Protocol Online logo
Top : Forum Archives: : Real-Time PCR

lower R square for my standard curve - (Oct/17/2007 )

unsure.gif Hi Everyone:

I just run my first standard curve to test TFF3 gene primers. five serial dilution of 5 fold. efficiency is good,0.97, however, R square(0.945) is lower than what was said in an online tutorial (>=0.985).

Just want to know what is the cutoff value you guys use for R square, and what can be done next to bring up that R square.

BTW, i am using SYBR green chemistry and concentration for both primers are 150nM and Mg concentration is 2.5mM.


Hi there,

i generally consider r2 values of >0.99 as fine. what you can try is to use another threshhold value, where the correlation of values is better (some software has a "best r2" feature for determination of that value) or you should have a look at your standard curve, if the curve flattens at either end, the linear area of your pcr is probably in between, so removal of the highest or lowest standards from calculation can help sometimes. Or you can try to concentrat more while pipetting smile.gif