Calibrator in qPCR - (May/23/2015 )
Hello again,
Could someone tell me what is a calibrator sample in qPCR and How do i prepare a calibrator sample for qPCR?
A calibrator is a control with a known concentration of an analyte used to verify an assay is functioning proper. It is also useful in qPCR to verify the reagents and standards are working well, and for normalization when running unknown samples on different plates. Standards and calibrators are similar because they both have known concentrations but they are different in practice because standards are run to establish your standard curve while the calibrator is run to verify the reagents, your standard curve and reproducibility between runs. Plus it helps you set your threshold. So basically you need a sample with a known concentration, preferably a stock of it that you can use for a period of time for consistency of results.
5280 on Sun May 24 01:55:27 2015 said:
A calibrator is a control with a known concentration of an analyte used to verify an assay is functioning proper. It is also useful in qPCR to verify the reagents and standards are working well, and for normalization when running unknown samples on different plates. Standards and calibrators are similar because they both have known concentrations but they are different in practice because standards are run to establish your standard curve while the calibrator is run to verify the reagents, your standard curve and reproducibility between runs. Plus it helps you set your threshold. So basically you need a sample with a known concentration, preferably a stock of it that you can use for a period of time for consistency of results.
Thanks :) So, i can prepare the calibrator with any one of the gene (maybe with the housekeeping gene) and have the Ct value and use it for further calculations?
You can use Ct values if you want but I prefer to use copy number as calculated from the standard curve. You should have a calibrator for each gene. The calibrator could be a plasmid containing the sequence for your gene and spiked into a buffer with some kind of carrier DNA. It could also be an actual sample where you verified the concentration of the unknown target, and it is preferable that you confirm this concentration with an independent methodology or different instrument/reagents. Either way, the reason you need a calibrator for each gene is because everything in the qPCR reaction is the same except the primers and standard curve so you will need a calibrator to verify their performance. It becomes cumbersome in research because you often measure expression of many different genes. If I planned to measure the gene expression of many samples over and over again then I would prepare or purchase a calibrator. Otherwise I would not make the effort.
If you want to be more stringent, then you could include two calibrators - representating low and high concentrations. If you only include one calibrator then make sure the concentration is in the range that you would see in a unknown sample. If gene x is usually between Ct 22-26 then make the calibrator around 24 Ct. You would not want to make the calibrator to have a Ct around 8-12. You can always dilute out the calibrator to put it in the ideal range.
Hope this helps!
5280 on Mon May 25 00:28:08 2015 said:
You can use Ct values if you want but I prefer to use copy number as calculated from the standard curve. You should have a calibrator for each gene. The calibrator could be a plasmid containing the sequence for your gene and spiked into a buffer with some kind of carrier DNA. It could also be an actual sample where you verified the concentration of the unknown target, and it is preferable that you confirm this concentration with an independent methodology or different instrument/reagents. Either way, the reason you need a calibrator for each gene is because everything in the qPCR reaction is the same except the primers and standard curve so you will need a calibrator to verify their performance. It becomes cumbersome in research because you often measure expression of many different genes. If I planned to measure the gene expression of many samples over and over again then I would prepare or purchase a calibrator. Otherwise I would not make the effort.
If you want to be more stringent, then you could include two calibrators - representating low and high concentrations. If you only include one calibrator then make sure the concentration is in the range that you would see in a unknown sample. If gene x is usually between Ct 22-26 then make the calibrator around 24 Ct. You would not want to make the calibrator to have a Ct around 8-12. You can always dilute out the calibrator to put it in the ideal range.
Hope this helps!
Thank you for the detailed explanation :) I appreciate it.
I am measuring data relatively, so do i need to have a standard curve? For calibrator, can i use a gene that has already been quantified and the ct value known? For example Gene x which was quantified and the ct value is 28 and i include this calibrator in every plate. OR i take a control sample (no treatment) and make a dilution (lets say 1:5 or 1:10) and use it as a calibrator?
Thanks :)
For a calibrator, you can use a sample the you previously used in a qPCR run.
In regards to the standard curve, you do not need to run a standard curve if you are measuring gene expression relative to a reference gene. Although, I would run a standard curve once just to test your primers and see if they perform well - e.g. they produce a nice standard curve based on your r value and efficiency (slope). For making a standard curve to test your primers, you can gel purify a PCR product, measure DNA concentration and then determine the copy number/ul of your eluate (use the formula below that is based on your PCR product size and the eluate DNA concentration). Then make your standards to cover a broad range from 10^10-10^1 copies /ul.
Gel purify the PCR product.
Quantitate the concentration using the spec.
Calculate the copy # by using the following formula:
Copies/ul = (ng/ul) * (10-9) * (6.02 * 1023)
(bp of PCR product) * 666
Starting from the obtained copy #, make a stock containing 1010 copies/ul.
Make serial dilutions (1:10) to get 109, 108, 107, 106, 105, 104, 103, 102, 10 copies/ul
Hope this helps!
5280 on Tue May 26 15:16:47 2015 said:
For a calibrator, you can use a sample the you previously used in a qPCR run.
In regards to the standard curve, you do not need to run a standard curve if you are measuring gene expression relative to a reference gene. Although, I would run a standard curve once just to test your primers and see if they perform well - e.g. they produce a nice standard curve based on your r value and efficiency (slope). For making a standard curve to test your primers, you can gel purify a PCR product, measure DNA concentration and then determine the copy number/ul of your eluate (use the formula below that is based on your PCR product size and the eluate DNA concentration). Then make your standards to cover a broad range from 10^10-10^1 copies /ul.
Gel purify the PCR product.
Quantitate the concentration using the spec.
Calculate the copy # by using the following formula:
Copies/ul = (ng/ul) * (10-9) * (6.02 * 1023)
(bp of PCR product) * 666
Starting from the obtained copy #, make a stock containing 1010 copies/ul.
Make serial dilutions (1:10) to get 109, 108, 107, 106, 105, 104, 103, 102, 10 copies/ul
Hope this helps!
Thanks a lot for the detailed explanation.
One more question is that, can i use a naive sample (no treatment) instead of an old sample that i used?
Thanks once again :)
You can use any sample you want as long as you have previous qpcr data for it.