I just want to check the expression of a particular mRNA throughout the different tissues (Liver, Kidney, Brain, Muscle, Gills, Intestine, Ovary, Skin, Spleen etc.) in a control/untreated fish. If I go through the Real-time PCR taking all the tissues together, then what method should I follow to meet my quiry?
Your main issue here is that you need a reference gene (ideally, two or three genes) that you trust to be valid for comparing expression in all your tissues of interest. This is non-trivial, especially for such a broad panel of tissues. I suggest (assuming you haven't done this already) you take a panel of tissues and a selection of candidate reference genes and establish suitable ref genes empirically (via geNorm, normfinder, etc -or ideally multiple assessment algorithms).
Once you have suitable reference genes, it's fine: you can run all your tissues on one plate quite happily (assuming by 'running all the tissues' you mean isolating RNA/synthesising cDNA from each tissue, then loading a different tissue cDNA in each series of wells).
If you have a lot of samples and you're forced into deciding between "all the samples on a plate, for one gene" and "some of the samples on one plate, for multiple genes", choose the former: always choose the former. Each of your samples (for a given genes) will be comparable to each of your other samples (for that gene) provided they are run on the same plate.
Together? Normally we must separate the tissues and do specific qPCR for each tissue separately. And last we compare the results between the different types of tissues.
Dear Gattai,
Is it theoretically/ practically correct if I run all the tissues (Untreated only) in a single plate for a particular mRNA along with endogenous control?
If I run separately (only the Untreated tissues) then what should be the basis of comparison? How I will compare?
For the comparison study, should I quantify the mRNA absolutely or should I follow the ∆∆Ct method?
Your main issue here is that you need a reference gene (ideally, two or three genes) that you trust to be valid for comparing expression in all your tissues of interest. This is non-trivial, especially for such a broad panel of tissues. I suggest (assuming you haven't done this already) you take a panel of tissues and a selection of candidate reference genes and establish suitable ref genes empirically (via geNorm, normfinder, etc -or ideally multiple assessment algorithms).
Once you have suitable reference genes, it's fine: you can run all your tissues on one plate quite happily (assuming by 'running all the tissues' you mean isolating RNA/synthesising cDNA from each tissue, then loading a different tissue cDNA in each series of wells).
If you have a lot of samples and you're forced into deciding between "all the samples on a plate, for one gene" and "some of the samples on one plate, for multiple genes", choose the former: always choose the former. Each of your samples (for a given genes) will be comparable to each of your other samples (for that gene) provided they are run on the same plate.
How to check tissue specific distribution of mRNA using Real-time PCR?
To best of my understanding of your question....if your are using qPCR machine with 96 well plate.....you can run all of your samples in the same run with endogenous control and then can compare the expression level by comparing delta Ct values...
Regards
This will depend on the number of samples you have. Because we use a 96 well plate for running qPRC, your samples should be within this number. One way is that you can run the qPCR of the different tissues together and compare their Ct values. You can also run them separately and compare the Ct vaues. There is no straight jacket way to perform this. You only need a good reference gene that has been tried and tested. It is usually ideal to use two or more reference genes in the case where you are not really sure of the quality of reference gene you are using
If you want to compare the abundance of mRNA between tissues, you can try absolute quantification, definitely you have to consider that you have to start from the same amount of tissue and adjust your results for the total amount of RNA isolated. Conversely, you can try several HK genes (5-6) and stay with the median value to normalize your target genes.
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