Hi Tuba Sena Ogurlu

I’ve used the same machine to run at different temperatures however, I’d advise to leave one or two spaces between temperatures.

Hi Tuba,

You have to set the temp according to the primer that have the lowest temp minus 3.

In another words, let us suppose you have three primers, their temp 56, 58, 55. Then you have to set up the temp at 52 c.

If you observe any sec. structure or primer dimers add DMSO 5% or set a 3 steps reaction or both at same time.

Best..

Hi Tuba,

are you running SYBR based assays? If so, I’d say it’s all about the efficiency of any individual reaction. You should run a serial dilution of your sample to calculate reaction’s efficiency at your specified cycling parameters. This will show how well the assay performs for the housekeeper at lower anneal temperature. If it’s not within the acceptable efficiency range (although it might be) this can justify shuffling the cycling parameters for different genes.

If there's a QuantStudio 5 around I would recommend you try that because it has a model where there are separate 6 temperature zones via VeriFlex Blocks.

https://tools.thermofisher.com/content/sfs/brochures/quantstudio-5-your-innovative-research.pdf (see fig 1 and 2)

It is generally recommended to amplify your targets at a single temperature in a qPCR experiment, as the efficiency of the polymerase reaction can vary with temperature and amplifying at a single temperature allows for more accurate and consistent results. However, if you have identified that the optimal annealing temperature for your target gene and housekeeping gene are different, it is possible to use a gradient temperature protocol in your qPCR experiment to optimize the amplification of each gene.

In this case, you can set up two separate qPCR reactions, one with a gradient temperature that is optimized for your target gene and one with a gradient temperature that is optimized for your housekeeping gene. You can then run these reactions in parallel, either in separate wells of the same qPCR plate or in separate qPCR plates, depending on the capacity of your qPCR machine. As long as the qPCR machine is able to read the signals from both reactions at the same time, it should not pose a problem.

It is important to note that using a gradient temperature protocol can be more time-consuming and may require specialized equipment. It is also worth noting that optimizing the annealing temperature of the primers is generally not necessary for most qPCR experiments, and a single temperature protocol is often sufficient.

Bertrand Cornu Can Kiessling Audrys G. Pauža Mohamed Khashan Dino Santos Matias Thank u all. After many trials I have decided to use single temperature. I have to admit that still qPCR experiment is not so objective to me (changes according to conditions very easily). However, since everyone use the same method, and what matters is to compare the mrna level for the same protein, I guess it is fine.