RT-PCR-CELL NUMBER - (Sep/03/2011 )
i have a small doubt.Should we take equal number of cells(invitro condition)/quantity of tissue for RNA isolation to convert to cDNA for real time pcr?
i have read many papers either they specify equal quantity of RNA or cDNA for expression studies.some of my friends suggest that we should also CONSIDER EQUAL NUMBER OF CELLS for RNA isolation is that correct?
i am little confused.could any one help me in this regard.
For real-time quantification you should have a reference gene(s) that is expressed equally in your samples, that way it compensates for small differences in concentration.
However it is still recomended to use the same amount of cDNA and put a same amount of RNA into RT reaction, since RT efficiency differs in samples that don't have the same concentration of RNA.
Usually RNA is measured and adjusted accordingly, sometimes there are different tissues and different RNA concentrations so I don't see much point in taking precisely the same number of cells, when the isolation process can differ too and you still have to measure RNA at the end and have to normalise to a stable reference gene.
thank you very much