Big Big problem with my Q-PCR results - (Nov/18/2008 )
Hi everybondy,
I am performing Q-PCR on osteoblastic cells in order to study the
effect of a new biomaterial on osteoblastic cell adhesion,
proliferation and differentiation. I have 2 groups, test and control
with 5 different time points (for each time point, Q-PCR is performed
in triplicates). When I analyse my results using the dilution values,
my HKG (GAPDH, Rsp 15) vary greatly, but when I use the C(t) values,
they are quite stable. I performed Q-PCR several times for each HKG and
then analysed the results with GeNorm. Is my approach correct?
Should I use the C(t) values or the dilution ones? I ran several (i mean many) PCR, from different RT, and I still have the same problem, so pleeeeeeeeeeeeeeeease help.
P.S. I am using SybrGreen from BioRad on Miniopticon.
Thank you very much for your help.
You can calculate delta delta Ct for your target gene and get expression change in fold. Based on the change in fold you can evaluate the effect of the method.
I am performing Q-PCR on osteoblastic cells in order to study the
effect of a new biomaterial on osteoblastic cell adhesion,
proliferation and differentiation. I have 2 groups, test and control
with 5 different time points (for each time point, Q-PCR is performed
in triplicates). When I analyse my results using the dilution values,
my HKG (GAPDH, Rsp 15) vary greatly, but when I use the C(t) values,
they are quite stable. I performed Q-PCR several times for each HKG and
then analysed the results with GeNorm. Is my approach correct?
Should I use the C(t) values or the dilution ones? I ran several (i mean many) PCR, from different RT, and I still have the same problem, so pleeeeeeeeeeeeeeeease help.
P.S. I am using SybrGreen from BioRad on Miniopticon.
Thank you very much for your help.
my HKG (GAPDH, Rsp 15) vary greatly, but when I use the C(t) values,
they are quite stable.
First I'm pretty confused by this, what are the analysis using dilution/Ct values?
There are basically two methods to analyse, one uses only Ct values - 2^delta-delta Ct, other uses efficiency-correction for each gene, efficiencies usually derived from dillution series.
Thank you microlight for your help. I will try to do it this way.
my HKG (GAPDH, Rsp 15) vary greatly, but when I use the C(t) values,
they are quite stable.
First I'm pretty confused by this, what are the analysis using dilution/Ct values?
There are basically two methods to analyse, one uses only Ct values - 2^delta-delta Ct, other uses efficiency-correction for each gene, efficiencies usually derived from dillution series.
Thank you Trof for your help. When I said dilutions I actually meant standard curves method.