Example of standard curve - qPCR

[newbie17425]

Hello,

 

Could someone give me an example of how to prepare a standard curve for qPCR? I read some protocols but got confused. It would really be helpful if someone could help me with an example.

 

Also, i read that i need to prepare a standard curve for each of my plate? Is it correct?

 

Lastly, can we run the housekeeping (HG) genes and gene of interest (GOI) on separate plates? Eg: i have 30 genes and i want to run it as triplicates. So, either i run all the GOI on one plate or omit some and run the HG with the GOI OR i could run the HG genes on a different plate. Is it correct that i need to run my HG genes for every new sample i run?

 

Thanks

[CharonY]

It depends a bit on the type of analysis (absolute vs relative), however it would be easier to explain if you outline what you have read and where the confusion is. I would also advise you to read up on MIQE standards (Bustin et al 2010, BMC Mol Biol 11:74, open source) and follow-up on that.

 

As a side note I would also like to point out that there are no true housekeeping genes, especially when considering conditions that severely affect growth. Many, if not most, ignore this point, but theoretically one has to establish stability of the HG for the condition(s) to be tested first.

It should be noted that the control is mostly for the biochemical part (i.e. extraction and assay setup). Theoretically, if you did not screw up the instrumental differences day-to-day will be much less than the biological + sample handling variance. With some training the latter will be minimized leaving you with the biological variance, which is really what is of interest to you.

[newbie17425]

It depends a bit on the type of analysis (absolute vs relative), however it would be easier to explain if you outline what you have read and where the confusion is. I would also advise you to read up on MIQE standards (Bustin et al 2010, BMC Mol Biol 11:74, open source) and follow-up on that.

 

As a side note I would also like to point out that there are no true housekeeping genes, especially when considering conditions that severely affect growth. Many, if not most, ignore this point, but theoretically one has to establish stability of the HG for the condition(s) to be tested first.

It should be noted that the control is mostly for the biochemical part (i.e. extraction and assay setup). Theoretically, if you did not screw up the instrumental differences day-to-day will be much less than the biological + sample handling variance. With some training the latter will be minimized leaving you with the biological variance, which is really what is of interest to you.

Thanks Charon for your quick response.

 

I am looking for relative quantification. I am confused (standard curve) if i have to mix all my sample cDNAs and then make a dilution series out of it (1:1,1:4,1:16,1:64 and 1:256) or i just take one sample make a dilution series out of it. Or i just make a dilution seris of only one sample.

[hypervalent_iodine]

It would be considerably easier for you to do serial dilutions.

[CharonY]

It depends a lot on the specific purpose of the curve and the experimental design. I.e. it depends what you want to use the curve for. The most common one being the analysis of the dynamic range and amplification efficiency of your assay and/or quality control purposes. However, if you are looking e.g. at samples with large variance in expression (as you mention housekeeping I assume you are measuring mRNA) a dilution series may also be appropriate to assess how much you have to dilute/concentrate your sample to be able to measure your various samples within a reasonable range of cycles (i.e. fitting your samples within your dynamic range). If you already have a good idea which make have the highest expression you could use that one to do the dilution series and gain both information of dynamic range and required dilution for comparative runs

 

However, in these examples the dilution series is base on using your gene of interest. A use of standard curves is for the actually quantification process. Here you require an experimental setup where you can prepare dilutions series of an independent sample (e.g. WT, untreated and so on). Here the independent sample is really used as calibrant against which expression (normalized against an internal control) is used.

 

So with regard to your question, it really depends on what you want the purpose of the particular dilution series is. Again, I would recommend going through your notes, evaluate what biological question you have (e.g. influence of mutation, influence of treatment etc.). Take a look of the required controls (with a look at the MIQE standards as a guideline) and design your experiment from the ground up. Typically it should become very clear what you need to combine (or not) to gain specific answers (e.g. if PCR efficiency has already been assessed you probably only need a one or two-point just to validate whether you have any issues, rather than a whole dilution series, for example).