I intend to validate the results of gene expression data from RNA sequencing/ microarray using Immunohistochemistry. In short I want to check the expression pattern on a protein level. How many slides (2 sections/slide) will I need to validate/ check the expression pattern of single gene?
Thanks,
Vrushali
More of a question of slides, I think this is a question of numbers of cell. I guess that RNA-seq / microarrays gave you some differentially expressed genes between different cell types and that you want to check if it's the case at the protein level. First IHC won't be quantitative, even if you have sections and cells on the same slide and processedc equally the same way. In the case of genes expressed in one cell type and not at all in others, you'll be able to confirm / infirm the RNA data, but if you're looking for cell types expressing a same gene at different (and not so far away) levels, it might be difficult to confirm it be IHC.
Good luck !
To avoid reporting results from adjacent tissue sections as independent observations, I suggest a minimum of 5 non-adjacent sections;10 sections over 5 sldes, considering either the first sectiond or the second section of every slide from a given sample. As objectively as possible, select 2-3 representative images from each set of 10+ sections and compare the representatives from each set of sections. So, 2-3 negative control, 2-3 positive control, 2-3 experimental sample selected from at least 5of your 10 or more sections. I hope that makes sense. Basically, avoid adjacent sections and select a few representative fields from a group of 5 or more slides. More is better but try to judge representative images from similar numbers of non-adjacent sections.
Best of luck!
Diane
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