method is that it corrects the pipetting/concentration errors by calculating the amount of your gene/ amount of a stably expressed gene. If you omit this test, you may well have false results.
The thing I would do, is to do the delta-delta ct method as usual. You only have to set the expression ratio of your gene/HKG before treatment to 1.
Hope I'm clear!
thank u for ur reply,but i still have some questions
: 1 as you said,"There are no reasons for the reaction to be different from a timepoint to another since the amplification of your gene and the housekeeping gene will be done with the same primers."do u mean that i can just do the validation in one sample at any time point by chance?in my opinion,the amplification efficiency of pcr is not only related to primers,but also the purity of the template,the buffer sysytem,etc.although i did RT of all time points samples at the same time,i am afraid the different RT efficiency among samples will influence gene's amplification efficiency,too.2 i am sorry for my haste,in fact, in paper mentioned above,they used UV absorbance to determine the amount of RNA added to a cDNA reaction.PCRs are then set up using cDNA derived from the same amount of RNA ,so they called it "external normalization" and this method derived from delta-delta ct method was called delta C't method.i don't know if this method is widely used,just put it here for correction of my last post.
best wishes!
Emily
I did mean that, in my view, you could do the validation for only one point. If the efficacity of the RT vary, the variation will be the same for the HKG and your gene, so the delta ct won't vary.
thank u,i think that's reasonable and i should put it into practice now:rolleyes: