I am currently analyzing the effects of a low protein diet (so compared with a normal protein diet) on the level of expression of certain genes expressed at several early developmental stages. Upon being collected at the pluripotent stage from the preimplantation blastocyst (E 3.5), the cells have been maintained in culture and subsequently neurally induced. I am testing actually how does a low protein diet during the preimplantation development only, affects neural induction in vitro. The genes that I am looking at include Nanog, Oct-4, Nestin, Sox2, Sox1, Pax6, Gata4, Brachyury, Map ii, Beta3- Tubulin. However, some of these genes will not be expressed in my samples (randing from a pluripotent stage until approximately day 5 of neural induction - Pluripotent stage, Day 1 of neural induction, Day 2, Day 3, Day 4, Day 5). Some of the genes would have appeared later if Neural induction would have been continued.
=====> Therefore, how do I get to calculate primer efficiencies for the primers pertaining to a gene that is not expressed in my samples? Because if I am not doing primer efficiency calculation, I won't be able to analyze my data using 1 of the 3 methods (Livak, Pfaffl, qBASE+).
I assume I should make use of some sort of positive control? I actually do require to present a positive control (apart from a no replicate -NRT- and no template controls -NTC) as well, right?
But is it okay to use another tissue to test your primer efficiency?
CRITICAL: How can I check gene expression across tissue for pluripotency-related genes such as Nanog? Obviously they would not be expressed in adult tissue.
ALSO CRITICAL: Is there any sort of Mouse gene expression Atlas that also includes the postnatal development? (I am aware of EMAGE that is about prenatal stages only)
CRITICAL: Did anyone heard about qBASE+ software?
THANK YOU!!
Sorry, I'm not going to answer all of your question, just some.
1) It is not okay to use another tissue to calculate primer efficiency, you will get different efficiencies depending on cDNA sample.
2) If your gene is not expressed, the best way to show it is to run an agarose gel of the qPCR sample after amplification. No band = no gene.
For your positive control and standard curve, you can just order some synthetic DNA matching your expected template as described in the MIQE guidelines: "Quantification calibrators may be purified target molecules, such as synthetic RNA or DNA oligonucleotides spanning the complete PCR amplicon, plasmid DNA constructs, cDNA cloned into plasmids, RNA transcribed in vitro, reference RNA pools, RNA or DNA from specific biological samples, or internationally recognized biological standards".
Maybe interesting for your other questions:
The NCBI gene page has some of the mouse ENCODE transcriptome data shown at :
https://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=DetailsSearch&Term=71950
Hi Diana, for your control you can use also Luciferase RNA that you add during your RNA extraction (as external gene to normalise with), you can use the same protocol for your genes that are not expressed (add some Nanog RNA if not expressed!), that's for your primers efficiency. Using RNA will avoid any bias of RNA extraction or cDNA synthesis.
Then, you can simply check the expression of Nanog in your adult tissue.
MGi, could be a good option for mouse gene expression or GEO from NCBI or even uniprot.
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