Quantification of gene expression by in situ hybridization: finding and using the raw values

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Hello,

My understanding is that the ISH data expressed as “expression energy” is not quantitative with respect to the actual amount of mRNA, because the relationship between color intensity and mRNA quantity is not linear.

  1. However, the data should be somehow linear within a certain range with the same probe. This would allow for relative quantitative comparisons of the gene expression in different brain areas within a certain range of expression. Is there any estimation of the range of expression energy values within which the signal can be considered fairly linear?

  2. I found that for some genes different probes have been used at different developmental stages of the mouse brain. In the case of the gene Cdh5, one probe was used for embryonic samples ( RP_080912_03_B02 ) and another for P56 ( RP_070219_02_C05 ).
    In this case the expression energy is quite different between the embryonic and postnatal samples.
    If a different probe was used, it is not possible to compare expression levels between stages.
    Is there any reason why two different probes were used?
    Has this been a common practice and should be checked for each gene?

Thank you for your help
Juan

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