I use the 3C_qPCR technique to study the physical interaction between two gene loci. for this I have designated the specific primers for these two regions; first of all, the standard curve must be drawn up using different concentrations diluted to 1/10 on the scale of 1 fmol/ul of DNA synthesized in vitro. For DNA concentrations of 1 fmol and up to 10-4 fmol, I have good CT proportional to the dilution and therefore an E close to 100% whereas from concentrations of 10-5, the CT are no longer in the curve while still DNA is detected which causes me problems at the E level.
I have to go down to 10-5 and 10-6 fmol because my 3C libraries that I want to locate their CT in the curve to determine the SQ give CT higher than those obtained for the range 1 up to 10- 4
my question: is it the problem of primers and how to solve the problem?
and can I determine the SQ of my 3C libraries even if their CT is outside the standard curve?
Hello Elias Gerges
The term "primers" typically refers to short nucleotide sequences used in molecular biology to initiate DNA amplification through techniques like PCR (polymerase chain reaction). If you're experiencing problems related to primers, it could indicate issues with primer design, quality, or experimental conditions. Here are some common problems related to primers and potential solutions:
Regarding your second question, if the cycle threshold (CT) values of your 3C libraries fall outside the range of your standard curve, it may be challenging to accurately determine the absolute starting quantity (SQ) of your target. Standard curves provide a reference for estimating the initial quantity of your target based on its CT value. If the CT values are beyond the range of your standard curve, it's difficult to make accurate quantifications using that particular curve.
However, you may still be able to make relative comparisons or qualitative assessments using the CT values. For example, you can compare the CT values of different samples to determine the relative abundance of your target sequence. Keep in mind that these comparisons are not absolute quantifications but can still provide valuable information about the relative differences between samples.
To accurately determine the SQ of your 3C libraries outside the standard curve, you may need to consider alternative methods such as digital PCR or qPCR with a new standard curve that covers the expected CT range. Alternatively, you could perform a dilution series of a known sample within the expected range and generate a new standard curve to quantify your samples accurately.
It's recommended to consult with experts in your specific field or molecular biology techniques for further guidance on addressing primer-related issues and quantification challenges.
[1]Rameshwar Gupta*,
Research Scholar,
Department of Lifelong Learning & Extension
CSJM University, Kanpur, U.P. India
Email id: [email protected]
Whatsapp/Mobile: +918630831266
https://orcid.org/0000-0003-3761-3591
[1]Ph.D. Student, Department of Lifelong Learning & Extension, CSJM University, Kanpur, U.P., India, Email id: [email protected], Mobile: 8630831266, https://orcid.org/0000-0003-3761-3591
*Single author