Hello everyone!
I have designed a few primers and then a tried optimized the conditions using reference RNA, diferente primer concentrations e annealing temperatures and then calculating the primer efficencies using a standard curve. Finally, i think i have my PCR conditions optimized and the efficiencies for each pair primer are > 90% and < 110% as recommended.
Now i want quantify these genes expression by qPCR in my samples. Do i always need to perform again a standard curve in each plate?
I'm using SYBR Green and differences in gene expression will be calculated using the Livak method.
Do you want to publish your results?
If yes, then you must include a standard curve and a full set of controls.
Hi Anna,
There will always be minor variations across experiments. Therefore, comparing standard curve generated from a different experiment/plate is not ideal. It is generally a good practice to include standards in each plate.
Good luck with your experiments!
Anoop
If you optimize your reaction conditions, then you need to include a control plate only. There is no need to repeat the standard curve again with each plate since the reason for doing that was to optimize the PCR conditions ( primer concentration, annealing temperature, cycling parameters, etc).
Hi Anna Oliveira
Taking into account that you have verified and optimized your primers and that everything is ok. If you are interested in the amount of nucleic acid per given amount of sample, you must make an absolute quantification and therefore need a standard curve per plate.
But if you want to know the increase or decrease of expression in your target gene, you need to do a relative quantification and to have equivalents amounts of cDNA between your samples, introducing NTC, NRT, ....you know... For the Livak Method (deltadeltaCt) You only need the Ct values in your target, housekeeping and calibrator genes.
Good luck!
Yes, you need to perform a standard curve. It is a good practice to keep your controls along with your experimental data.
Yes, with each plate control need to be added. Standard curve is not essential.
Thank you all for your answers.
Angel del Marco I want to check if there is up or dowregulation of several genes in my experimental groups using the livak method. That's why i am wondering why it is always necessary a standard curve in each place since primer efficiency has been calculated before.
@Anna Oliveira since you are looking for up or down downregulation, you need to have a negative control.
@°Anna Oliveira Negative controls include, No RT and other one with No template. Let me know if you require further clarification. All the best
Hi anna,
U only need to do the standard curve for each gene once. Because the purpose of having a standard curve is to determine whether your primer is efficient enough to duplicate your gene of interest. And you only need to the determine the value of efficiency (90 -110%) and use it for your calculation, that's all..
Good luck!
Hello Anna,
Depends on the type of analysis performed. For the livak method you only need to do it once to check the efficiency of your primers. If they have similar efficiencies, you can do the Delta delta Ct without redoing the standard curve each time. While if you use the relative standard curve method then you need to redo the standard curve each time.
No, you need to do standard curve standarization conditions for each primers in the beginning according to the purpose of qRt PCR
I agree with dr Charbel Alfeghaly
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