Im new to DEGs and PCR data and i received a RT-qPCR data of interpolate corrected CT values of around 50 microRNAs . The dataset contains 5 different conditions each condition contains :
- 3 different concentrations and a control
- 3 replicates for each concentration and for the control
The goal is to find within each condition which microRNAs are differentially expressed compared to the condition.
I have several questions:
1. Can i proceed without having any reference housekeeping genes, and does the interpolate correction of the CT values help in that case?
2. Should i calculate the delta CT or delta delta CT for each microRNA based on each condition seperatly or all the conditions together, and how to do it if i have no reference genes?
3. I am planning on using excel and/or R , and PRISM, any other software or tool recommendations?
Thanks a lot in advance :D
Dear colleague:
Answer 1:
If you are new in the qPCR world there are some recomendations to start on it:
First you need to determine if you are going to use absolute or relative quantification. If you want to compare different conditions to a control, I strongly recommend you the relative quantification method. Next, you need to evaluate at least three different candidates for house keeping genes. Search in papers, search for the most similar papers to your model, and pick the most appropiate candidates. Once you have picked them, you must to perform a qPCR including all your conditions, and then analize the results using the 2^-ddCt, choosing one of your candidates at time, comparing it with the others. You must to perform this comparison for each of your candidates. Whit this procedure it is easy to identify wich is not appropiate to use as a HK gene, because is going to produce variations in your measurements each time that you include it in your analyses.
If you do not have any HK gene, the 2^*dCt quantification could be altered by systematic errors; that is why HK gene is important, it helps you to correct pipeting errors (for example). Otherwise, if you do not want to use a HK gene, you can use a standard curve with a purified (and quantified) PCR product of your gene of interest.
Answer 2:
Using de 2^-ddCt you can compare all your conditions to your controls, and all of your genes of interest to the control at the same time, only if all of them are included in the same qPCR experiment (the same plaque, depending of your equipment). It is not correct to compare two different qPCR experiment due to the fluctuations between each experiment.
Answer 3:
I don´t have a specific answer for that. I recomend you to use the most confortable software for you. In my case, I use Excel, Prism and R.
If you have more questions, I attached two documents that we use a lot in our laboratory when we begin with the qPCR experiments.
Best regards!!!!!
You really do need to include a housekeeping gene to account for differences in cDNA preparation between your samples.
Here are the publication guidelines for qPCR experiments. I'd strongly suggest you read through them before you plan out your experiment.
Good luck!
Article The MIQE Guidelines: minimum Information for Publication of ...
Hello Rayan
Without a reference gene all of your calculations would have a very large error. Reading the MIQE guidline is essential as Katie A Burnette said.
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