Dear all,
I am new in relative quantification and gene expression field and I will appreciate very much if you could advice me for my situation.
I am dealing with fish parasite and I want to check the expression of Gene A in different developmental stages of this parasite. For checking the relative expression of my Gene A I need a good housekeeping gene. I have chosen 10 candidate genes ( suggested as a housekeeping genes) for similar organisms and I would like to validate or estimate which one from these 10 would be the best for me. My whole problem is that my parasite is always mixed with the host (fish ) blood or tissue and basically I do not know the exact amount of my input parasite RNA (or CDNA). Does anyone deal with similar case or could you advice any paper that would be useful for my case.
Many thanks
Anush
In your case, if you can measure the concentrations of each of your total RNA extracts prior to reverse transcription, then you will want to use the exact same amount of total RNA (which would be fish RNA + parasite RNA) per each RT. After RT, you would assume that each cDNA is of equal concentration. Then, in the qPCR, you would load the exact same amount of cDNA to all reactions (after you have found out what that good dilution is by having done a preliminary 15-point 1:2 dilution series of a representative sample for establishing your valid dilution range for your relative dilution standard curve and so on).
In the end here, then, you would not be using reference/housekeeping genes at all, but would merely be using equal cDNA sample loading into all the qPCReactions as your normalizing tool.
Choosing a housekeeping/reference gene that would address blood and tissue content equally across all samples would have to be done carefully if you choose to go that direction.
But, here, I am merely pointing out a possible alternative for you.
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