I've run some qRT-PCR experiments looking at expression of TH (tyrosine hydroxylase) and a number of other genes of interest in mRNA sections from human brain tissue. I have already run delta delta Ct analysis to look at the change in expression of my genes using a housekeeper gene. However my question is whether it is possible to use Ct values of a gene which is stably expressed in a particular cell population of interest (e.g. TH expression in dopaminergic cells) as a housekeeper to find the relative expression of genes on interest in that particular population? Or is my only other option is to perform laser capture microdissection to isolate this particular cell population from the tissue and extract mRNA from it to it? I.e. is there any other way to look at the expression of genes in a particular population of interest if the mRNA sample is contaminated with many other cell types? All suggestions greatly appreciated!
In our lab we usually look at whole tissue mRNA expression by qPCR then look at protein expression by immunohistochemistry to confirm that there are changes at the protein level and locate the activity.
To look at RNA in specific cells you could try in situ hybridization to look at where your RNA is present in tissue sections.
https://www.lifetechnologies.com/au/en/home/life-science/cell-analysis/cellular-imaging/in-situ-hybridization-ish.html
Whatever you do, bear in mind that you should use more than one reference gene for normalisation and that the amplification efficiencies of target and reference genes should be similar. Search for publications by M Pfaffl and J Vandesompele for further explanations and read the MIQE guidelines published in Clin Chem in 2009. Laser capture micro dissection works well and, provided you're sure RNA quality is acceptable, can give reliable results.
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