Fpkm and log2fold change and pcr validation

Question

[mounavisirekolam]

For a gene(xist) that I am trying to study. I have four cell stages (esc,normal,cond1,cond2).

When I compare fpkm values across these conditions cond1 had fpkm value 1.5 times than normal.

I performed pcr for normal stage and cond1 but I got band in normal stage that is thick enough but in cond1 its very less.

Why is that ?

Answer 1

[bioruffo]

Endpoint PCR is qualitative (semi-quantitative at best when you really try for it), and it isn't really the correct method to determine a 1.5x fold change. Differences in the thickness of the amplicon band may reflect variations in RNA extraction, cDNA synthesis, contaminants, or sample differences. This could all cause variations in amplification; and all in all, the plateau phase of PCR ends up flattening differences anyways. So, unfortunately endpoint PCR isn't the way to go. And in truth, RT-qPCR would also have some difficulty in determining a 1.5x difference. You're up for a bit of a challenge here.

[mounavisirekolam]

when comparing across cell differentiation status is it good to trust fpkm value or log2fold change