I have done the qRT-PCR, where I took the same concentration of total RNA for both the leaf and root tissues of same plant event. I got the different expression of same gene in same event means I got more expression in leaf compare to root.
I want to know that is it due to the variation in mRNA concentration or anything else?
Thanks in advance!
Alkesh Hada For performing qRT-PCR you must have prepared primer (specific for template) If you are getting more expression it means that desired template is more.
Both root and leaf cells have different function so the expression level of a same gene can be different wrt requirement.
It is quite likely that their expressions will be different since the activation profile of genes involved are different on different parts of the same plant. Although I do not work with plants but I can surely suggest you to use at least one endogenous marker (house-keeping gene) in the same context and check its expression.
The expression of house-keeping gene(s) should be same in every case. If you find any discrepancy in the expression of house-keeping genes, then there must be an error in the actual experiment you have designed, be it a mechanical error or a physiological one. If you find that the expression of house-keeping gene(s) is/are same and yet there is/are change in expression in other gene(s), then it must be due to their different activation profiles.
Different tissue types, even within the same organism, can have different inhibitors of cDNA synthesis and polymerase chain reactions (PCR), which can affect qRT-PCR results. Different levels of DNA contamination in your total RNA extracts also could alter your mRNA:total nucleic acid measures. You need to include some housekeeping genes (genes consistently expressed under all conditions and in all tissues!) with which to standardize levels of your gene of interest.
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