I am attempting to plan a qPCR experiment in which I evaluate the expression of multiple genes in various brain regions of fetuses, as a function of location in utero (intrauterine position, whether the fetus is next to a male or female).
Samples are obtained through routine culling efforts, and due to these field conditions, there is slight variability in the pregnancy age of each litter.
I understand that the best way to plan qPCR is to evaluate the expression of one gene per plate. Am I able to check the expression of one gene per plate, with multiple fetuses from different litters with differing pregnancy ages? Should I attempt to group litters in certain pregnancy ages? Additionally, does anyone have insight on the best way to standardizing different plates with housekeeping genes in order to compare results from different plates?
Does anyone have experience with dPCR (digital PCR) and would know to answer if it would solve my problem?
Hi Ariel Yael as I understand you plan gene expression analysis with qPCR.
1. Before you start design primers and probes (TaqMan chemistry i recommend), please find full information about your target genes. Sometimes you may find only 1 mRNA for gene. But now, regarding the NGS, you may find 5, 10 or more alternative transcript variants. Some of them could have different effects! Be careful. For example: you plan to study 2-3 genes and it is probably 2-3 mRNAs or 20-30 mRNAs. in the last case NGS is preferable.
2. Regarding qPCR data normalization. Reference genes. before you start the experiment, please use commercial kit for determknation of the most stable genes under your experiment conditions. Do not use reference gene without it, from example from the research of another author. Check it preciously.
3. Use triplicates for all samples.
4. Do not use multiplex.
5. Assess reaction effectiveness with standard curves. At least once before experiment. Use for it dilutions of your sample cDNA.
6. Regarding comparison of different plates...it depends on your hands and other factors. If possible use robotic station.
7. Try to work with homogeneous samples. I.e. the same time of sample collection and handling, the same conditions and time for storing, the same chemistry and equipment and so on.
At least.
Good luck in your interested research
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