Hi every body,
I have the following queries please guide me in simple and step wise,
1) Which type of gene is required for knowdown experiment in cells, I mean how to select a gene?
2) How to measure the expression level of a gene in a specific age (i.e. 1week)in cell?
Several criteria are used to identify a gene for "knockdown" in any cell type, including whether a knockdown would inhibit cell proliferation. In this case, a stable knockdown would not be possible. One can use PCR approach or immunoblotting to assess the knockdown.
While using primary cells, you have to complete the knockdown and assessment of phenotype within the next 1-2 passages to look at the effects prior to loosing the characteristics of primary cell type during passage. To do this, use adenoviral expression system as they are quick and efficient. When you want to select some novel targets, screen for genes associated with the function that you are looking for. For example, when you want to assess cell proliferation, go for cyclins, p18, p21, p27, p53, etc. If you want to assess differentiation (say for eg. adipocyte - assess PPARgamma) and likewise any specific gene that mediates the process for other properties of cells.
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