I have problems determining the reliable dilution for my qPCR. I have repeated the experiment 3 times, it seems that I have gotten different results for every experiment. However by comparing the cycle number, it didn't have any significant different from each experiment.
My question is how to determine the most reliable dilution factor for my template to run with this primer.
*I have used gAPDH to normalize my data (you may see the attachment below).
Thanks
The expression of you target gene is significantly lower than the reference gene, variation will tend to increase with higher Ct values and in my experience data gets very noisy after cycle 33, for this reason I would advise you to user the template at a concentration range of 50-12.5 ng/ul.
It is not uncommon to run a couple of housekeeping genes to normalize your data. GAPDH tends to be quite questionable when metabolic differences occur in the cells. (Post drug treatment or in cancerous cells). We have moved to using 18s as the control in our qRT-PCR to compensate for this. Another issue that tends to occur is the efficiency of your gene of interest may be quite low if you have a large amount of variability between experiments. To best recognize this by using a normal sample serially diluting it 2-fold for five or more dilutions.
This is a link to a way to calculate primer efficiency
http://www.thermoscientificbio.com/webtools/qpcrefficiency/
Your Ct values are very high, and your gene seems to be expressed at really low levels relative to GAPDH. Anything >30 tends to get noisy, because your starting template is present in such low amounts, so you're more susceptible to random variations in the amount. If you try diluting your cDNA less, the results will probably be more reliable.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques